Identification of a single amino acid as a primary determinant of prohormone convertase 1 cleavage specificity

Abstract

Prohormone convertase 1 (PC1), present in a number of endocrine and neuronal cell types, is a serine endoproteinase that cleaves at specific dibasic sites to convert peptide precursors to their active form. Using a monomeric version of human proinsulin (DKP-hPI) as a model, we discovered that an acidic cluster carboxy-terminal to the cleavage site (cleavage sequence RR-EAED, represented as P2P1-P1′ P2′ P3′ P4′ with cleavage occurring between the P1 and P1′ amino acids) is required for efficient processing of the B chain/C peptide junction. Single amino acid deletions suggested that the acidic-neutral sequence at P1′ P2′ is the most significant specificity determinant at this site. To continue our investigation, we replaced the P1′-P4′ region with alanine residues (either individually or collectively) or with the P1′-P4′ sequence from glicentin, a prohormone that is normally cleaved by PC2, but not by PC1. The alanine-containing proinsulins were cleaved with efficiencies similar to the parent compound, while the glicentin-substituted DKP-hPI was cleaved quite poorly. To further define the specificity requirements, additional mutants were created. A reverse alanine mutation (A34E, introducing an acidic amino acid into the P2′ site) completely prevented PC1 cleavage at the B/C junction. In addition, sequential substitution of the glicentin sequence into the P1′-P4′ region indicated that the P2′ amino acid (introduced as E33H/A34S-DKP-hPI) is responsible for diminishing the ability of PC1 to cut glicentin-substituted proinsulin. We conclude that the presence of an aliphatic amino acid at the P2′ position is a major determinant of PC1 specificity. Supported by NIH grant DK-52085.