Evidence for redundancy in propeptide/prohormone convertase activities in processing proglucagon: an antisense study.

Abstract

To further examine the physiological roles of the neuroendocrine prohormone convertases (PCs) in proglucagon processing, alpha TC1-6 cells were transiently transfected with PC1/3 and PC2 expression vectors containing either antisense or sense encoding cDNAs. PC1/3- and PC2-directed RIAs were used to determine that the PC1/3 antisense transfections lowered endogenous levels of PC1/3 by 40 +/- 7.9% but did not alter the levels of PC2. The PC2 antisense transfections decreased the endogenous levels of PC2 by 91 +/- 11.7% without affecting the levels of PC1/3. To quantitate the levels of proglucagon and proglucagon-derived products, transfected cells were metabolically labeled with [3H]tryptophan, and extracts were chromatographed by reversed-phase HPLC. Recovered peptides were then subjected to peptide mapping analyses, allowing precise quantification of 3H-radioactivity incorporated into proglucagon and its cleavage products. Product-precursor ratios were determined, and percent change in the proportion of products generated in antisense-transfected vs. sense-transfected cells was calculated. The decrease in PC1/3 after antisense treatment significantly reduced the amounts of glicentin produced and partially reduced the levels of all other proglucagon cleavage products. PC2 antisense treatment significantly reduced the levels of glicentin and 9K glucagon generated but had no significant effect on the remainder of the proglucagon-derived peptides. These results suggest the existence of redundant mechanisms that ensure the production of each of the intermediate and product peptides derived from proglucagon. PC1/3 is potentially an important enzyme in the processing of most proglucagon-derived peptides, whereas PC2-processing activity appears to predominate at only two of the four potential cleavage sites.

Severe Defect in Proglucagon Processing in Islet A-cells of Prohormone Convertase 2 Null Mice

“…There is no detectable production of glucagon, oxyntomodulin, GLP-1, or other smaller processing products. Although other investigators have raised questions as to whether PC2 acting alone is capable of cleaving glucagon from proglucagon, especially cleavage at the N terminus of the glucagon sequence in the precursor (20,21), these results indicate that the action of another convertase is not likely. If another convertase were required for cleavage after GRPP (see Fig.…”

“…Proglucagon processing has been examined in a number of cell lines expressing PC2, PC3, or both convertases with proglucagon by coexpression techniques, and these studies have suggested that PC2 generates the A-cell-processing phenotype (12,13). However, other investigators have reported that PC2 acting alone is not able to generate mature glucagon (20,21). Some of these discrepancies may have arisen because of low levels of expression of PC2 obtained in transfection experiments or low levels of activity in partially purified preparations of the recombinant enzyme used for in vitro studies.…”

Cellular Localization and Role of Prohormone Convertases in the Processing of Pro-melanin Concentrating Hormone in Mammals

“…Indeed, PC1/3 and PC2 cleave pro-opiomelanocortin at distinct pairs of basic residues in established cell lines (19) and in a temporal order in the corticotrophs (20). Specific patterns of cleavage by either PC1/3 or PC2 have also been demonstrated for proglucagon (21)(22)(23), proneurotensin (24), pro-thyrotropin-releasing hormone (25), proenkephalin (26,27), and procholecystokinin (28,29). The functional relevance of the proconvertases in a whole animal model was recently established by disrupting the corresponding genes such as these encoding PC2 (30) and PC4 (16).…”

Defective prohormone processing and altered pancreatic islet morphology in mice lacking active SPC2

“…SPC2 is also expressed at high levels in the islet alpha cells, where it selectively processes proglucagon to liberate only glucagon as the major active hormone from this large multihormone precursor (19). On the other hand, in the neuroendocrine L cells of the gut, proglucagon is processed at different sites by SPC3 to liberate mainly two glucagon-like peptides-GLP1 and GLP2 (20)(21)(22). GLP1 enhances insulin secretion from the beta cells in response to glucose, whereas glucagon counters the hypoglycemic action of insulin by triggering hepatic glycogenolysis and enhancing hepatic gluconeogenesis to raise blood glucose levels (23)(24)(25).…”