Processing of Proinsulin by Furin, PC2, and PC3 in (Co) Transfected COS (Monkey Kidney) Cells

Abstract

The enzymology of proinsulin conversion was studied in COS cells by cotransfection of three species of proinsulin and each of three conversion endoproteases (furin, PC2, and PC3). In addition to the parts of basic residues linking the B-chain to C-peptide (Arg31-Arg32) and C-peptide to the A-chain (Lys64-Arg65), which were present in all three proinsulins studied, human proinsulin presents a P4 basic residue (four residues NH2-terminal to the point of cleavage) only at the former junction (Lys29) and rat proinsulin II only at the latter (Arg62). Human proinsulin Arg62 (prepared by site-directed mutagenesis of human proinsulin) contains a P4 basic residue at both junctions. Transfected cells were incubated for four successive 2-h periods. The media were pooled, and pro-insulin, conversion intermediates, and insulin were separated by reverse-phase high-performance liquid chromatography to monitor conversion activity. There was little conversion of any proinsulin in COS cells without cotransfection of an exogenous endoprotease. When furin or PC3 was cotransfected with any of the three proinsulins, there was extensive processing, with insulin as the major conversion product. PC2, by contrast, failed to cleave human proinsulin but was able to cleave both human proinsulin Arg62 and rat proinsulin II. Cleavage by PC2 of these proinsulins was predominantly at the C-peptide-A-chain junction, generating the conversion intermediate des-64,65-split proinsulin as the major product and only very small amounts of insulin itself.

Isolation and characterization of VGF peptides in rat brain. Role of PC1/3 and PC2 in the maturation of VGF precursor

“…Expression vectors for mouse PC1/3 and human PC2 under the control of the cytomegalovirus promoter (CMV‐mPC1/3 and CMV‐hPC2, respectively) have been previously described (Vollenweider et al . 1995).…”

“…Expression vectors for mouse PC1/3 and human PC2 under the control of the cytomegalovirus promoter (CMV-mPC1/3 and CMV-hPC2, respectively) have been previously described (Vollenweider et al 1995). The beta galactosidase expression vector used as a control (pcDNA3.1/His/lacZ) was purchased from Invitrogen (San Diego, CA, USA).…”

Capillary electrophoresis for simultaneous quantification of human proinsulin, insulin and intermediate forms

“…The expression of human proinsulin (hPI) gene is restricted to the beta cells of the islets of Langerhans [6], and hPI conversion to mature human insulin (hI) and C-peptide occurs by specific endoproteases (PC3 and PC2) [7]. Besides insulin and C-peptide, four intermediate forms (split 32-33, des 31, 32; split 65-66 and des 64, 65) are produced in the hPI proteolitic cleavage [8]. In the context of an insulin gene replacement therapy, human primary fibroblasts (HPF) were initially transfected with the wild hPI gene.…”

Adenovirus‐mediated preproinsulin gene transfer into adipose tissues ameliorates hyperglycemia in obese diabetic KKAy mice

“…2B clearly shows the immunoblot protein band of proinsulin and insulin in the adipose tissues. Although proinsulin‐converting enzymes PC2 and PC3 are not expressed in adipocytes, furin expressed in adipocytes (data not shown) can process proinsulin to mature insulin, although with low efficiency [19]. The introduction of furin‐cleavable consensus sequence into pchi may cause the efficient conversion of proinsulin to insulin in adipose tissue.…”