ROLE OF PROHORMONE CONVERTASES IN PRO-NEUROPEPTIDE-Y PROCESSING - COEXPRESSION AND IN-VITRO KINETIC INVESTIGATIONS

Proneuropeptide Y (ProNPY) undergoes cleavage at a single dibasic site Lys38-Arg39 resulting in the formation of 1-39 amino acid NPY which i s further processed successively by carboxypeptidase-like and peptidyl glycine alpha-amidating monooxygenase enzymes. To investigate whether prohormone convertases are involved in ProNPY processing, a vaccinia v irus derived expression system was used to coexpress recombinant ProNP Y with each of the prohormone convertases PC1/3, PC2, furin, and PACE4 in Neuro2A and NIH 3T3 cell lines as regulated neuroendocrine and con stitutive prototype cell lines, respectively. The analysis of processe d products shows that only PC1/3 generates NPY in NIH 3T3 cells while both PC1/3 and PC2 are able to generate NPY in Neuro2A cells. The conv ertases furin and PACE4 are unable to process ProNPY in either cell li ne. Moreover, comparative in vitro cleavage of recombinant NPY precurs or by the enzymes PC1/3, PC2 and furin shows that only PC1/3 and PC2 a re involved in specific cleavage of the dibasic site. Kinetic studies demonstrate that PC1/3 cleaves ProNPY more efficiently than PC2, The m ain difference between the cleavage efficiency is observed in the V-ma x values whereas no major difference is observed in K-m values. In add ition the cleavage by PC1/3 and PC2 of two peptides reproducing the di basic cleavage site with different amino acid sequence lengths namely (20-49)-ProNPY and (28-43)-ProNPY was studied. These shortened ProNPY substrates, when recognized by the enzymes, are more efficiently cleav ed than ProNPY itself. The shortest peptide is not cleaved by PC2 whil e it is by PC1/3. On the basis of these observations it is proposed, f irst, that the constitutive secreted NPY does not result from the clea vage carried out by ubiquitously expressed enzymes furin and PACE4; se cond, that PC1/3 and PC2 are not equipotent in the cleavage of ProNPY; and third, substrate peptide length might discriminate PC1/3 and PC2 processing activity.