Characterization of PC2, a mammalian Kex2 homologue, following expression of the cDNA in microinjected Xenopus oocytes

Abstract

A human insulinoma cDNA (PC2) that encodes a protein homologous to the KexZ/subtilisin-like proteinases has recently been described [1990. J. Biol. Chem. 265.2997-30001. In order to characterise the associated proteinase activity, mRNA encoding PC2 was synthesised in vitro and microinjected into Xcnopus oocytes. The proteinase activity released into the media from oocytes microinjected with PC2 mRNA was assayed using small peptide Ruorogenic substrates. Boc.Gln.Arg.Arg aminomethyl coumarin was hydrolysed in a Ca*+-dependent manner. but substrate analogues bearing a single basic aminodcid were not. The substrate specificity, inhibitor profile. and pH optimum of 5.5 were compatible with an involvement of PC2 in prohormone processing in mammalian cells.

Sorting and processing of secretory proteins

“…This unusual processing pattern may be peculiar to this particular, and same cleavage site was identified in CHO cells expressing a mutant Sorting and processing of secretory proteins 11 unnatural, setting. With this proviso in mind, it is nonetheless interesting to note that in the same study it was found that mutation of Asp167, which lies at the heart of the putative PC2 active site, failed to affect the processing of the PC2 precursor, suggesting that in oocytes such processing is not autocatalytic [269]. It was, finally, also shown that the PC2 precursor was enzymically inert [269].…”

“…There is a precise alignment of Asp, His and Ser in the catalytic domains of the different members, although a highly conserved Asn residue has been replaced by Asp in PC2, an interesting mutation since this residue has been shown to play an important role in catalysis in the bacterial subtilisins [227,228]. PCl and PC2 have an optimal enzyme activity at acidic pH [229][230][231][232], while furin has a broad, neutral pH optimum [233]. This family of enzymes has also in common the fact that Ca2l is an essential requirement, although the concentration dependency varies greatly [181].…”

“…Note that these sequences would be suitable for conversion by furin [233,237]. When PC2 was expressed in Xenopus oocytes, it was found that the pro-region was initially cleaved at Lys-ArgArg-Arg8l to yield an intermediate of 71 kDa, which was further cleaved to a 68 kDa form only after secretion [269]. This unusual processing pattern may be peculiar to this particular, and same cleavage site was identified in CHO cells expressing a mutant Sorting and processing of secretory proteins 11 unnatural, setting.…”

The neuroendocrine chaperone 7B2 can enhance in vitro POMC cleavage by prohormone convertase PC2

“…Cleavage of POMC in the presence of the processed form of 7B2 was partially blocked by EDTA (Fig. 1A, lane 11), an agent known to inhibit many proteolytic enzymes, including PC2 [3]. Boiling of processed 7B2 did not affect its potency to enhance POMC cleavage (Fig.…”

“…The activity of these subtilisin-like enzymes in the secretory pathway of neuroendocrine cells is regulated at multiple levels. PC1 and PC2 exhibit an acidic pH optimum and Ca 2+ dependence which limits their activity mainly to the transGolgi network (TGN) and the secretory granules [3][4][5][6]. Furthermore, PC1 and PC2 are synthesized as proenzymes and their activation involves proteolytic removal of the N-terminal proregion, a process that is thought to be autocatalytic [7][8][9].…”