w:u.-.. r... ~ magma-rm 1 . " .' it “no 0.4 y «.5! .- "- H a ‘ I n 3 O 3 . f" 4 .J i . r .- -.-. 2 a» .vn' '1. '5' P: f‘ (3.! 33".? .112N...A‘.-.- r14»; {ow-«lawn. w l D . l '. «I. “my“ was ‘_ :5). J ' '. ‘vr . ‘ ‘14:)9'4 ‘4 no: Veda This is to certify that the dissertation entitled The Rea u l a hora Role cl: 3" HadVOXj'S“ Mekhijlalutohjl Coent. me A Reduct—OSC l“ 533 Production by Schist'osown W3 presented by Elizabeth Ann \étnclelA/aq has been accepted towards fulfillment of the requirements for P1"- D‘ degree in Pharmacology +Toxicol037 Major professor Date 6 q MS U i: an Affirmative Action/Equal Opportunity Institution 0-12771 MSU LIBRARIES 4—3—— RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. THE REGULATORY ROLE OF 3-HYDROXY-3-M ETHYLGLUTARYL COENZYME A REDUCTASE IN EGG PRODUCTION BY SCHISTOSOMA MANSONI by Elizabeth Ann VandeWaa A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Pharmacology and Toxicology 1986 ‘1 (O -J ABSTRACT The Regulatory Role of 3-Hydroxy-3-Methylglu taryl Coenzyme A Reduc tase in Egg Production by Schistosoma mansoni by Elizabeth Ann VandeWaa The schistosome egg is responsible for most of the pathology associated with schistosomiasis. Eggs deposited in host tissues elicit an immune response, resulting in granuloma formation with subsequent tissue fibrosis, which can ultimately produce death in the host. In studies on the regulation of schistosome egg production, it was found that mevinolin, a competitive inhibitor of the enzyme 3-hydroxy-3—methylglutaryl coenzyme A (HMG CoA) reductase was able to significantly reduce egg production by the parasite both in v_itr3 and in M. The in m effect of the drug was reversible by mevalonate and farnesol, both products of the HMO CoA reductase-catalyzed reaction. Furthermore, these lipids were also able to stimulate oviposition in the schistosome. The presence of HMG CoA reductase activity was identified in this parasite, in microsomal fractions prepared from worm homogenates. In the assay system used, mevinolin at 10 uM was found to reduce parasite enzyme activity by 7596. HMG CoA reductase activity is known to be both suppressible as well as inducible by mevinolin. To measure if induction of the enzyme occurred in the schistosome, parasites exposed to low concentrations of the drug in m were assayed for activity. Here, a doubling of HMG CoA reductase activity was measured. When these parasites were incubated in drug-free medium for a Elizabeth Ann VandeWaa determination of egg production, schistosomes exposed to low doses of mevinolin i_r_i_ iii? produced five times more eggs i3 m than did control parasites. Mevinolin disrupts egg production in the schistosome and affects HMG CoA reductase activity in this parasite, indicating that a product in the pathway catalyzed by this enzyme is essential for egg production by the worm. When products of HMG CoA were analyzed in the parasite following mevinolin exposure ill £92: it was found that the polyisoprenoid lipids were diminished in the parasite by 70%. Polyisoprenoid lipids function in glycoprotein synthesis to transfer carbohydrate to newly formed proteins. Since glycoproteins are major constitu- ents of the schistosome egg, the effect of mevinolin on HMG CoA reductase may be reflected in a reduction in parasite egg production. Therefore, HMG CoA reductase may be playing a regulatory role in the production of eggs by _S_. mansoni. To my husband John, whose love, encouragement and sense of humor helped me to maintain my perspective and continue to enrich my life. ii ACK NOW LEDG EM ENTS I would like to acknowledge the support and guidance of my advisor, Dr. James L. Bennett. Dr. Bennett's confidence in me was instrumental in initiating and directing my graduate career, and I greatly appreciate his encourgement and commitment to my training. Iam particularly grateful for the many opportunities I have had in Dr. Bennett's lab which have contributed to my scientific development. I would also like to thank those who have given me their time and knowledge, contributions which were vital to the completion of this thesis. They include my committee members: Dr. Jeff Williams, Dr. Emmett Braselton, and Dr. Theodore Brody. I would like to thank Dr. Gary Mills for his interest and efforts in this project, as well as for his friendship. Special thanks also to Helen Cirrito whose assistance was excellent and always cheerfully given. Finally, I gratefully acknowledge the love and support given me by my parents, Alice and Cornelius DeRuiter. Their faith in me and their interest in my education have been inspirational and invaluable. iii TABLE OF CONTENTS LIST OF FIGURES LIST OF TABLES LIST OF ABBREVIATIO NS INTRODUCTION A. 903 F. The Parasite: Schistosoma mansoni 1. The life cycle of _S_. mansoni 2. Egg production in S. mansoni The Role of the Egg in the Pathology of Schistosomiasis Chemotherapy of Schistosomiasis by Suppression of Egg Production 1 . Past studies on the inhibition of schistosome egg production 2. The effect of mevinolin on schis tosome egg production HMG CoA Reduc tase 1. Effects of mevinolin on HMG CoA reductase 2. Induction of HMG CoA reductase by mevinolin 3. Regulation of HMG CoA reductase The Role of Nons terol Lipid Metabolites of Mevalonate 1. Characteristics of the lipids involved in glycoprotein synthe- sis 2. The role of polyisoprenoid lipids in the glycosylation of proteins Summary and Proposed Research MATERIALS AND METHODS A. B. Schis tosomes Incubation of Parasite E Vitro: Egg Production Assays 1. Exposure to mevinolin i_n vitro iv Page vii ix aural-a (13's) 10 15 l7 18 19 20 21 27 28 28 29 TABLE OF CONTENTS (continued) C. Administration of Mevinolin to Schistosome-Infected Mice D. Histological Preparations of Liver Tissue E. Hydroxymethyl Glu taryl Coenzyme A Reduc tase Assays 1. Preparation of microsomal frac b’ons 2. Standard HMG CoA reduc tase assay 3. Inhibitor studies F. Radiolabehng of Parasite Lipids I__n Vi__tr__o G. Characterization of Radiolabeled Lipids 1. Extraction and isolation of labeled schis tosome lipids 2. Purification of labeled lipid components H. Determination of the Effect of Mevinolin on Lipid Intermediates, Lipid Oligosaccharides and Glycoproteins of S. mansoni 1. Preparation of reaction mixtures for the isolation of lipid intermediates, lipid Oligosaccharides and glycoproteins 2. Isolation of lipid intermediates and lipid Oligosaccharides 3. Kinetic studies 1. Protein Determinations J. Liquid Scintillation Counting K. Statistical Methods RESULTS A. Regulation of Schistosome Egg Production: _Ir_1_ Vitro Studies with Mevinolin 1. Effect of mevinolin on i_n_ v_i___tro egg production by _S_. mansoni 2. Effect of metabolites of HMG CoA on schistosome egg production 3. Reversal of mevinolin's effect in vitro by mevalonate and farnesol 4. Stimulation of egg productoin by _S_. mansoni following i_rl vi tro or i_n_ vivo exposure to mevinolin B. Regulation of Schistosome Egg Production: 111 Vivo Studies with 2. Addition of lipids to the culture media 3. Determination of egg production Mevinolin Page 29 29 29 30 31 31 32 33 33 34 34 35 36 36 37 38 38 40 41 41 41 41 43 47 52 TABLE OF CONTENTS (continued) HMG CoA Reduc tase Assays 1. Characteristics of the HMG CoA reductase-catalyzed reac- tion in S. mansoni Inhibition of Schistosome HMG CoA Reductase in the Enzyme Assay System Q 1’32 Effects of Mevinolin on HMG CoA Reductase Activity in S. mansoni The Effects of Mevinolin on Lipids of S. mansoni 1. Normal distribution of 14C-labeled lipids in the parasite 2. The effect of mevinolin on the distribution of labeled lipids in S. mansoni 3. The e)ffect of mevinolin on lipid intermediates (polyisoprenoid lipids DISCUSSIO N A. 9‘2“” P9?” The I_n Vitro Effects of Mevinolin on S. mansoni 1. Effect of mevinolin on i_n v_itro egg production by S. mansoni 2. Effects of metabolites of— HMG CoA on egg production by S. mansoni in vitro Reversal of mevinolin's effect on schistosome egg production 3. 4. Stimulation of egg production by S. mansoni following i3 vitro or ir_1_ vivo exposure to mevinolin The I_n_ V__i_vo Effects of Mevinolin on S. mansoni Egg Production Isolation of Schis tosome HMG CoA Reductase Inhibition of Schistosome HMG CoA Reductase by Mevinolin in the Enzyme Assay System _111 Xiy_o Effects of Mevinolin on Schis tosome HMG CoA Reductase The Effect of Mevinolin on the Lipids of S. mansoni The Potential Role of Polyisoprenoid Lipids in the Synthesis of Schistosome Eggs SUMMARY AND CONCLUSIONS BIBLIOGRAPHY vi Page 54 54 63 63 68 68 77 82 92 92 92 93 95 96 98 100 102 103 104 106 111 10 11 12 13 14 15 16 re LIST OF FIGURES External characteristics of male and female S. mansoni Life cycle of S. mansoni Anatomical features associated with egg production in female S. mansoni Chemical structure of mevinolin Structures of HMG CoA and the acid form of mevinolin Domain map of HMG CoA reductase Catalytic role of HMG CoA reduc tase The branched pathway of mevalonate metabolism Biosynthesis of lipid-linked intermediates Role of lipids in the glycosylation of proteins Formation of complex glycoproteins Cellular sites of glycoprotein synthesis Flow diagram for the isolation of 14C-mannose-labeled lipid intermediates, lipid Oligosaccharides and glycoproteins of S. mansoni ~ Effect of mevalonate on i3 vitro egg production by S. mansoni exposed to mevinolin Effect of farnesol on i3 vitro egg production by S. mansoni exposed to mevinolin Gross pathology of livers removed from vehicle- and mevinolin-treated mice vii Page 11 12 13 14 16 22 23 25 26 39 45 48 55 LIST OF FIGURES (Continued) Fiflre 17 18 19 20 21 22 23 24 25 26 27 Histological preparations of livers from vehicle- and mevinolin-treated mice Histological preparations of livers from vehicle- and mevinolin-treated mice The effect of various amounts of microsome on HMG CoA reduc tase activity in S. mansoni Time-dependence of the HMG CoA reductase—catalyzed reaction in microsomes prepared from S. mansoni Effect of mevinolin on HMG CoA reduc tase activity Effect of IE \Liyg doses of mevinolin on schistosome HMG CoA reduc tase Radioscans of TLC plates spotted lvxith extracts from 14C- acetate labeled schistosomes, C- evalonate labeled schistosomes, and media containing C-acetate but no parasites Radioascans of the lipid classes of S. mansoni The incorporation of 14C-acetzate into total lipids of S. mansoni following exposure to mevinolin or its vehicle Radioscans of lipid intermediates of S. mansoni Radioscans of lipid intermediates of S. mansoni exposed to mevinolin versus control viii Page 58 59 61 64 66 69 72 73 83 86 88 Table 10 11 LIST OF TABLES Effect of mevinolin on ir_1_ vitro egg production by S. mansoni Effects of mevalonate and farnesol on 111 vitro egg produc- tion by S. mansoni Effect of dolichols on egg production by S. mansoni in the presence and absence of mevinolin Effect of mevinolin on i_n vitro schistosome egg production following i2 vitro exposure to the drug Effect of mevinolin on i3 vitro egg production by S. mansoni following i3 vivo exposure to the drug Percent distribution of label into neutral lipids of S. mansoni Percent distribution of label into polyisoprenoid lipids of S. mansoni Percent distribution of label into polar lipids of S. mansoni Percent distribution of label into lipids of S. mansoni exposed to mevinolin Percent distribution of label into total lipids of S. mansoni under various incubation conditions Effect of mevinolin on the formation of lipid intermediates and glycoproteins in S. mansoni homogenates ix Page 42 44 50 51 53 76 78 79 80 81 90 Asn DPG EDTA G DP HC HMG CoA LPC LPE MG-DG MVA NADPH PA PC PE PI PI-l,2 ,3 PL-l, 2,3 PS RNA SD SE SEM Ser Sph TG TLC UDP LIST OF ABBREVIATIONS asparagine diphosphatidylglycerol ethylenediaminete traacetic acid guanosine diphospha te hydrocarbon 3-hydroxy-3-methylglutaryl coenzyme A lysophosphatidylcholine lysophosphatidylethanolamine mono-, diglyceride mevalonate nicotinamide—adenine dinucleotide phosphate phosphatidic acid phosphatidylcholine phosphatidylethanolamine phosphatidylinositol polyisoprenoid l, 2, 3 polar lipid 1, 2, 3 phosphatidylserine ribonucleic acid standard deviation sterol ester standard error of the mean serine sphingolipids triglyceride thin-layer chromatography uridine diphosphate INTRODUCTION The schistosome is a helminth parasite which currently afflicts more than 300 million people worldwide. Schistosoma mansoni, one of the three major schistosome species, is endemic to much of Africa, as well as to parts of South and Central America. The schistosomes as a group differ from other trematodes in that these parasites exist as separate sexes within the definitive host. Schisto- soma mansoni dwells in the portal and mesenteric blood vessels of its host, wherein the larger male parasite folds its body around the slender female forming the gynecophoral canal in which the female lies during copulation (Figure 1). The pairing and copulation of the adult parasites in the host results in the production of up to 300 eggs per worm pair per day (Moore and Sandground, 1956). The formation of eggs by S. mansoni is not only essential to the maintenance of the life cycle of this parasite, but the eggs are also responsible for much of the pathology associated with bilharziasis (schistosomiasis). A. The Parasite: Schistosoma mansoni 1. The life cycle of S. mansoni The life cycle of the schistosome is depicted in Figure 2. The life cycle of this parasite is complex, involving an intermediate host and a definitive host, the latter is in a mammal in which the parasite reaches sexual maturity. Within the definitive host, the sexually mature female produces eggs which are deposited into the venules of the mesentery. The eggs secrete lytic enzymes Figure 1. Drawing illustrating the external characteristics of male and female S. mansoni. OS, oral sucker; VS, ventral sucker; GC, gynecophoral canal. MAMMAL FRESH WATER (in feces) /' @ egg \ H ‘ miracidium (free—living) paired adults (mesenteries) l T sporocysts ”—7( (in snails) ! i lung stage 1 schistosomulum \ cercaria @ skin (free-living) F penetration skin stage schistosomulum Figure 2. Life cycle of S. mansoni which help them to rupture the venule and penetrate the intestine. Once in the lumen of the intestine, the eggs are carried out of the host via the feces. The eggs contain a larval form of the parasite known as the miraci- dium. Upon contact with fresh water the eggs hatch, releasing the free-swimming miracidia. The miracidia then penetrate a snail which serves as the intermediate host in which the miracidia multiply, asexually, and differentiate into cercariae (Faust and Hoffman, 1934). The cercariae are released by the snail, and subsequently penetrate the skin of man (or other mammal), burrowing down to the peripheral capillary bed. Once in the bloodstream, the parasites are carried to the right heart and lungs, from which they migrate to the portal circulation. After a maturation period within the portal vessels, the adolescent worms migrate into the mesenteric venules where they pair, and oviposition begins. The adult parasites may live in the blood vessels of man for as long as 30 years actively producing eggs (Faust g1 31:, 1934). 2. Egg Production in S. mansoni The production of eggs by S. mansoni is considered an important process for two reasons. First of all, egg production is necessary to maintain the life cycle of the parasite, as discussed above. Secondly, the deposition of eggs in host tissues by S. mansoni is the primary cause of the pathology associated with schistosomiasis. Egg production by the parasite is a complex biological process, about which little is known. It has been established that the female schistosome contains an organelle called the ootype in which vitelline cells are combined with the ovum (egg). The vitelline cells release droplets which coalesce to form a thin membrane around the uterus, at which time egg shell formation occurs (Stephen— son, 1947; Smyth and Clegg, 1959) (Figure 3). The egg shell, which consists primarily of cross—linked proteins, is highly resistant to immunolytic (Stenger e_t 131., 1967) and chemical degradation (Stephenson, 1947). Furthermore, the Ill. 1 .Sma Jamccoo E9; @3363 EomcwE .m 23:8 5 couozvoa mam 5:5 a8308$ 893mg BEEch< .m 229m n 955 goat . a: . 22:15:52 proteinaceous shell possesses no antigenic activity and thus plays little role in the induction of pathology associated with schistosomiasis (Boros and Warren, 1970; Lichtenberg and Raslavicious, 1967). Rather, the egg shell seems to function only to protect the developing miracidium from the host defense mechanisms, allowing the egg to safely pass to the external environment. In chronic schistosomiasis, more than fifty percent of the eggs produced by the worms may remain within the definitive host, being carried by the portal circulation to the liver, where they become trapped (Warren, 1978). Eggs which remain within the host are ultimately responsible for the pathology of schistosomiasis. B. The Role of the Egg in the Pathology of Schistosomiasis The pathology resulting from infections with S. mansoni may be divided into three general stages (Brown, 1973). The first of these is the pathology associated with the penetration of the cercariae through the skin and subsequent develop- ment of the adult worms. Pathological signs here consist mainly of mild cutaneous lesions and petechial hemorrhages as the developing parasites migrate through host tissues. The second stage of schistosome-associated pathology manifests itself upon active egg production and extrusion. Here, eggs, via the secretion of collagenase- like enzymes, penetrate through the vascular endothelium, causing local foci of inflammation and immune cell infiltration. Penetration of the eggs into other tissues (such as the organs of the digestive tract) causes a similar response in these tissues. The production and penetration of schistosome eggs leads directly to the third stage of the pathological response, that of proliferation and repair. As stated earlier, most of the eggs produced by the schistosome are retained by the host, wherein they may become trapped in host tissues, particularly in the liver. Here, the presence of the egg elicits the formation of granulomas or non- necrotizing pseudotubercles. Granulomas consist of layers of epithelioid cells, fibroblasts and giant cells, surrounded by plasma cells and eosinophils. Formation of the granuloma is instigated by the secretion of a number of soluble antigens by the intact schistosome eggs (Pelley g; 9.__1., 1976; Hamburger gt _al_., 1976). These proteinaceous antigens are produced by the miracidium developing within the egg shell, and diffuse through the shell into the surrounding tissues, thereby triggering granuloma formation (Hang e_t a_1., 1974). The immunological response of the host to the egg antigens is vigorous, resulting in the formation of granulomas up to 100 times the volume of the egg itself (Winslow, 1967). The result of these large areas of cellular proliferation and infiltration in the liver is manifest as an occlusion of portal blood flow to these regions, which compromises liver function. Several other clinical signs and symptoms are characteristic of schistoso- miasis. These include hepatomegaly, splenomegaly, esophageal varices and portal hypertension (Warren, 1978). Again, these overt disease symptoms are due largely to the presence of schistosome eggs within the host and, more specifically, to the immunological response of the host to the eggs. Recognizing the major role which the egg plays in schistosomiasis, a number of investigators have attempted to suppress oviposition in this parasite. Clearly, the inhibition of egg production by the parasite presents an attractive chemotherapeutic target whereby schisto- some-induced pathology can be reduced. C. Chemotherapy of Schis tosomiasis by Suppression of Egg Production 1. Past studies on the inhibition of schistosomeggg production Attempts to inhibit oviposition in S. mansoni have been aimed at reducing or eliminating schistosome-associated pathology known to be caused by the eggs. One such attempt focused on inhibiting schistosome phenol oxidase, the enzyme which serves to cross-link the protein in the eggshell, causing eggshell hardening (Seed gt a_1., 1979). Inhibition of parasite egg production by the suppression of phenol oxidase proved to be of little therapeutic value however, when it was discovered that the most active inhibitors of the enzyme were copper chelators which affected various mammalian copper-containing enzymes as well. Similar results were seen with various metabolic inhibitors (Lee and Michaels, 1968) which also adversely affected the host as well as the parasite. Other attempts to interfere with schistosome egg production have focused on the endocrinology underlying oviposition. It is well accepted that stimulation of the development of the female reproductive tract of the schisto- some is brought about through a close physical relationship with the male parasite (Sagawa 91gb 1928; Severinghaus, 1928). It was thought therefore, that the male worm may be secreting a hormone-like substance which would regulate fecundity in the female. To assess this possibility, several antispermatogenic compounds (Jackson gg a_1., 1968; Davies and Jackson, 1970) and steroid hormones (Morrison e_t a_1., 1986) were examined for their effects on schistosome egg production. In all of these studies, none of the compounds tested were efficacious in disrupting oviposition in the schistosome. 2. The effect of mevinolin on schistosome gggproduc tion In the process of determining the effects of steroid hormones on schistosome egg production, a steroid synthesis inhibitor, mevinolin, was examined for its effects on parasite oviposition. Mevinolin is a potent inhibitor of the enzyme 3—hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA reduc tase) (Alberts gt a_l_., 1980) which regulates the metabolic pathway for the production of cholesterol and several non-steroid lipids such as terpenes, dolichols and ubiqui- I'IOIIBS. 10 Chemically, mevinolin belongs to the class of substituted hexahydro- naphthalene lactones (Figure 4). It is a small molecule, with a molecular weight of 404 daltons. Mevinolin was first isolated from die fungus Monascus ruber by Endo (1979) who called it monacolin K. Later, the same compound was isolated as a metabolite of the fungus Aspgrgiuus terreus by Alberts and coworkers at Merck, Sharp and Dohme Research Labs (1980). The active form of mevinolin is the hydroxy acid (opened lactone) structure. The structure of the acid form of this agent contains a portion that resembles the HMG portion of HMG CoA, perhaps contributing to is ability to inhibit HMG CoA reductase (Figure 5). Although there exist several structural analogs of mevinolin, it is the most potent inhibitor of HMG CoA reductase in this series of compounds. Results with mevinolin in the schitsosome indicate that it is a highly efficacious inhibitor of egg production in S. mansoni both i3 vitro and i3 vivo. D. HMG CoA Reductase HMG CoA reductase (E.C. 1.1.1.34) is a transmembrane glycoprotein of the endoplasmic reticulum, consisting of a soluble and a membrane bound domain (Figure 6). HMG CoA reduc tase has a subunit molecular weight of 90 kilodaltons and, although it is a glycosylated protein, is carbohydrate moieties are not required for is enzymic activity (Liscum e_t a_1., 1983). This enzyme has a short half-life, turning over within 2-4 hours i_rl 1’22 (Edwards and Gould, 1972). The regulation of HMG CoA reductase is currently an area of intensive study because of the important role this enzyme plays in the synthesis of sterols in mammals. Specifically, HMG CoA reductase catalyzes the rate-limiting step in the synthesis of the steroid nucleus, catalyzing the conversion of 3—hydroxy—3- methylglutaryl coenzyme A to mevalonate (Figure 7). Mevalonate is then converted, in several steps, to farnesyl pyrophosphate which serves as a precursor 11 MEVINOLIN Figure 4. Chemical structure of mevinolin. 12 "0 coon OH 0 /\ru\o H 3c CH, .. cc / H,c SCoA Figure 5. Structures of HMG CoA (left) and the acid form of mevinolin (right). l3 SOLUBLE DOMAIN MEMBRANE BOUND *—-——— 531100 DOMAIN “—62 ROG ~; NH2 4% aa ag/ZE’ a S : ]'-COOH & c c CHO Figure 6. Domain map of HMG CoA reductase (adapted from Luskey, 1985). 14 CH CH NADPH NAOP+ CH l 3 I \/ I 3 HOZC - CH2 - E - CH2 - f‘- SCoA eear- HOZC - CH2 - C - CH2 - CHZOH OH H OH 3-Hydroxy-3—methylglutaryl SCoA Mevalonate Enzyme: HMGCoA Reductase (EC l.l.l.34) Regulation of Enzyme: Cholesterol (and non—sterol products??) produced by this pathway in- fluences the rate of transcription of the reductase gene (Luskey gt_gl,, 1983). Figure 7. Catalytic role of HMG CoA reductase. 15 to a variety of compounds (Figure 8). In mammals, the major product of this pathway is cholesterol, however, the other producs derived from mevalonate may be essential for a variety of cellular functions in various organisms. 1. Effecs of mevinolin on HMG CoA reductase The inhibition of HMG CoA reductase by mevinolin is competitive with respect to HMG CoA, and non-competitive with respect to NADPH, a necessary cofactor for the reaction (Endo gt a_1., 1976; Endo, 1980). As stated earlier, the structure of mevinolin contains a portion which resembles the HMG moiety of HMG CoA (see Figure 5), this structural similarity is thought to contribute to the compeh'tive nature of the inhibition of HMG CoA reductase by the drug. Furthermore, mevinolin, which has two methyl groups on is decaline ring moiety is a more potent inhibitor of the enzyme than are structural analogs of the drug with one or no methyl groups on the decaline ring (Endo gt 1%, 1979). Additional- ly, HMG alone is insufficient to inhibit HMG CoA reductase (Brown _e_t_ EL: 1978), indicating again that the decaline ring is prominently involved in the inhibition of the enzyme. Therefore, it appears that both the HMG-like moiety as well as the decaline ring structure contribute to the inhibitory action of mevinolin on the reductase. The precise mechanism involved in the inhibition of HMG CoA reduc tase by mevinolin is not entirely understood. The reaction catalyzed by the enzyme involves a two-step process. First, the enzyme interacs with HMG CoA to form a binary complex, this structure then forms a ternary complex with one molecule of NADPH (Tanzawa and Endo, 1979). It has been suggested (Rogers and Rudney, 1982) that mevinolin binds to the enzyme causing a conformational change in is tertiary structure. This structural change prevens the enzyme from forming a ternary complex with HMG CoA and NADPH and, as a result, the subsequent formation of mevalonate from HMG CoA is reduced. 16 Acetyl CoA HMG CoA Mevinolin HMG CoA Reductase I1 Mevalona te I Mevalonate Pyrophospha te I Isopentenyl Pyrophosphate l Geranyl Pyrophospha ta 1 Insec t Plan t Caro tenoids Juvenile Farnesyl 4. Chlorophylls Hormones ‘* . Pyrophosphate Gibberellic Acids (terpenes) Squalene Ubiquinones Sterols Dolichols Figure 8. The branched pathway of mevalonate metabolism. 17 2. Induction of HMG CoA reduc tase by mevinolin The discovery that HMG CoA reductase is an inducible enzyme was made by Goldstein and Brown (1977), who showed that when cells dependent on cholesterol were deprived of this lipid, HMG CoA reductase activity was increased. Since mevinolin deprives cells of mevalonate and is metabolites, including cholesterol, the ability of this drug to induce HMG CoA reductase was examined. Although mevinolin is a potent inhibitor of HMG CoA reductase, it has also been shown that this drug can induce enzyme activity in cells exposed to it. When human fibroblast cells in culture were exposed to compactin (a structural analog of mevinolin) a 15-fold increase in enzyme synthesis was seen within a 24- hour period (Brown _e_t a_1., 1978). In i3 \_ri_vg studies, ras administerd 40 mg/kg compactin or mevinolin in their diet showed a significant induction of liver HMG CoA reductase (Endo e_t g, 1979; Tanaka e_t a_l_., 1982). The mechanism of induction of this enzyme is not yet clearly understood. In experimens by Sinensky and Logel (1983), the increased amount of enzyme detected following exposure of Chinese hamster ovary (CHO) cells to mevinolin was attributed to a decreased rate of enzyme degradation. Thus, mevinolin was found to extend the half-life of the enzyme in these studies. The vast majority of research into the induction of HMG CoA reductase, however, seems to indicate that the increased amount of the enzyme seen upon induction is due to increased enzyme synthesis, not decreased degradation in the presence of inducers. Specifically, rats fed mevinolin showed a significant induction in HMG CoA reductase activity which was correlated with an increase in messenger RNA (mRNA) for the enzyme (Clarke gt a_1., 1983; Liscum gt g, 1983). Therefore, mevinolin both inhibis and induces HMG CoA reductase activity. It inhibis the enzyme by binding to it, altering is conformation as discussed earlier. The ability of mevinolin to induce HMG CoA reductase suggests 18 that the drug, by inhibiting the enzyme, blocks the synthesis of a product which may normally feed back to regulate it. The question of the regulation of HMG CoA reduc tase is considered further below. 3. Regulation of HMG CoA reduc tase The HMG CoA reductase-catalyzed reaction is responsible for the production of lipids may be involved in several cellular functions. Because of the importance of these lipids to the cell, the regulation of HMG CoA reductase is a tightly controlled cellular process, dependent on the balance between the rates of synthesis and degradation of the enzyme. The rates of synthesis and degradation of HMG CoA reductase are thought to be controlled by a multivalent feedback mechanism via producs in the metabolic pathway catalyzed by the enzyme. In mammalian systems, it has been found that increased dietary cholesterol caused a decrease in the rate of enzyme synthesis (Erickson gt 51., 1975) subsequent to a decrease in mRNA for the enzyme (Chin e_t 111,, 1982). Cholesterol has also been found to cause the HMG CoA reduc tase which is present in the cell to be catalytically less active (Edwards _e_t 31., 1980; Arebalo e_t 31:, 1982). Furthermore, mevalonate, the immediate product of the HMG CoA reductase catalyzed reaction has been shown to have a similar inactivating effect on the enzyme in ras (Arebalo e_t g_l., 1982), although this effect is most likely due to the metabolism of mevalonate to cholesterol in these animals. While the feedback control of cholesterol on HMG CoA reductase has been extensively studied, the regulation of the enzyme by other metabolic producs in the enzyme pathway has also been examined. In these studies, mevinolin or compactin was used to determine what other producs of the pathway could exert regulatory control on HMG CoA reductase. Carson and Lennarz (1979) noted that compac tin caused abnormal gastrulation of embryos of 19 the sea urchin, Strorgylocentrotus mirpuratus. This effect was reversible by supplementing the embryos with dolichols, however, cholesterol supplementation did not prevent or reverse abnormal gas trulation in the presence of compactin. In another study where nonsterol regulators of HMG CoA reductase were examined, CHO cells, whose growth was inhibited by compactin or mevino- lin, were used (Brown and Goldstein, 1980). Here, the effect of these drugs on CHO cell growth was completely reversed by the addition of mevalonate to the cell culture, while the addition of squalene or cholesterol had no ability to prevent the growth inhibition. These studies, combined with the experimens on a variety of organisms from insect larvae (Monger gt a_l_., 1982) to plant seedlings (Bach and Lichtenthaler, 1982), in which cholesterol was unable to reverse the effects of mevinolin or compactin, suggest that different cell types vary in their regulatory mechanisms of HMG CoA reductase, relying on a variety of metabolites of mevalonate for this purpose. In conclusion, it is known that HMG CoA reductase is regulated by a feedback mechanism, but the product of this enzyme's metabolic pathway which exerts this regulation is not necessarily cholesterol. With respect to the schistosome, this is a very important concept, for the schistosome lacks the ability to synthesize cholesterol gig EV—O (Meyer _e_t_ $1., 1970). Thus, the effect of mevinolin on parasite egg production and the regulation of HMG CoA reductase in S. mansoni must center around a nonsterol lipid. The synthesis of this lipid, therefore, may be crucial not only to the regulation of parasite HMG CoA reduc tase, but may also be essential for the production of eggs by S. mansoni. E. The Role of Nonsterol Lipid Metabolites of Mevalonate Although the schistosome is incapable of cholesterol biosynthesis, mevinolin, a sterol synthesis inhibitor, was able to exert an effect on parasite egg 20 production. Thus, some other product formed in the HMG CoA reductase catalyzed pathway must be critical to oviposition in the parasite. While HMG CoA reductase catalyzes the rate-limiting step in the synthesis of cholesterol, studies by Mills and Adamany (1978), wherein inhibition of the enzyme resulted in decreased dolichol and subsequent glycoprotein synthesis in aortic smooth muscle cells, suggested that HMG CoA reduc tase may also catalyze the rate-limiting step in dolichol synthesis. Inhibition of dolichols, which are polyisoprenoid lipids involved in glycoprotein synthesis, by mevinolin would, therefore, affect the glycosylation of proteins. The effect of mevinolin or compactin on the synthesis of these lipids occurs at concentrations of these agens higher than those needed to completely block sterol synthesis (Filipovic and Menzel, 1981). In the schistosome, where sterol synthesis does not occur, the ability of mevinolin to interfere with polyisoprenoid lipids and glycoprotein synthesis can be studied without the concern that other parasite lipids (of cholesterol synthesis and metabolism) are also being affected by the drug. 1. Characteristics of the lipids involved in glycoprotein synthesis The polyisoprenoid lipids are known to function as lipid intermediates in the synthesis of glycoproteins. Specifically, these lipids serve as carriers of multiple sugars, which they subsequently transfer to forming peptides. Chemical- ly, these specialized polyisoprenoid lipids are referred to as dolichols, and exist as phosphorylated 111-saturated polyprenols, varying in chain length from 80 to 110 carbons or more (Hemming, 1974). The basic unit of these large carbon chains is a five-carbon isoprene, several of which are linked together to form the hydropho- bic dolichol molecule. Dolichols are embedded in the surface of the endoplasmic reticulum, where glycosylation of proteins takes place. Polyisoprenoid lipids such as the 21 dolichols are known to be involved in the synthesis of several carbohydrate- containing molecules in many systems. They are involved in the synthesis of bacterial extracellular polysaccharides (Osborn, 1969), cell-wall mannan—protein complexes in yeast (Parodi, 1977), yeast cell walls (Hopp e_t $1., 1978), and plant and animal glycoproteins (Waechter and Lennarz, 1976; Elbein, 1979). Several examples of these lipids may be seen in Figure 9. 2. The role of polyisoprenoid lipids in the glycosylation of proteins As stated above, polyisoprenoid lipids function as carriers of carbo- hydrates, transferring these sugars to peptides in the formation of glycoproteins. In this capacity, these lipids accept sugars from sugar—nucleotides until a large lipid-oligosaccharide is formed. The oligosaccharide portion is then transferred from the lipid to the polypeptide e_n_ 9122 (Behrens, 1974; Elbein, 1979). In this manner, it is speculated, these lipids act as intermediates, aiding the transport of hydrophilic sugars into or through the membranous environment of the endoplas- mic reticulum (ER) so that polymerization can occur (Elbein, 1979). The scheme illustrating the role of these lipids in glycoprotein synthesis is depicted in Figure 10. In the early portion of the scheme (upper left), the phosphorylated lipid acceps two N-acetylglucosamine residues from the nucleotide. This is followed by the stepwise addition of nine mannose residues, with the subsequent addition of three glucose residues (Schachter and Roseman, 1980). At this point, the oligosaccharide is ready to be transferred to the protein in the ER. The oligosaccharide is generally linked to the amino group on the side chain of an asparagine (Asn) residue of the protein (Kornfeld and Kornfeld, 1976). These asparagine—linked Oligosaccharides, referred to as N-linked oligosaccha- rides, are the most prevalent in glycoproteins. However, Oligosaccharides linked to the protein via the hydroxyl group on the side chain of a serine, threonine, or 22 Involvement of polyisoprenoid alcohols in the synthesis of pepfidoglycan (Hopp), Salmonella O-antigen (Osborn), and yeast mannans (Parodi). as General Structure: H(CH2 - C = CHCH2)110H Waechter and Lennarz demonstrated that animal tissues produce lipid-linked Oligosaccharides as intermediates in glycoprotein synthesis. CH3 CH3 1(1) General Structure: H(CH2) - c = CHCH2)nCH2CHCH2CHZO - I" - o' o' dolichylmonophosphate n = 15-20 Monoglycosyl derivatives of dolichylmonophosphate exist (Waechter and Len- narz). O 0 II II General Structure: GICNAcO - 1" - O - P - ODol l O O a—linked Man-O-fi-ODOI O B -linked Figure 9. Biosynthesis of lipid-linked intermediates. 23 The Role of Lipids in Protein GlycosLlation - cow r /‘ M—GN-CN-P-PLip id . . M—M—M ' QI-m-P-P-Llpid P-M UDP-(3N M’M\M d ‘M—Q‘Jm-P-P-Lipi GN-P-P-Lipid M'M/ M-M—M DP—G UDP- M'M\M M__M/ ‘M—QI-CN-P-P-Lipid 9-1.1me ocean-M my 1 M-M’/\M-CN-G‘I . -XIO(AsnXSerXIO(- M—M-M -A.snXSer- Wmse Muetylglucosanire G-Glucose Xsknim acid Figure 10. Role of lipics in the glycosylation of proteins. 24 hydroxylysine (O—linked Oligosaccharides) may also be found in glycoproteins on occasion (Wagh and Bahl, 1981). The glycosylation of a protein in the ER is the first step in the synthesis of complex glycoproteins. In order to modify the glycoprotein for is specific purpose, processing and remodeling of the oligosaccharide occurs. An example of the processing of the oligosaccharide is shown in Figure 11. The first step in this process, which occurs exclusively in the Golgi apparatus, is the trimming of the three terminal glucose residues. The oligosaccharide is then generally processed further to form an inner core structure, consisting of two N- acetylglucosamine and three mannose residues, still linked to asparagine. The terminal region of the oligosaccharide may now be modified by the addition of a variable number of different sugars, including N—acetylglucosamine, fucose, galactose and sialic acid (Hubbard and lvatt, 1981), all added by their appropriate glycosyl transf erases. Once the glycoproteins are synthesized, they are packaged into secretory vesicles and released from the cell if they are secretory proteins. Glycoproteins may also be retained by the cell to serve in a structural capacity, or to act as enzymes. Figure 12 presens a summary of glycoprotein synthesis, along with the cellular sites for each step in this process. In summary, polyisoprenoid lipids located in the ER play a vital role in the synthesis of glycoproteins; without these lipids, the formation of N—linked Oligosaccharides would be impossible. Thus, these producs of the HMG CoA reduc tase catalyzed pathway perform a key cellular function and, in certain cells they may be involved in the regulation of HMG CoA reductase as well. 25 O]. igornarmos idic Chains M=Mamrnse M-Acetylglucosamine G=Glucose N-L-cm-ik F /M-C.N—ci1-Asn N—L—GN—M N==N-Acetyl.nalraminic A: id L=Calac tose F=Eucose Figure 11. Formation of complex glycoproteins. 26 .mfimficzm £39583» Ho mafia 32:30 .2 9:63 \ Equamltli 180‘ ( \Il..(\lI\/~uz 1000 \ , co <).\ 2095 ii £2 Bans r558 .n “259. $0.5m .e \ \ \ \ a 121/)... emoE 2: 3 man A<>$c mus—835:. .31 20595 50:8 25:: Ho 8583 o5 E REESE?” 2m: mcowwnsofi .::oE>oE 8 comoaxo EOmcwE .m an cob 262a wmo ob; c|_ co 855—39: .«0 “ovum .3 2:9,.“ 46 3 955 099-2952 E : -2 .2 r O O F /6 3663 'ON 91 noq u icon 47 was able to restore egg production to control levels in the presence of 10 11M mevinolin (Figure 15). The egg production in these parasites was again signifi- cantly higher than that seen in worms incubated only in 10 11M mevinolin (p< 0.01). Therefore, the MVA and farnesol data indicate a true ability of these lipids to reverse mevinolin's effect on schis tosome egg production. In a third study, dolichols, a downstream metabolite of farnesol, were examined for their ability to stimulate egg production or reverse mevinolin's effect. In these studies, dolichols at concentrations shown to be effective in other i2 _v_i_tr_c_>_ systems (Carson and Lennarz, 1979) were unable to reverse the effect of mevinolin, and also had no ability to stimulate egg production in the parasite (Table 3). Since radiolabeled dolichols were unavailable, it is unknown whether the failure of dolichols to exert an effect was due to an inability of the parasite to use these lipids, or due to lack of penetration of these lipids into the parasite in culture. 4. Stimulation of egg production by S. mansoni following in vitro or in vivo exposure to mevinolin The enzyme HMG CoA reduc tase has been shown to be highly inducible by low doses of mevinolin or its structural analog, compactin (Brown 93 Q” 1978; Skalnik 91 al., 1985). To determine whether or not this induction phenomenon also occurred in the schistosome, assays were carried out which measured i2 Q32 egg production by the parasite followng acute exposure to mevinolin i3 \_ri_tr_g or i_g Egg. In the i_n_ :13 studies, paired schistosome were exposed to 10 11M mevinolin for 24 h, after which they were placed in drug-free media for an additional 72 h incubation. Following incubation, it was noted that parasites exposed to mevinolin acutely produced twice as many eggs as did the controls, and four times as many eggs as did parasites exposed to mevinolin for the entire incubation period (Table 4). 48 40.5.8 @323an So...“ 353:6 Ewart—Ea 8306.: xm€3m< .mcoB 335$ awn 23m fines—>2: 2: 3 min _omocawm 3:5 93qu .mcowwEEzflg m “mam: aw mo ..Q m + con 9: 3:82am: :5 zoom £22.39: 2: 3 man 3858 .31 3025 .838 £52? .«o 8533 2: E poeotoa 0.63 98.5355 .c:oE>oE 9. @0898 EOmcwE .w .3 5.52603 wwo oh; :I_ :o .0853 .«o «8.2m .3 FEMS 49 1504 100~ O ID 8111 u / oxsooa 200 80 40 20 FARNESOL [uM ] Figure 15 50 Table 3 Effect of Dolichols on Egg Production by S. mansoni in the Presence and Absence of Mevinolin [Dolichol] ug/ml [Mevinolin] 11M No. Eggs/Female/72 h o o 51.7: 5.9b 0 10 9.3: 5.7 10 o 64.1: 9.3 10 1o 17.4:15.7 20 o 58.6:11.3 20 10 15.8: 6.3 aPaired schistosomes were incubated in the presence or absence of dolichols or mevinolin as described in the Methods. bData are presented as mean number of eggs : S.D. 51 Table 4 Effect of Mevinolin on E Vi tro Schistosome Egg Production Following g Vitro Exposure to the Drug8 . Concentration zéggmvfrfi—o of Mevinolin No. Eggs/Female/72 h in Culture Media Vehicle 0 63.0:28.1b Mevinolin (10 11M) 0 117.8:32.3° Mevinolin (10 11M) 10 11M 29.3:22.7c aPaired schistosomes were incubated in the presence or absence of mevinolin after in vi tro exposure to the drug or its vehicle. bData are presented as mean number of eggs _+_ S.D. cSignificantly different from control, p < 0.05. 52 In a second group of studies, mice 35 days post-infection were dosed daily with 50 mg/kg mevinolin orally until day 45, when the parasites were retrieved. Here, parasites removed from mice treated with the drug and placed in drug-free media demonstrated a significant increase in egg production over parasites from mice treated only with vehicle (p< 0.01, Table 5). Schistosomes from either in v_ivg group always produced fewer eggs when maintained in 10 11M mevinolin in \_Iitr_o. Thus, it appears that schistosome HMG CoA reductase is inducible by mevinolin and this induction appears to be correlated with egg production i_n vitro. B. Regulation of Schistosome Egg Production: In Vivo Studies with Mevinolin The in y_i_vg effects of mevinolin were studied by dosing §. mansoni-infected mice with the drug utilizing several dosing regimens. It was found that daily doses of 50 mg/kg or 100 mg/kg mevinolin did not significantly reduce worm burden or pathology (as assessed by granuloma content in the liver) compared to mice dosed with vehicle on a comparable regimen. When mice were dosed from days 35-45 post-infection with 250 mg/kg mevinolin however, a significant reduction in pathology was noted while worm burden was not significantly changed. Pathological changes associated with mevinolin treatment at 250 mg/kg were assessed in two ways. First, gross liver pathology was compared between control and treated mice, and numbers of eggs deposited in each group of livers was determined. Secondly, livers from vehicle- and mevinolin-treated mice were sectioned and examined histopathologically. Changes in gross pathology between control and mevinolin-treated mouse livers were readily apparent upon sacrifice of the animals. Livers from mevinolin-treated mice were smaller (20-3096, based on weight) and, upon gross 53 Table 5 Effect of Mevinolin on E Vi tro Egg Production by S. mansoni Following Q Vivo Exposure to the Druga- In Viv 0 Concentration Treatment in ($332133; ia No. Eggs/Female/72 h Vehicle 0 60.4:32.6b Vehicle 10 11M 10.7: 7.3c Mevinolin (50 mg/kg) 0 321.8:9o.4° Mevinolin (50 mg/kg) 10 11M 6.3: 4.1c aParasites were incubated in the presence or absence of mevinolin following i_n_ vivo exposure to the drug or its vehicle. bData are presented as mean number of eggs : S.D. 0Significantly different from control, p < 0.01. 54 examination, appeared to contain fewer granulomas (Figure 16). Homogenization of livers from each group (n=6 livers per group per experiment) yielded an average of 45.496 fewer eggs from livers of the mevinolin-treated mice. Thus, the reduction in gross pathology seemed to be correlated with reduced egg deposition in the livers of treated mice. To better examine the pathological changes induced by mevinolin, histologi- cal sections were taken of livers from mice of both treated and control groups. Histopathologically, livers of mevinolin-treated animals were again strikingly different from controls. In one respect, the granulomas in livers of mevinolin- treated animals were much smaller than in vehicle-treated controls (Figures 17 and 18). This was probably attributable to the observation that in the drug- treated animals, generally speaking, only one egg was at the center of each granuloma. In the control animals, however, several eggs were involved in each area of granulomatous inflammation (Figures 17 and 18). Furthermore, in the drug-treated mice, histopathological examination by a trained pathologist revealed an average of 50% fewer mature miracidia in the deposited eggs than in granulomas of control animals. These results indicate an ability of mevinolin to interfere with schistosome egg production i_n \Lvo, an effect correlated with an amelioration of schitosome-associated pathology. C. HMG CoA Reduc tase Assays 1. Characteristics of the HMG CoA reductase-catalyzed reaction in S. mansoni Schistosome HMG CoA reductase activity has previously not been identified or characterized. However, using the method of Alberts e_t g. (1980) for measuring HMG CoA reductase activity in rat liver, some purification and preliminary characterization of schistosome HMG CoA reductase was accom- plished. It should be pointed out that the studies on this enzyme in the 55 Figure 16. Gross pathology of livers removed from vehicle-treated and mevinolin-treated mice. Mice were dosed daily with 250 mg/kg mevinolin or its vehicle, days 35-45 post-infection, after which their livers were removed. Top two livers were removed from mevinolin-treated animals, bottom liver was removed from a vehicle-treated control mouse. Magnification is 2X. 56 Figure 16 57 Figures 17 and 18. Histological preparations of livers from control and mevinolin-treated mice. Mice were dosed daily from days 35-45 post-infection with 250 mg/kg mevinolin. Tissue sections were made of livers from vehicle- treated infected mice (top photos) and drug-treated infected mice (bottom photos). Tissues are stained with hematoxylin and eosin, magnification is 100x. 58 Figures 17 59 Figures 18 60 schistosome were limited by the amount of activity recoverable from this parasite. The main objective, therefore, was to demonstrate the presence of this enzyme in _S_. mansoni and determine its response to mevinolin i_n m and in yi_vo. In rat liver, HMG CoA reductase activity has been isolated in microsomal fractions (Kleinsek e_t g, 1977). In the schitosome studies, parasite fractions including a "crude" pellet (resulting from 8,000 x g centrifugation of worm homogenate), a microsomal pellet (100,000 x g) and the supernatant remaining after ultracentrifugation of the microsome were assayed for HMG CoA reductase activity using the Alberts assay. The 8,000 x g pellet and the microsomal supernatant demonstrated no measurable HMG CoA reductase activity (data not shown); the enzyme activity was confined to the microsomal pellet. Once the enzyme activity was localized to the microsome, enzyme assays were run testing three different amounts of microsome, 5 ul, 10 111, and 20 111 (50, 100 and 200 ug of protein, respectively), for enzyme activity during a ten- 14 minute incubation. Each microsomal fraction was incubated with 5 uCi C-HMG CoA and the reaction mixture described in the Materials and Methods, and the generation of 14 C-labeled products of the reductase-catalyzed reaction was monitored. Radiolabeled products were eluted from Dowex-1 columns which retained the unreacted substrate, and fractions were counted with liquid scintilla- tion spectrometry. Counts per minute were converted to picomoles of radio- labeled product generated per milligram of microsome protein. The data from these experiments are presented in Figure 19. In order to standardize the assay, 10 111 (100 ug protein) was chosen as the amount of microsome used in all further enzyme assays. The time-dependence of the HMG CoA reductase activity in the microsome was next determined. Here, 10 ul worm microsome was reacted with l4C-HMG CoA for 1, 5, 10, 20 or 40 minutes, after which the reaction was 61 Figure 19. The effect of various amounts of microsome on HMG CoA reductase activity in S. mansoni. Enzyme activity was recorded after a 10 min incubation. Each bar represents the S.D. for at least 6 determinations. 62 400- 200“ 5305 9.52505 .25. ul Microsome Figure 19 63 terminated with 5 M HCl. The results of these assays are shown in Figure 20. In these studies, the HMG CoA reductase-catalyzed generation of product was linear for at least 10 min, after which products continued to be formed, but at a slower rate. D. Inhibition of Schistosome HMG CoA Reduc tase in the Enzyme Assay System Mevinolin has been shown to affect schistosome egg production in concen- trations ranging from 1 uM to 100 uM (see Table 1). To determine whether this range of concentrations was also effective to inhibit enzyme activity in the assay previously described, mevinolin, in concentrations of 0.1, 1.0, 10, and 100 uM, was included in the assay. The vehicle for mevinolin in these studies was 100% ethanol, as it was discovered that a DMSO vehicle completely abolished all enzyme activity whether or not mevinolin was present. Mevinolin in EtOH was added to the reaction mixture in a volume not greater than 196 of the total reaction mixture. All control assays (0 uM Mevinolin) contained the appropriate amount of EtOH. The sensitivity of schistosome HMG CoA reductase to mevinolin is shown in Figure 21. When plotted on a semi-logarithmic scale, the IC50 for the reaction was found to be approximately 7.5 uM mevinolin. Concentrations from 1 uM to 100 uM mevinolin all significantly inhibited the formation of radiolabeled products relative to the control (p < 0.05). E. In Vivo Effects of Mevinolin on HMG CoA Reductase Activity in S. mansoni That HMG CoA reductase appears to be highly sensitive to mevinolin has been shown. The data thus far have also demonstrated the ability of mevinolin to either inhibit or stimulate schistosome egg production, depending on dose or duration of exposure (Tables 4 and 5). It was, therefore, attempted to correlate 64 Figure 20. Time-dependence of the HMG CoA reduc tase-catalyzed reaction in microsomes prepared from S. mansoni. Each bar represents the S.D. of at least 6 determinations. 1000* 800-1 .5 2 2 600* n. m E g 400‘ .u 2 n. '5 200- E n. 65 minu tes Figure 20 40 66 Figure 21. Effect of mevinolin on HMG CoA reductase activity. Schistosome microsomes were incubated 30 min in the presence of various concentrations of mevinolin. Bars represent S.D. for at least 6 determinations. 67 L0 10 uM Mevinolin OJ r0 600‘ _ 0 0 4 £203 mE\.o=uoE .95. 200‘ Figure 21 68 the effects of mevinolin on egg production with effects on schistosome HMG CoA reductase activity. In these studies, the enzyme assay was run as previously described, utilizing 10 ul (100 ug protein) worm microsome per assay tube during a 30-min incubation time. The parasites used to prepare each microsome were treated differently, however. For one group of parasites, mice were closed from days 42-45 post- infection with 250 mg/kg mevinolin, after which the worms were removed from the mice, and microsomes prepared. In a second group, mice were dosed as above (250 mg/kg mevinolin for 3 days), but here, the parasites were aseptically removed from the mice and were incubated for 24 h in drug-free culture medium. In a third group, mice were dosed for 3 days with 50 mg/kg mevinolin. Finally, mice dosed with mevinolin's vehicle (2596 glycerol-196 Cremophor EL) were considered to harbor control parasites. Following microsome preparation from each group of worms the enzyme assay was run. Enzyme activity was found to be significantly lower than control activity (p< 0.05) in microsomes prepared from worms exposed to 250 mg/kg mevinolin in y_i_v_9_ (Figure 22). However, if similarly- treated parasites were incubated in drug-free medium for 24 h, enzyme activity was stimulated above control. Furthermore, schistosomes exposed to 50 mg/kg mevinolin i3 flv_o showed a siginficant (p< 0.05) induction of enzyme activity compared to that seen in control parasites. These data suggest that schistosome HMG CoA reduc tase activity can be induced or inhibited by mevinolin, and these changes in enzyme activity may affect schistosome egg production. F. The Effects of Mevinolin on Lipids of S. mansoni 1. Normal distribution of 14C-labeled lipids in the parasite In order to assess what effects, if any, mevinolin was exerting on parasite lipids, the normal pattern of lipid distribution in the schistosome was 69 Aobcoo E9: 3:98.26 EwocEmfi Ecomoaoe meBm< .mcouw:_E._8oo o :82 a: mo .Q.m “comma? 5:5 4.265 m2: wx\mE omS 5:832: : em a 9.35:3 .5 .waBE on .meE emu Jobcoov :oEEv: wEmoc 2: .63: 38388:: :23on .556 was 3350: oEzucm .o_o_:o> m: .5 53:39: mime: cm 5:839: wig: saw 5:: :26: 2o; 8:: @8035 .85 262 <00 02m oEOmBmEom :o 5:532: .«o memo: a :J «o 38.5 .mm v.59: i n L L .1 ‘- 71 examined. In these studies, parasite lipids were metabolically radiolabeled by incubating worms for at least 24 h in the presence of [U-14C1acetate or [2- 14Clmevalonl'ite. Following incubation, the radiolabeled lipids synthesized from these precursors were extracted and isolated. Radiolabeled lipids were purified by column and thin-layer chromatography (TLC) as described in the Materials and Methods. The TLC plates were scanned for radioactivity to determine the location and relative amounts of the radiolabeled lipids. A profile of the distribution of radiolabel into the lipids of S. mansoni is shown in Figure 23. As a control, media with radiolabel but no parasites were assayed to assure that the parasite, and not a contaminant in the culture, was responsible for the labeling pattern (bottom trace of Figure 23). For identification of the schistosome— synthesized labeled lipids, unlabeled authentic lipid standards were co- chromatographed with the parasite lipids; this allowed for tentative identification of some of the lipids synthesized by the parasite. The lipids of S. mansoni were chromatographically separated into 3 classes: neutral lipids, polyisoprenoids, and polar lipids (Figure 24). Each class was then further purified, so that the individual components of the classes could be separated and identified. The distribution of radiolabel into the neutral lipids is shown in Table 6. Identification of the lipid components was based on co- chromatography with lipid standards. The bulk of the labeled acetate was incorporated into the triglycerides (72.4%), while the labeled mevalonate was incorporated into a sub-class of compounds which behaved chromatographically like hydrocarbon—alcohols. No labeled mevalonate was associated with sterol esters, triglycerides, or mono- and diglycerides. The distribution of labeled acetate and mevalonate into the polyiso- prenoids and the polar lipids was also determined by TLC. While identification of the polyisoprenoid lipids was not complete, these lipids were shown to contain 72 .5050: 55 E “5955: E89? 0.; 05 E @2555: 5:: 83on :55 ScaembEoEo Bow 055m 5:: xocwzaom 5:: 8:5: was 59:5 2: 5:: c<> fig _ocwfioeuEhouoeoEo 5:5 :8 owbxo v.53 555E .5 moEomBmEom $8623 0: :5 358510 wEEmEoo 2:9: 5:: .5588 mEom 5282 8:555:70 .moEomBmEom 85:2 BSooinvaH Eoé flowbxo 5:5 83on 8qu 0.: .5 9582me .mm 5.5mm 73 2 952: ((((( 74 .83: :2: 305:: 5 8:82: :5 5.. 55:8 :55 5.53 :82: 655:2 :5 E .5555: 555»: 5:20: 5 5:555: 5:: m8 :5 0.; 55555 :0 585%. :05 .>:Q:._m8:Eo.Eo 5:35: :5: 055m 5:: 55:55 5 52.2.5: :55 5:5 :5: 5:: 5:: :8 85x: :53 55: £055.55 .55.? 45:50:10 55558 :5mE 5 c «a :8 53555 :53 856:5: .5852: .w 5 8:55 :5: :5 5 5:085:%H .3 5.5.9.: 75 5:5 MOE... ¢JOn £25 maze; J<¢h=mz 76 Table 6 Percent Distribution of Label into Neutral Lipids of S. mansoni Lipid l4C-Acetatea l4C-Mevalonateb Sterol Esters 21.3 __- Triglycerides 72.4 --- Mono-, Diglycerides 6.3 --- HC-Long chain alcohols -—- 100.0 aMean incorporation of label from 8 experiments. bMean incorporation of label from 6 experiments. 77 carbohydrate using the method of Dubois g a_l. (1956). Both acetate and mevalonate were incorporated ito the same three polyisoprenoid lipids (Table 7). At least nine polar lipids were labeled by 14C-acetate; these lipids were all shown to contain phosphorous by the method of Bartlett (1959), and were subsequently identified using lipid standards (Table 8). Radiolabeled mevalonate was incorporated into three polar lipids of S. mansoni. Two of these were present in relatively minor amounts, and did not co—chromatograph with any available standards. The major mevalonate-labeled polar lipid co-chromatographed with phosphatidyl choline, however, choline analysis of this lipid using the method of Wagner 2: a_l. (1961) and Beiss (1964), which utilizes the Dragendorff stain (bismuth nitrate and potassium iodide) to stain for choline, was negative for this lipid. Thus, while the mevalonate-labeled lipids were not identificable, the acetate—labeled lipids proved less difficult to identify. 2. The effect of mevinolin on the distribution of labeled lipids in S. mansoni 14C-acetate into parasite lipids was monitored in The incorporation of the presence of 10 11M mevinolin. In these studies, schistosomes were incubated for 24 h in 50 110i [U-14Clacetate plus 10 uM mevinolin or its DMSO vehicle. After incubation, the labeled lipids were divided into classes as described earlier. The effect of mevinolin was seen primarily on the polyisoprenoid lipids. Label incorporation into this class of lipids was reduced by approximately 70% in parasites exposed to mevinolin during incubation. The effects of mevinolin on each of the three polyisoprenoids can be ssen in Table 9. The neutral and polar lipid classes were not significantly affected by the drug. A summary of the distribution of label into each of the lipids of S. mansoni under each of the incubation conditions described is shown in Table 10. While the polyisoprenoid lipids and the mevalonate-labeled polar lipids remain 78 Table 7 Percent Distribution of Label into Polyisoprenoid Lipids of S. mansoni Lipid 14C—Acetatea 14C—Mevalonateb Polyisoprenoid 1 48.3 23.5 Polyisoprenoid 2 37.7 61.0 Polyisoprenoid 3 14.0 15.5 aMean incorporation of label from 8 experiments. bMean incorporation of label from 6 experiments. 79 Table 8 Percent Distribution of Label into Polar Lipids of S. mansoni Lipid 14C-Acetatea 14C-Mevalonateb Phosphatidylcholine 48.8 --- Lysophosphatidyl- 1.0 --- chofine Sphingolipids 1.1 _.._ Phosphatidylethanol- 18.3 ——- amlne Lysophospha tidyl- 7 . 2 --- ethanolamine Phosphatidylserine 10.5 --- Phosphatidic Acid 2.7 --- Phosphatidylinositol 3.2 -—- Diphosphatidyl- 7.2 --_ glycerol Unidentified Polar Lipid 1 --- 81.2 Unidentified Polar Lipid 2 --- 2.5 Unidentified Polar Lipid 3 --- 16.3 aMean incorporation of label from 8 experiments. bMean incorporation of label from 6 experiments. 80 Table 9 Percent Distribution of Label into Lipids of S. mansoni Exposed to Mevinolin Lipid A B Neutral Lipids 37.715.4 36.013.7 Polyisoprenoid 1 5.514.5 0.910.6" Polyisoprenoid 2 4.313.3 1.610.8‘I Polyisoprenoid 3 1.611.3 0.410.2“ Polar Lipids 50.917.5 61.1139 A. Parasites were incubated in [U-MCJacetate. 5 B. Parasites were incubated in [U-14C1acetate plus 10' M mevinolin. Asterisk indicates significance from control. 81 Table 10 Percent Distribufion of 14C—Label into Total Lipids of _S_. mansoni Under Various Incubation Conditions 14 Lipid“ 14C-Acetatea Meviggfifififikb 14C-Mevalonatec SE 7.2+1.o 3.1+o.2 --- TG 27.6+4.o 3o.1+2.4 --— MG-DG 2.9+o.4 1.5+o.1 --- HC-Long chain --- —-— 43,5+5,5 alcohols PI—l 5.5+4.5 0.8+0.4* 3.8+1.0 PI-2 4.3+3.3 1.8+0.6* 11.5+2.9 PI—3 1.6+1.3 o.5+o.2* 3.3+2.6 PC 23.4+5.5 25.1+1.5 --- LPC o.5+o.2 o.5+o.2 —-- Sph 0.6+o.1 o.1+o.o --- PE 1o.4+4.4 15.4+4.2 -- LPE 4.1+1.7 5.2+1.5 —-- PS 5.5+2.2 4.9+1.8 —-- PA 1.4+0.5 5.4+2.o — PI 1.6+0.4 0.3+0.1 -..- DPG 3.5+0.8 5.3+o.3 --- PL-1 --— --- 25.9+3.1 PL—2 --- --- o.s+o.7 PL-3 --- --- 5.2+1.6 aMean 1 S.D. for 8 experiments. bMean 1 S.D. for 5 experiments. cMean 1 S.D. for 6 experiments. * Key to Abbreviations: SE = Sterol esters; T6 = Triglycerdies; MG-DG = Mono-and Diglycerides; RC = hydrocarbons; PI 1,2,3 = Polyisoprenoid 1, 2, 3; PC = Phosphatidylcholine; LPC = Lysophosphatidylcholine; Sph = Sphingolipids; PE = Phosphtaidylethanolamine; LPE = Lysophosphatidylethanolamine; PS = Phospha- tidylserine; PA = Phosphatidic acid; P1 = Phosphatidylinositol; DPG = Diphospha- tidylglycerol; PL-1,2,3 = Polar lipid 1, 2, 3. Asterisk = significantly different from control. 82 unidentified, lipids which co-chromatographed with known standards and were identifiable by their chemical characteristics were tentatively named. Finally, it was shown earlier that HMG CoA reductase activity in the schistosome is inducible (see Figure 22), and the induction of activity after 13 M exposure to mevinolin was correlated with increased egg production (Table 4). To measure if i3 Ligg induction of the enzyme caused increased label incorporation into total lipids of the schistosome, parasites were exposed to 10 11M mevinolin for 24 h after which they were removed from the drug, and were placed in culture with [U-14C]acetate for up to 72 h. The results of this experiment showed that by 24 h, the amount of label incorporated into total lipid was significantly lower (p<0.05) in mevinolin-exposed worms as compared to control parasites (Figure 25). However, by 48 h, the drug-treated worms had recovered, and had begun to incorporate a significantly higher amount of labeled acetate into the total lipid. By 72 h, the time point at which egg production is usually measured, the amount of radioactive acetate incorporated into schisto- some lipids remained significantly higher than in the control worms. Egg production at this time point was double in the mevinolin-treated worms as compared to control parasites (data not shown). 3. The effect of mevinolin on lipid intermediates (polyisoprenoid lipids) The effect of mevinolin on lipid intermediates (i.e., the polyisoprenoid lipids) in §. mansoni was assessed by measuring the ability of these lipids to serve as carriers for sugars in the synthesis of glycoproteins before and after exposure to mevinolin. In order to do this, it had to be established that the schistosome contained lipids capable of carrying sugars. For these studies, homogenates were prepared from paired parasites, and GDP-[U-14C1-mannose was incubated with the homogenate. After a 30-min incubation, the incubation mixture was ex- tracted with chloroformzmethanol (2:1 v/v) to remove the lipid intermediate 83 Figure 25. The incorporation of 14C-acetate into total lipids of S. mansoni following 24 h exposure to 1 uM mevinolin (dashed line) or its vehicle (solid line). Fifteen paired parasites (n=6) were incubated in the presence of mevinolin (1 11M) or DMSO for 24 h, afterlwhich they were removed from the drug and placed in media containing 5 uCi C—acetate. At various time points, the parasites were extracted with chloroform:methanol (2:1 v/v), and the extract was counted by liquid scintillation spectrometry. Bars represent S.D. of six determinations. Asterisk represents significant difference from control. cpm into lipid 5000- 2500‘ 84 l I 48 72 hours after drug exPosure Figure 25 85 (lipids carrying 1 mannose residue), then the mixture was re-extracted with chloro form:methanol:water (1:1:0.3 v/v/v) to remove lipid oligosaccharides (lipids carry- ing 2 or more mannose residues). The extracts were spotted onto TLC plates which were developed as described in the Materials and Methods. Upon scanning the plate for lipids carrying radioactive mannose residues, a single lipid inter- mediate (Rf 0.73) was found (Figure 26, upper trace). Similarly, a single lipid- oligosaccharide was detected when the l:1:0.3 extract was scanned for radioacti- vity (Rf 0.41, Figure 26, lower trace). Since these sugar-carrying lipids were detected in the schistosome, the sensitivity of these lipids to mevinolin was next determined. Here, homogenates were prepared from parasites incubated for 24 h 12 £92 in the presence of 10 uM mevinolin or its vehicle. The homogenates of these parasites were then incubated in the presence of GDP-[U-MCI-mannose, and were extracted and spotted as above. Scanning of the TLC plates in this case revealed a marked reduction in the amount of labeled mannose incorporated into the lipids of the mevinolin-treated worms (Figure 27). Specifically, the mevinolin-treated parasites incorporated 6696 less mannose into the 2:1—extractable lipid, and 7696 less mannose into the 1:1:0.3-extractable lipid. These results indicate a greatly reduced amount of these lipids in the drug-treated schis tosomes. The rate of transfer of the mannose residues from the lipids to the forming protein in control and mevinolin-treated worms was next examined. Here, aliquots of the incubation mixtures for each group of parasites were removed and analyzed for radioactivity in each of the lipid fractions and in the remaining protein residue, which was solubilized and counted by liquid scintilla- tion spectrometry. As the reaction progressed, the incorporation of 14C-mannose was virtually abolished in the lipid-oligosaccharide fraction of the mevinolin- treated worms (Table 11). While label incorporation was also lower into the lipid 86 Figure 26. Radioscans of lipid intermediates of _S_. mansoni. The lipid intermediate (2:1) and the lipid oligosaccharide (1:1:0.3) were labeled with GDP- [U- Cl-mannose. SF =solvent front. 87 211 CPM 121:0.3 8,041 Figure 26 88 —N Z.JOZ.>N§ .o .CQFZOO .233 .532 can Loan: 29.: 2525 DS wobucaoE>oE can 36:3 .832 EB .63: So: 35:00 E 3:28.. 32:85: 9.3.58 33: L3 8588 983 meow—Q 0.: 405:8 329, 5.055.: B 8898 EomcwE .w No mBammmELBE Ba: «0 £832va .2“ magma 89 3 2:5 i 4.112 m6”: Z..._OZ_>m_2 II > «ton—”P 1.0m #200 WdO 58> NISIHO)» TN Z...OZ_>m_2 NISIUO)» TN JOthOO :18” WdO 90 Table 1 1 Effect of Mevinolin on the Formation of Lipid Intermediates and Glycoproteins in _S_. mansoni Homogenates Incubation time (min): 2.5 5.0 10.0 20.0 Sample cpm/mg protein homogenatea 2:1 Lipid Intermediate Control 178 978 990 1,024 Mevinolin 64 76 608 644 1:1:0.3 Lipid Oligosaccharide Control 3,956 5,034 13,784 24,732 Mevinolin 480 496 712 1,184 Protein Control 49,978 134,644 224,482 334,754 Mevinolin 37,554 80,322 174,436 202,512 8Data are expressed as mean cpm incorporated per mg protein homogenate for at least 6 determinations. 91 intermediate and protein residue of the drug-treated worms, the lipid-oligosac- charide seemed particularly sensitive to the effects of mevinolin. These results again indicate that mevinolin is acting relatively specifi- cally to deplete the schistosome of polyisoprenoid lipids. These lipids may be necessary for glycoprotein synthesis in the parasite and glycoproteins, in turn, are necessary for the synthesis of eggs. Therefore, mevinolin, by inhibiting schisto— some HMG CoA reductase, effectively disrupts egg production in Schistosoma mansoni. DISCUSSION Knowledge of the complex biochemical processes underlying egg production by Schistosoma mansoni is highly relevant to any efforts toward the amelioration of pathology associated with schistosomiasis. Unfortunately, the understanding of the process of parasite egg production is quite limited. It is the purpose of this dissertation to further elucidate the events involved in schistosome egg produc- tion. For this purpose, mevinolin, a sterol synthesis inhibitor, has been used as a tool with which the biochemistry involved in parasite fecundity could be studied. Using this agent, aspects of the regulation of schistosome egg production have been uncovered which may aid in the development of chemotherapeutic drugs for schistosomiasis. A. The In Vitro Effects of Mevinolin on S. mansoni 1. Effect of mevinolin on in vi tro eggproduc tion by S. mansoni The finding that mevinolin, an inhibitor of sterol biosynthesis, de- creased egg production in S. mansoni in m (Table 1) was surprising in light of studies where steroids and other steroid inhibitors were shown to have no effect on the parasite (Morrison e_t a_1., 1986). From these studies and those in which it was shown that the schistosome is incapable of d_e m sterol synthesis (Meyer e_t a_1., 1970), it was concluded that steroid hormones were probably not involved in parasite fecundity. However, the i3 gill-3 effect of mevinolin, which inhibits HMG CoA reductase, on the process of egg production indicates that some product in the metabolic pathway in which this enzyme is involved, plays a role in oviposition 92 93 in S. mansoni. Since the sterol synthetic pathway does not exist in this parasite, the metabolite of HMG CoA being affected by mevinolin is most likely to be a product between mevalonate and farnesyl pyrophosphate, or a polyprenol, such as ubiquinone or the dolichols, since the synthesis of all of these compounds is dependent on HMG CoA reductase (see Figure 8). This lipid seems to exert a significant influence on schistosome egg production since the effect of mevinolin Was highly specific for this process, affecting no other parameters of parasite physiology during i_n m incubations. Thus, a mevinolin-sensitive metabolite of HMG CoA appears to play a prominent role in schis tosome egg production. 2. Effects of metabolites of HMG CoA on egg production by S. mansoni in vitro Mevinolin, by competitive inhibition of HMG CoA reductase, starves cells for the metabolic products of HMG CoA. Since mevinolin was able to inhibit egg production by the schistosome, some of the metabolic products of HMG CoA were studied for their ability to influence parasite oviposition. The first of these lipids tested was mevalonate, the immediate product of the HMG CoA reductase- catalyzed reaction. Mevalonate was added to schistosome cultures in concentra- tions ranging from 0 to 200 uM (Table 2) and an interesting effect of this metabolite on schistosome fecundity was noted. At lower concentrations (10 uM or 25 uM) mevalonate exerted a slight to a significant effect on egg production, increasing egg number over two-fold at 25 uM. However, at a higher concentra- tion (200 uM), mevalonate significantly suppressed egg production in the parasite, reducing it by 7596 from the control value. At fliis concentration of mevalonate, worm pairing and adherence to the glass substrate (the incubation flask) was no different than that of the control parasites. Thus, it appears that at high concentrations, mevalonate or a schis tosome-derived metabolite of mevalonate, is able to exert a negative effect on parasite egg production, analogous to the effect seen with 10 uM mevinolin. 94 To assess whether metabolites of mevalonate could influence schisto- some fecundity, farnesol and dolichols were added to the culture system. When farnesol was added to the schistosomes in culture, an effect similar to that seen with mevalonate was observed (Table 2). Specifically, lower concentrations of this lipid (20 uM or 40 uM) caused a two-fold stimulation in egg production. However, 80 11M or 200 uM farnesol significantly reduced the number of eggs produced during the 72 h incubation period. Here, the reduction in egg number was approximately 75% at either concentration of farnesol. Again, this reduction in egg production is comparable to that seen in parasites incubated in the presence of 10 11M mevinolin. The effect of dolichols on S. mansoni egg production (Table 3) was measured using two concentrations of these lipids (10 ug/ml and 20 ug/ml) proven effecfive in other systems (Carson and Lennarz, 1979). In these studies on the schistosome, however, dolichols at either concentration were ineffective at stimulating schistosome egg production. The lack of an effect observed with the dolichols could be attributable to several factors. First of all, it could be that the schistosome is unable to use these lipids or cannot take them up in culture. Radiolabeled dolichols were unavailable, and therefore uptake of these lipids by the parasite was not monitored. Secondly, dolichols are very hydrophobic, while the culture medium for the parasites is aqueous. The addition of hydrophobic dolichols to this aqueous environment lessens the chance that these lipids would be in a form which the parsites could utilize. Thus, the lack of effect of the dolichols on schis tosome egg production is not unexpected. In summary, low concentrations of either mevalonate or farnesol were able to stimulate egg production in the schistosome. It is interesting that higher concentrations of either of these lipids reduced the number of eggs produced, but not in a graded, dose-dependent manner (i.e., 80 uM farnesol had the same effect 95 as 200 uM farnesol). This seems to suggest that a critical concentration of these lipids or their metabolites is necessary to stimulate and maintain egg production in the schistosome. In the case of both of these lipids, that concentration is approximately 25 11M. As stated earlier, higher concentrations of these lipids have an effect similar to 10 uM mevinolin in the schistosome. While mevinolin is a potent inhibitor of HMG CoA reductase (Alberts g1 a_1., 1980), mevalonate and its metabolites are also known to exert negative feedback control on HMG CoA reductase both i3 _v_i_\1c_>_ (Arebalo gt a_1., 1980; Beg and Brewer, 1981) and i_n gi_tr_g (Gibson e_t 31., 1982; Parker _e_t a_1., 1983). This may also be occurring in the schistosome, where lower concentrations of the products of the HMG CoA reduc tase-catalyzed reacfion may be optimal for the production of eggs by the parasite. High concentrations of these lipids may, however, be inhibitory to egg production due to negative feedback regulation of these lipids on HMG CoA reductase. This study on schistosome egg production is therefore unique, in that it relates the regulation of this enzyme by nonsterol lipids in the parasite to a physiological process, the production of eggs by the schistosome. 3. Reversal of mevinolin's effect on schistosome egg production Since mevalonate and farnesol were effective in stimulating egg production in S. mansoni, their ability to "rescue" the parasite from the effect of mevinolin was examined. When parasites were co-cultured with both mevalonate and 10 11M mevinolin, 25 uM mevalonate appeared to be the optimum concentra- tion at which mevinolin's effect could be reversed (Figure 14). Here, egg production was restored to the control level, which was significantly higher than that seen in parasites incubated in 10 uM mevinolin alone. This same experiment was repeated in the presence of farnesol and dolichols. Farnesol at 80 uM was able to reverse mevinolin's effect, restoring eg number to control values (Figure 15). Dolichols were unable to cause a significant reversal of the effect of 10 uM 96 mevinolin on the parasite (Table 3). Considering the inability of these lipids to stimulate egg production, their lack of effect in the presence of mevinolin is not surprising. The ability of metabolites of HMG CoA to reverse mevionlin's effect has been intensively studied using cells in culture. Here, growth inhibition of Swiss 3T3 cells (Habenicht _e__t_ a_1., 1980) and cultured mouse spleen lymphocytes (Perkins e_t a_1., 1982) by mevinolin or compactin was shown to be reversible by mevalonate. However, the ability to reverse mevinolin's effect in a whole organism, such as the schistosome, allows one to relate the reversal of the drug's effect to a physiological process. For in the schistosome, the effect of mevalonate or farnesol on parasites incubated in the presence of mevinolin, involves restoration of the process of egg production. Therefore, the metabolites of HMG CoA may not only play a significant role in the regulation of HMG CoA reduc tase in the schistosome, but they may also be necessary for the production of eggs by this parasite. 4. Stimulation of 5% production by S. mansoni following in vitro or in vivo exposure to mevinolin HMG CoA reduc tase is known to be a highly inducible enzyme, stimulated after exposure to low doses of mevinolin or compactin (Brown e_t 31., 1978; Skalnik _e_t 51., 1985). The effect of mevinolin on oviposition in S. mansoni, if mediated through HMG CoA reductase, should also prove to be inducible to an extent. To test this possibility, both _i_n v_it_r_o and i3 xiv—o exposure of the parasite to mevinolin was carried out, followed by incubation in the typical egg—laying system so that egg production could be monitored. The results of i_n 1192 exposure to mevinolin (Table 4) indicated that a slight induction of HMG CoA reductase activity concomitant with an increase in egg production may occur after 13 1i_tr_o exposure to 10 11M mevinolin. Here, parasites exposed to the drug for 24 h produced twice as many eggs as controls, whereas parasites maintained in 97 mevinolin for the entire incubation period showed markedly depressed egg production. This finding is suggestive of a dual effect of the drug namely, an induction of HMG CoA reductase after acute exposure to mevinolin, but an inhibition of the enzyme (even when induced) as long as the drug is present. The induction effect of mevinolin on egg production was even more dramatic in parasites exposed to the drug i3 [i_vg (Table 5). Here, parasites exposed to 50 mg/kg mevinolin jg 1’12: when placed in culture, produced five u'mes as many eggs as controls. Again, the presence of mevinolin (10 uM) in the culture was able to suppress this effect. Although induction of HMG CoA reduc tase activity has been found in several types of cells exposed to mevinolin or compac tin, the studies with mevinolin on schistosome egg production are the first to show an effect on the enzyme with a subsequent physiological response in an intact organism. Thus, not only is mevinolin exerting an effect on schistosome egg production that can be described as inhibitory i3 m, but it also appears that this agent can induce egg production in the parasite under the appropriate conditions. The differences in the extent of the induction of this process following i3 Mg versus i_n yiyg exposure to mevinolin is striking. This difference in the stimulation of egg producfion (a 2-fold increase in worms exposed to mevinolin i_n_ \1i_t_r£ versus a 5-fold increase in worms exposed to the drug i3 v_iyg) can best be explained by differences in the amounts of drug which the parasite encounters in each situation, and the duration of this exposure. In the i3 giv_o situation, the parasite probably experiences a lower dose of the drug for a shorter period of time -- a situation which may be more conducive to induction of the enzyme than 24h exposure to 10 11M mevinolin. Furthermore, the parasites exposed to mevinolin i3 £92 were maintained in culture for an additional 24 h, a condition more stressful to the parasite than maintenance within the host. 98 However, either set of experimental conditions resulted in a significant increase in egg production by the schistosome following exposure to mevinolin. B. The In Vivo Effects of Mevinolin on S. mansoni Egg Production Recognizing the i_n £32 effect of mevinolin on schistosome egg production, the i3 mo effect of this drug was examined in mice infected with these parasites. The schistosome-infected mouse lends itself well to the study of the effects of mevinolin on the parasite, since the mouse is refractory to the cholesterol- lowering effects of the drug (Alberts e_t al_., 1980). Thus, the metabolic or dietary status of the mouse host is not a consideration in examining the effects of mevinolin on the parasite in treated mice. A significant effect on schis tosome-induced pathology in the mouse was not seen until a daily dose of 250 mg/kg mevinolin was used. Since the LD50 of this drug in mice is greater than 1000 mg/kg (Endo, 1979), no toxic or lethal responses were seen in any of the animals during treatment with mevinolin. At a dose of 250 mg/kg however, schistosome-associated pathology appeared to be greatly altered in mevinolin-treated animals. Pathology was assessed in two ways, the first of these being the observation of gross liver pathology in drug-treated and control animals, with subsequent retrieval of eggs from these livers. In these studies, gross liver pathology was markedly reduced in the mevinolin-treated infected mice when compared to controls (Figure 16). This result was related to the isolation of 45% fewer eggs from livers of the drug-treated mice. While liver size and egg burden were significantly reduced in treated mice, a clearer picture of the effect of mevinolin on the liver pathology was seen in the second assessment of pathology, the examination of thin sections of liver tissue from both groups of animals. 99 When thin sections were made of livers from mevinolin— and vehicle-treated mice, there was a readily apparent difference between the two groups (Figures 17 and 18). Specifically, in the mevinolin-treated mice, the granuloma volume was obviously smaller than in livers from the control animals. This is most probably due to two factors. First of all, the granulomas in the drug-treated livers almost invariably contained only one egg, while the granulomas in the control livers involved at least 2-3 eggs per area of granulomatous infiltration. Generally speaking, the number of eggs deposited in a given area of the liver is directly related to the granuloma volume, thus the decrease in granulomatous tissue in the mevinolin-treated mice is probably partly due to the deposition of fewer eggs by the parasite in these animals. Secondly, it was found that the eggs in the livers of mevinolin-treated mice contained 50% fewer mature miracidia than did the eggs in livers of control animals. Since it is known that the miracidia secrete antigens which instigate granuloma formation (Hang e_t 11., 1974), eggs containing imma- ture miracidia would be less likely to cause a vigorous immune response. Therefore, mevinolin seemed to affect two aspects of egg production i3 v_iyg, both reducing egg number and affecting the production of the eggs which were formed, such that development of the miracidium within the egg was altered or slowed. It is interesting to note that although mevinolin disrupted schistosome egg produc- tion, parasite number or maturity was not significantly different in vehicle-versus drug-treated mice. This again suggests that the effect of mevinolin, even at high concentrations i_n m, is rather specific for schistosome fecundity. Furthermore, the ability of mevinolin to reduce schistosome-associated pathology i3 ‘LLVE by affecting egg production, again suggests that HMG CoA reductase is an important enzyme in the formation of eggs by this parasite. 100 C. Isolation of Schistosome HMG CoA Reductase The effect of mevinolin on the schistosome both _i_n m and i3 v_i_vg is, as discussed above, quite specific for parasite egg production. Specifically, mevino- lin did not cause a general depression of all parasite functions, rather, it seemed that the drug was targeted to a single biological process. Since mevinolin is known to inhibit HMG CoA reductase, and since it also inhibits schistosome egg production, an attempt was made to find this enzyme in the parasite in order to determine its characteristics and its role in schistosome egg production. Mammalian HMG CoA reductase is a microsomally-bound enzyme (Rodwell e_t a_l_., 1973), thought to be associated with the membranes of the rough endoplasmic reticulum (ER) (Guder e_t a_1., 1968; Shapiro and Rodwell, 1971), although the enzyme has also been isolated from smooth ER membranes (Gold- farb, 1972). Thus, in the search for this enzyme in the schistosome, microsomal fractions of parasites were prepared. The process of solubilization of the enzyme from the microsomal fractions has been an area of intense research, attempting to optimize enzyme activity from the microsomal pellet. While the microsomal- ly-bound enzyme is highly labile to cold temperatures (Kleinsek and Porter, 197 9), a modification of the freeze-thaw technique devised by Heller and Gould (1973) has been found to be an effective means of solubilizing the enzyme. Thus, schistosome microsomes were frozen overnight, then thawed at room temperature prior to use. Because the enzyme could be solubilized by this relatively mild treatment, it has been referred to as a peripheral ER protein. However, other studies using Triton X-100 to solubilize microsomes showed a 70% solubilization of membrane proteins while less than 5% of the HMG CoA reductase was solubilized (Ness e_t a_1., 1981). Thus, the enzyme may have some properties normally associated with integral proteins of the ER. The purification of HMG CoA reductase from microsomes has been complicated by this seeming dual 101 nature of the enzyme, as well as by the lability of this enzyme to temperature, and by inactivating enzymes (particularly HMG CoA lyase) released during the homogenization of tissue. However, purification of the enzyme has been accomplished from rat liver (Ness e_t a_1., 1979; Rogers 91 a_1., 1980; Gil e_t_ g” 1981), and specific acivity of the pure enzyme was reported to be between 10,000 and 20,000 nmol mevalonate synthesized per minute per mg protein. Specific activity of HMG CoA reductase is determined using an enzyme assay (Alberts 31 14C_ gl., 1980) which is based on the generation of radiolabeled mevalonate from HMG CoA substrate by microsomal fractions containing the enzyme. While the recovery of pure enzyme from rat liver resulted in HMG CoA reductase with very high specific activity, the isolation procedures for the enzyme involved several extensive purification steps and large amounts of starting material. In the studies with the schistosome, where material was limited, only a crude purification of the enzyme was accomplished using the freeze-thaw technique to solubilize the enzyme as described above. Like the mammalian enzyme, schistosome HMG CoA reductase activity was localized to the microsome. In the first study of this enzyme in the parasite, various amounts of the microsomal preparation were assayed for enzyme activity (Figure 19). Ten microliters of worm microsome (equal to approximately 100 pg protein) was used as a standard amount of enzyme in all subsequent assays when it was found that 10 ul microsome generated enough radiolabeled product to be quantified. Thus, in order to conserve parasite material, radiolabeled substrate, and to quantify the assay, 10 ul of parasite microsome were routinely used. The time-dependence of the HMG CoA reduc tase-catalyzed reaction in the schistosome was next determined (Figure 20). In these studies, the activity of the enzyme in parasite microsomes was linear for 10 minutes, a finding in accordance with assays using rat liver, where the reaction was found to be linear for at least 102 5-10 minutes (Kleinsek e_t_ a1, 1977; Alberts e_t 51;, 1980). Thus, excepting the purity of the enzyme preparation from the schistosome, which undoubtedly contributed to its lower specific activity in these preparations, schistosome HMG CoA reductase behaved like the mammalian enzyme in the localizah’on of the enzyme and its rate of catalysis. D. Inhibition of Schistosome HMG CoA Reductase by Mevinolin in the Enzyme Assay System The presence of HMG CoA reductase activity in the schistosome, and its similarlity to the mammalian enzyme in some respects raised the question of whether the schistosome enzyme, like the mammalian enzyme, was inhibitable by mevinolin in the assay system. To measure the effect of mevinolin on the enzyme activity, the same concentrations of mevinolin was those utilized in the i_n_ v_itr_o egg-laying studies (0.1 uM to 100 uM mevinolin) were used in the enzyme assay. It is interesting to note that 10 uM mevinolin, which caused a 70.8% decrease in schistosome egg production (Table 1), was able to inhibit HMG CoA reductase activity by 71.5% in the enzyme assay (Figure 21). The relationship between the mevinolin-induced effect on egg production at 10 uM and the inhibition of HMG CoA reduc tase activity by this concentration of the drug in the enzyme assay is excellent, even though 10 uM mevinolin is a high concentration of the drug, pharmacologically. However, although mevinolin has been shown to be a potent inhibitor of sterol biosynthesis, the HMG CoA reductase-catalyzed synthesis of other lipdis (such as dolichols) is more resistant to the drug. For instance, mevinolin or compactin was able to inhibit the conversion of 14C-acetate into sterolds by 50% at concentrations of 1 nM in rat hepatocytes (Alberts e_t_ 131., 1980), while the incorporation of 3H-mannose into dolichol-linked oligosaccharides was inhibited by 50% only in the presence of 5 uM of these drugs, a concentration where sterol synthesis from l4C-acetate was almost completely blocked (Filipovic 103 and Menzel, 1981). Since the schistosome cannot synthesize sterols, its HMG CoA reduc tase may be more resistant to mevinolin, thus explaining why higher concentrations of the drug were needed to exert an effect on schis tosome enzyme activity i3 m. In this respect again, schistosome HMG CoA reductase may be similar to the mammalian enzyme. E. In Vivo Effects of Mevinolin on Schis tosome HMG CoA Reductase A final study of schistosome HMG CoA reductase activity involved the determination of whether the parasite enzyme, like enzyme purified from other systems, was inducible by mevinolin. For the purpose of these studies, parasites were exposed to mevinolin i3 m, as this seemed to be an effective means of inhibiting egg producfion at high doses (Figure 15) while inducing egg producfion at low doses (Table 5). The goal of these experiments was to correlate mevinolin's effects on egg production with HMG CoA reductase in parasite microsomes. When schistosomes were exposed to 250 mg/kg mevinolin i3 m for 3 days, their HMG CoA reductase activity was markedly suppressed (Figure 21). How- ever, when parasites treated the same way in \1v_o_ were incubated for 24 h in drug-free media prior to the preparation of the microsomes, enzyme activity as measured in the enzyme assay was restored to above control levels. This result was indicative of an ability of the enzyme to rebound quickly following drug exposure, suggesting perhaps that the enzyme is inducible in the schistosome. The ability of mevinolin to induce parasite HMG CoA reductase was confirmed by using 50 mg/kg mevinolin in infected mice for 3 days. Here, parasites exposed to the drug showed a doubling of enzyme activity compared to that in controls (Figure 21). Although this level of induction is not as great as that seen in cultured cells exposed to compactin or mevinolin, where induction of activity ranged from 3.5- to 15-fold (Brown e_t $1., 1978), the induction of this enzyme in 104 the schistosome, and the correlation between this induction and a physiological response, egg production, is unique. Specifically, induction of HMG CoA reduc tase activity in this parasite seems to be related direclty to an induction of egg production by the worm. In conclusion, HMG CoA reductase activity in S. mansoni appears to have characteristics similar to those of enzyme preparations from diverse mammalian systems. These characteristics include similar rates of catalysis, subcellular location, sensitivity to the inhibitory effects of mevinolin, as well as the ability to be induced by this drug. An additional feature of this enzyme in the schistosome is the ability to ascribe to it not only a metabolic role, but also a physiological funcu'on in the parasite. Since the production of eggs gy the worm closely parallels the activity of the enzyme, HMG CoA reductase may regulate this Lrocess via the production of lipid metabolites necessary for parasite fecundity. F. The Effect of Mevinolin on the Lipids of S. mansoni A nonsterol product of the HMG CoA reductase-catalyzed reaction is obviously involved in the production of eggs in S. mansoni. This lipid may serve to affect egg production by acting in two capacities; first, it may actually play a functional role in the synthesis of egg components. Secondly, this lipid may exert regulatory control over HMG CoA reductase, influencing the rate or number of eggs produced. Studies described earlier with mevalonate and farnesol (Table 2), suggest that this second role for a lipid metabolite of HMG CoA may exist in the schistosome. Whether this lipid could also be acting in a functional capacity in the synthesis of schistosome eggs could be best assessed by examining the lipids which the parasite synthesizes and determining which, if any, of these lipids are sensitive to mevinolin. 105 14c- By far the most prevalent lipids synthesized by the schistosome from acetate were the triglycerides and phosphatidyl choline (Table 10). This finding is consistent with that of Smith and coworkers (1970) who labeled parasite lipids with both 14 C-acetate and tritiated free fatty acids, and recovered the bulk of the label in the above two lipids. In the present studies, radiolabeled mevalonate was incorporated primarily into lipids which were unidentifiable, however, these lipids could be classified into groups referred to as hydrocarbons-long chain alcohols, polyisoprenoids, and polar lipids. While the lipids into which mevalonate was incorporated were of the most interest in the determination of lipid 14C-mevalonate could not be used to determine which metabolites of HMG CoA, lipids were being affected by mevinolin, as it is the product of the reaction which the drug inhibits. For the purpose of determining the effect of mevinolin or lipids in S. 14C-acetate in the presence of the drug. mansoni, parasites were incubated with The result of these studies indicated a profound, selective effect of mevinolin on the synthesis of polyisoprenoid-type lipids (Table 9). These lipids were identified as polyisoprenoids based on their behavior on various chromatographic columns and thin—layer chromatography. The fractions from which these lipids were isolated generally may contain glycolipids, cerebrosides, or glycosylated isopre- 14C-acetate-labeled schisto- noid-type lipids (Carroll and Serdarevich, 1967). The some lipids in this fraction did not co-chromatograph with various glycolipids or cerebrosides in several chromatographic systems. Rather, these lipids ran slightly behind farnesol and slightly ahead of a mixed dolichol standard in the thin-layer systems tested. This suggests that these lipids may be isoprenoids of chain length between that of farnesol (which contains 15 carbons) and the long chain dolichols. Additionally, although minor amounts of ubiquinone have been detected in this 106 parasite (Folkers g g, 1983), none of the schistosome-labeled lipids co- chroma tographed with a ubiquinone standard. Mevinolin at 10 uM exerted a 70% decrease in labeled acetate incorporafion into the polyisoprenoid lipids as a class. This, again, correlates nicely with the effects of this compound on i_n £92 egg production and HMG CoA reductase activity at this concentration. Thus, the effect of mevinolin, as expected based on the pathway of mevalonate metabolism (Figure 7), was confined to the synthesis of polyisoprenoid lipids, and, more specifically, the effect of this drug is probably limited to metabolites in the pathway involving mevalonate, farnesol, and the dolichols. G. The Potential Role of Polyisoprenoid Lipids in the Synthesis of Schistosome Eggs Polyisoprenoid lipids are known to function as lipid intermedites in the synthesis of glycoproteins, transferring carbohydrates to forming peptides in the endoplasmic reticulum (Waechler and Lennarz, 1976). In order to determine if the polyisoprenoids in the schistosome could function to carry sugars, a special procedure to isolate these lipids was used (Rumjanek and Smithers, 1978). The result of this procedure was the isolation of two polyisoprenoid lipids capable of carrying radiolabeled mannose residues (Figure 26). One of these lipids, the lipid intermediate, was found to carry one mannose residue, while the second lipid, the lipid-linked oligosaccharide, was found to carry two or more of these sugars. Once it was established that schistosome polyisoprenoids could function to carry sugars, the effect of mevinolin on these lipids was determined. Parasites incubated in mevinolin contained significantly lower amounts of these lipids (Figure 27), as measured by a decrease in the ability of these lipids to incorporate radiolabeled sugars. Thus the effect of mevinolin on the parasite seems to involve a depletion of polyisoprenoid lipids which function to carry sugars. 107 The ramifications of a depletion of lipid intermediates in S. mansoni could be significant, since the parasite relies heavily on glycoprotein synthesis, particu- larly for their incorporafion into eggs (Pelley e_t al_., 1976). To assess the effect of mevinolin on the incorporation of 14C-mannose into glycoproteins, a kinetic study was carried out in which the radioactivity incorporated into parasite lipid intermediates and glycoproteins was measured (Table 11). The results of this study indicate a particular sensitivity of the lipid oligosaccharide to the presence of mevinolin. One would expect that since this lipid functions to transfer the sugars to the protein, a proportional decrease in radioactivity incorporated into protein would be seen. This, however, was not the case, as radiolabeled mannose incorporation decreased by 39.5% into protein, while incorporation is reduced by 95.2% into the lipid-linked oligosaccharide. This discrepancy may be the result of a high level of O-linked glycosylation, which does not function through a lipid intermediate, in the schistosome, or may be due to the addition of more mannose residues to the glycoprotein following ER processing. Nonetheless, the suppres- sion of the formation of the lipid intermediates and the subsequent decrease in mannose incorporation into glycoprotiens of the parasite exposed to mevinolin could seriously affect processes in the parasite which are dependent on glycopro- tein synthesis. Since the parasite egg is highly proteinaceous, containing a significant amount of glycosylated proteins, it is very likely that mevinolin, by inhibiting parasite HMG CoA reductase, deprives the schistosome of lipid inter- mediates necessary f or the synthesis of egg glycoproteins. SUMMARY AND CONCLUSIONS The effect of mevinolin, an inhibitor of HMG CoA reduc tase, was examined in the schistosome. Specifically, it was found that this drug inhibits schistosome egg production 13 1193. at concentrations of 10 11M or greater. Furthermore, this effect on egg production was shown to be reversible by the addition of mevalonate or farnesol, metabolites of HMG CoA to the parasite culture. These lipids were also shown to have stimulatory effects on egg production at low concentrations, while at higher concentrations, they inhibited it to a degree similar to that seen with 10 uM mevinolin. The effect of mevinolin on the schistosome in culture, coupled with the results on egg production obtained with the metabolites of HMG CoA, opened two further areas of research, that involving an invesu'gation for HMG CoA reductase in the parasite, and a determination of a role for the lipid metabolites of HMG 00A in the schistosome. Schistosome HMG CoA reductase was found to resemble the mammalian enzyme in several respects. For instance, the parasite enzyme found to be associated with the microsome, had a similar rate of catalysis as well as similar sensitivity to the inhibitory effect of mevinolin as the enzyme isolated from rat liver. Perhaps the most interesting similarity between the enzymes derived from both sources was the inducibility of enzyme activity following low dose or acute exposure to mevinolin. The mechanism for this induction remains unknown, but it is probably mediated by a product of the HMG CoA reductase metabolic pathway, which feeds back to exert regulatory control over the enzyme. The process of control of this enzyme may involve enhanced rates of transcription or of 108 109 translation of the message for the enzyme when it is induced. Currently, this remains an area of active research. In the schistosome, the induction of HMG CoA reduc tase activity paralleled an induction of egg production, thereby confirming a role for this enzyme in parasite oviposition. The presence of HMG CoA reduc tase in S. mansoni suggests that products of the metabolic pathway in which the enzyme is involved must also be present in the parasite. These products, polyisoprenoid lipids, were found to be synthesized 14C-acetate and 14 by the parasite from C-mevalonate, and this synthesis was highly sensitive to the presence of mevinolin. Mevinolin caused a selective reduction in the polyisoprenoid lipids of S. mansoni, leaving the other lipid classes of the parasite unaffected. A function of polyisoprenoid lipids in the schis tosome which could be related to the effect of mevinolin on egg production was their role in glycoprotein formation. Schistosome polyisoprenoids were found to be capable of carrying sugars and transferring these to protein. Parasites exposed to mevinolin did not have the ability to carry this process out efficiently, presumably due to a depletion of the polyisoprenoids in these schistosomes. As a result, the glycosyla- tion of proteins in mevinolin-treated worms was markedly reduced. Due to the prevalence of glycoproteins in schistosome eggs, a reduction in protein glycosyla- tion could greatly affect egg production, accounting for the ability of mevinolin to inhibit mis process i2 £22 and in v_iyg. The effect of mevinolin i_n yi_v_o was striking, with doses of 250 mg/kg greatly reducing schistosome—induced pathology. While the low toxicity of this agent as well as its efficacy in reducing the pathology of schistosomiasis make it attractive chemotherapeutically, its use has limitations. For instance, in the human mevinolin would cause a significant effect on cholesterol metabolism -- indeed, this drug will probably find most use as a cholesterol-lowering agent. 110 Furthermore, since HMG CoA reductase is inducible by mevinolin, a chronic dosing regimen would be necessary to continuously suppress egg producfion (and not induce it) in the parasite. 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