Proinsulin, Insulin and C-Peptide Antibodies

Proinsulin Processing

Fig. 1 Proinsulin processing. (Kay RG.; et al. Clin Chem. 2021)

The human insulin prohormone consists of a signal peptide, the A and B chains of insulin, and the connecting peptide, or C-peptide. Within the endoplasmic reticulum of the pancreatic β-cells, the signal peptide is cleaved, and the cysteine residues of the insulin A and B chains are oxidized to form disulfide bonds between the two chains. The prohormone is then exported to the Golgi apparatus where it is packaged into secretory vesicles. Within these secretory vesicles, the prohormone is proteolytically cleaved by prohormone convertase 2 at site 65, 66 and by prohormone convertase 1/3 at site 32, 33, forming distinct insulin and C-peptide molecules. Lastly, the B chain of insulin and C-peptide are further processed by carboxypeptide E to remove terminal basic residues and form the mature insulin and C-peptide peptides. Insulin and C-peptide are stored in the secretory vesicles until stimulation of the pancreatic β-cells by elevations in extracellular glucose concentration. The amino acid sequence of C-peptide, a single polypeptide chain, is present in proinsulin, des 31, 32 proinsulin, and des 64, 65 proinsulin such that these precursors have the potential to cross-react in C-peptide immunoassay.

Structural and Interspecific Differences

Fig. 2 Structure of proinsulin and its amino acid sequences (Imai S.; et al. Anal Biochem. 2015)

Insulin is phylogenetically ancient, being found in mammals, reptiles, birds and fish. Their sequences all share some degree of homology. However, C-peptide, shows a greater degree of interspecies variability. Rats and mice produce two different non-allelic proinsulins (I and II) which are processed to insulin I and insulin II and C-peptides I and II, which differ from each other in two or three amino acid residues.

As shown in Figure 2 (A), proinsulin is an 86-residue-long single polypeptide and it is processed by proteolytic cleavage at the B–C junction (32R–33E) and C–A junction (65R–66G) to generate C-peptide and insulin (comprises A-chain and B-chain). In (B), we can see that there are some differences in the amino acid sequence around the B-C junction of the mouse and rat proinsulin molecules, proinsulin I and proinsulin II. These differences are important when considering assay specificity.

Creative Diagnostics provides a series of monoclonal antibodies specific to different parts of rat C-peptides I and II. Thoroughly chosen epitopes for selecting specific monoclonal antibodies have produced specific antibodies which make C-peptide detection possible without cross-reactivity with native proinsulin or other known forms of partially processed proinsulin. Please see the product list for the specificity details.