DIRECT BINDING OF THE GOLGI CARGO RECEPTOR MUCLIN TO PANCREATIC ZYMOGENS AT MILDLY ACIDIC pH.

Abstract

Sorting and packaging of regulated secretory proteins involves protein aggregation in the trans Golgi network and secretory granules. We characterized the pH-dependent interactions of pancreatic zymogens with Muclin. Muclin is a ~300 kDa sulfated, O-glycosylated protein that is a major component of the mouse zymogen granule. Muclin is derived from pro-Muclin, a type I membrane protein which is proposed to be a Golgi cargo receptor Acini were incubated with [35S]sulfate to label Muclin which was purified to near homogeneity on preparative SDS-PAGE. In solution, [35S]-Muclin co-aggregated with isolated zymogens but did not self-aggregate at mildly acidic pH. In an overlay assay using zymogens immobilized on PVDF membranes, Muclin exhibited direct binding to several zymogens at mildly acidic pH. The Muclin binding partners were identified by N-terminal sequencing and mass spec analysis to be: amylase, pro-lipase, pro-carboxypeptidase A1, pro-elastase II, chymotrypsinogen B, and Reg1. Muclin contains numerous intrachain disulfide bonds in its SRCR and CUB domains which are important for the three dimensional structure of these domains. To test whether the protein conformation is important for Muclin binding to zymogens, Muclin was treated with reducing agents to denature the protein backbone. Reduced Muclin bound as well as untreated Muclin. Pronase digestion of Muclin was used to produce glycopeptides containing only the sulfated, O-glycosylated domains. The glycopeptides bound to zymogens in the overlay assay, and unlabeled glycopeptides competed with binding of labeled intact Muclin. These data demonstrate that the sulfated, O-glycosylated groups are responsible for the pH-dependent interactions of Muclin with the zymogens. The behavior of Muclin fulfils the requirement of a Golgi cargo receptor to bind to regulated secretory proteins under the mildly acidic pH conditions that exist in the trans Golgi network. Supported by NIDDK 55998.