Albumin Glycation Affects the Delivery of C-Peptide to the Red Blood Cells

Abstract

Serum albumin is a prominent plasma protein that becomes modified in hyperglycemic conditions. In a process known as glycation, these modifications can change the structure and function of proteins, which decrease ligand binding capabilities and alter the bioavailability of ligands. C-peptide is a molecule that binds to the red blood cell (RBC) and stimulates the release of adenosine triphosphate (ATP), which is known to participate in the regulation of blood flow. C-peptide binding to the RBC only occurs in the presence of albumin, and downstream signaling cascades only occur when the albumin and C-peptide complex contains Zn²⁺. Here, we measure the binding of glycated bovine serum albumin (gBSA) to the RBC in conditions with or without C-peptide and Zn²⁺. Key to these studies is the analytical sample preparation involving separation of BSA fractions with boronate affinity chromatography and characterization of the varying glycation levels with mass spectrometry. Results from this study show an increase in binding for higher % glycation of gBSA to the RBCs, but a decrease in ability to deliver C-peptide (0.75 ± 0.11 nM for 22% gBSA) compared to samples with less glycation (1.22 ± 0.16 nM for 13% gBSA). A similar trend was measured for Zn²⁺ delivery to the RBC as a function of glycation percentage. When 15% gBSA or 18% gBSA was combined with C-peptide/Zn²⁺, the derived ATP release from the RBCs significantly increased to 113% or 36%, respectively. However, 26% gBSA with C-peptide/Zn²⁺ had no significant increase in ATP release from RBCs. These results indicate that glycation of BSA interferes in C-peptide and Zn²⁺ binding to the RBC and subsequent RBC ATP release, which may have implications in C-peptide therapy for people with type 1 diabetes.

Figure 1

Mass spectra of albumin. Examples of control gBSA and hyperglycemic gBSA mass spectra to determine the glycation percentage. (a) Healthy control gBSA sample at 11% glycation and (b) enriched gBSA sample at 48% glycation. Labeled peaks include BSA, +G (glucose), +K (potassium), and +C (cysteine).

Figure 2

Binding of the glycated albumins to RBCs with extreme glycation percentages. The specific binding of 11% gBSA (triangles) and 48% gBSA (circles) to RBCs in the presence of C-peptide and Zn²⁺ (n ≥ 4, error = SEM, *p < 0.05).

Figure 3

Binding of the glycated albumin to RBCs in the presence or absence of C-peptide and Zn²⁺. The specific binding of >45% gBSA (a), 23% gBSA (b), and 17% gBSA (c) to RBCs in the presence (closed) and absence (open) of C-peptide and Zn²⁺ (n = 4, error = SEM, *p < 0.05, **p = 0.05).

Figure 4

Binding of C-peptide to RBCs with different percent gBSA samples. In samples containing 13% gBSA and 17% gBSA, there was statistically the same amount of C-peptide bound to RBCs. There was a significant decrease in C-peptide binding when 22% gBSA and 50% gBSA were present in comparison with both 13% gBSA and 17% gBSA (n = 4–6, error = SEM, *p < 0.05 to 13% gBSA, **p < 0.05 to 17% gBSA).

Figure 5

Binding of Zn²⁺ to RBCs with different percent gBSA samples. Samples signifying controls at 14% gBSA had a significant increase in Zn²⁺ binding when compared to all other glycation percentages. There was a significant increase in Zn²⁺ binding in 18% and 23% gBSA samples compared to the 49% gBSA sample. However, 18% gBSA and 23% gBSA had statistically the same amount of Zn²⁺ binding (n = 4, error = SEM, *p < 0.05 from 14% gBSA, p < 0.05 from 18 and 23% gBSA).

Figure 6

ATP release from the RBCs with various gBSA percentages with and without C-peptide and Zn²⁺. The RBC-derived ATP release was significantly higher in the control conditions with the 15% gBSA samples when C-peptide and Zn²⁺ were present. There was a significant increase in ATP from RBCs and18% gBSA with C-peptide and Zn²⁺ compared to those without C-peptide and Zn²⁺. There was no statistical increase in ATP in 26% gBSA and 56% gBSA samples with and without C-peptide and Zn²⁺ (n = 3–6, error = SEM, striped bars denote the samples containing C-peptide and Zn²⁺, *p < 0.05 for all gBSA samples, **p < 0.05 for all samples except 15% gBSA c/z).