EXPERIMENTAL EVIDENCE FOR A C-PEPTIDE COMPLEX RECEPTOR ON RED BLOOD CELLS

Pancreatic β-cells secrete insulin and C-peptide, a 31-amino acid peptide, in a 1:1 mole ratio. People with type 1 diabetes (T1D) require exogenous insulin to survive due to damaged or destroyed β-cells. However, even with regular insulin treatments, patients develop complications such as retinopathy, neuropathy, and nephropathy, and it has been proposed that poor blood flow could be causing these complications. Prior research has shown that C peptide increases microvascular blood flow, therefore, it could be a useful therapy to prevent these complications. The unknown C peptide receptor and mechanism has been a major roadblock in utilizing C-peptide as a therapeutic. Our group has reported that approximately 1,800 C peptide molecules bind per red blood cell (RBC) in the presence of albumin, while there was no detectable C-peptide binding per RBC without albumin. Thus, it is hypothesized here that C-peptide binds to RBCs through an albumin/C-peptide complex receptor, as opposed to binding to the RBC alone. The work in this dissertation focuses on analyzing the binding of bovine serum albumin (BSA) to RBCs using a radiolabeling method to attach technetium-99m (Tc99m) to BSA for gamma decay detection. A binding saturation experiment was conducted to examine the BSA specific binding to RBCs with and without C-peptide. The specific binding curves revealed that albumin saturates at 14,021 (±1,489) BSA molecules/RBC with a Kd of 1.14 (±0.07) x 10-7 M. Whereas, in the presence of C-peptide and Zn2+, albumin saturates at 16,695 (±1,479) BSA molecules/RBC with a Kd of 2.00 (±0.05) x 10 7 M. At saturation, the additional 2,700 BSA molecules/RBC in the presence of C-peptide and Zn2+ indicates that not only does an albumin receptor exist on RBCs, but also a separate receptor for an albumin/C peptide complex. Due to its role in the delivery of C-peptide, the molecular state of albumin may be critical. Albumin is more glycated in individuals with T1D in comparison to healthy controls. This dissertation utilizes the radiolabeling saturation experiment to analyze the effects of glycation on BSA binding to RBCs. Varying percentage of glycation (11-48%) were analyzed with and without C-peptide and Zn2+. As the glycation percentage increased, the number of BSA molecules binding per RBC increased; however, the difference between samples containing C-peptide to those without C-peptide decreased. In fact, at the higher glycation levels, there is more albumin binding in the absence of C peptide. Importantly, the amount of RBC C-peptide uptake decreased when carried by glycated BSA compared to normal BSA. Abnormal albumin delivery may not only be a determinant of disease in people with T1D. Albumin delivery of C-peptide was also examined in people with multiple sclerosis (MS, an autoimmune disease). When comparing BSA binding to MS RBCs to healthy control RBCs, more BSA molecules bound per MS RBC. Previous research in our lab has shown that MS RBCs binds more C peptide and release higher concentrations of ATP than healthy controls. This suggests that in unhealthy conditions, the albumin/C peptide/Zn2+ complex is binding differently than in healthy conditions, which results in abnormal downstream effects.