Differences between the catalytic properties of recombinant human PC2 and endogenous rat PC2

Abstract

Human prohormone convertase PC2 was expressed in Xenopus oocytes and its properties were compared with those of the Type-2 endopeptidase of rat insulin secretory granules, previously identified as PC2 [Bennett, Bailyes, Nielson, Guest, Rutherford, Arden and Hutton (1992) J. Biol. Chem. 267, 15229-15236]. Recombinant PC2 had the same substrate specificity as the Type-2 endopeptidase, cleaving at the CA-junction (Lys64, Arg65) of human des-31,32-proinsulin to generate insulin; little activity was found toward human des-64,65-proinsulin or proinsulin itself. Recombinant PC2 was maximally active in 5-7 mM Ca2+ (K0.5 = 1.6 mM) whereas the Type-2 endopeptidase was maximally active in 0.5-1 mM Ca2+ (K0.5 = 40 microM). Both enzymes had a pH optimum of 5.0-5.5 but the Type-2 endopeptidase was active over a wider pH range. Two molecular forms of recombinant PC2 (71 kDa and 68 kDa) were found, both had an intact C-terminus but differed by the presence of the propeptide. The endogenous PC2 comprised several overlapping forms (size range 64-68 kDa), approximately two-thirds of which lacked C-terminal immunoreactivity. Part of the size difference between recombinant and endogenous PC2 was attributable to differences in N-glycosylation. The different post-translational proteolytic modifications of recombinant and endogenous PC2 did not account for the different pH and Ca2+ sensitivities shown by the enzymes. A modulating effect of carbohydrate on enzyme activity could not be excluded.

Identification of a Transferable Sorting Domain for the Regulated Pathway in the Prohormone Convertase PC2

“…The maturation of PC2 in the stable transfected AtT-20 cells was consistent with the reported proteolytic cleavage of pro-PC2 at both tetrabasic sites Lys-Arg-Arg-Arg 81 or Arg-Lys-LysArg 109 to produce the mature secreted forms (Bailyes et al, 1995). The intracellular localization of propeptide cleavage has not been determined unequivocally, although the present consensus favors a late compartment, probably the TGN or immature granule.…”

“…2A). Western blotting experiments (data not shown) using a panel of antibodies directed against the propeptide (aa 87-104; Bailyes et al, 1995), the catalytic domain, and the carboxyl terminus of PC2 revealed that the 76-kDa band represents pro-PC2, the 68-kDa form is probably cleaved after the first tetrabasic site Lys-Arg-Arg-Arg 81 , and the 64-kDa form is mature PC2 probably cleaved after the second tetrabasic site Arg-Lys-LysArg 109 (Bailyes et al, 1995). Analysis of the N-linked glycans showed that the 76-kDa pro-PC2 was endo H-sensitive, whereas the mature 64-kDa form produced two bands after endo H treatment, both of which were larger than the single band detected after endo F treatment but smaller than the untreated protein (Fig.…”

Activation and Routing of Membrane-tethered Prohormone Convertases 1 and 2

“…Unlike sPC2, PC2-Myc-CD exhibited TGN accumulation similar to PAM-1, suggesting more rapid exit of PC2-Myc-CD from the ER than sPC2. It has previously been reported that soluble PC2 can undergo C-terminal truncation within insulin secretory granules (84). Although the current data do not support C-terminal cleavage of sPC2, limited cleavage of the PC2 domain within the PC2-Myc-CD chimera cannot be discounted.…”

Specificity of Prohormone Convertase 2 on Proenkephalin and Proenkephalin-related Substrates

“…Studies of recombinant PC2 cleavage on natural substrates include proglucagon (18,19), cholecystokinin-33 (20), and prodynorphin (21). Comparative work on both enzymes includes reports on the cleavage of proneuropeptide Y (22) and proinsulin (23,24). Taken together, this work supported the idea that both prohormone convertases prefer paired basic cleavage sites containing a P4 basic residue and can cleave at single basic residues given the presence of additional amino-terminal basic residues.…”