The Prohormone Convertase Enzyme 2 (PC2) Is Essential for Processing Pro-Islet Amyloid Polypeptide at the NH₂-Terminal Cleavage Site

Abstract

Impaired processing of pro-islet amyloid polypeptide (proIAPP), the precursor of the β-cell peptide islet amyloid polypeptide (IAPP) (amylin), has been implicated in islet amyloid formation in type 2 diabetes. The prohormone convertase enzymes PC3 (also known as PC1) and PC2 are localized to β-cell secretory granules with proIAPP and proinsulin and are responsible for proinsulin processing. To determine whether PC2 might be essential for proIAPP processing, we performed Western blot analysis of freshly isolated islets from normal mice and mice lacking active PC2. As expected, the primary species of IAPP immunoreactivity in islets from wild-type mice was fully processed (4-kDa) IAPP, with only small amounts of the 8-kDa precursor (unprocessed proIAPP) present. Islets from heterozygous PC2 null mice were identical to wild-type animals, suggesting that half the normal complement of PC2 is sufficient for normal proIAPP processing. By contrast, in islets from homozygous PC2 null mice, the predominant IAPP-immunoreactive form was of intermediate size (∼6 kDa), with no detectable mature IAPP and slightly elevated amounts of the 8-kDa precursor form present. Thus, in the absence of PC2, proIAPP processing appears to be blocked at the level of a proIAPP conversion intermediate. Immunofluorescence of pancreas sections and immunoblotting using antisera raised to the NH₂- and COOH-terminal flanking regions of mouse proIAPP demonstrated that the 6-kDa intermediate form was an NH₂-terminally extended proIAPP conversion intermediate (processed only at the COOH-terminus). These data indicate that PC2 is essential for processing of proIAPP at the NH₂-terminal cleavage site in vivo and that PC3 is likely only capable of processing proIAPP at the COOH-terminal cleavage site.