Sorting and packaging of regulated secretory proteins involves protein aggregation in the trans-Golgi network and secretory granules. In this work, we characterized the pH-dependent interactions of pancreatic acinar cell-regulated secretory proteins (zymogens) with Muclin, a putative Golgi cargo receptor. In solution, purified Muclin co-aggregated with isolated zymogens at mildly acidic pH. In an overlay assay, [35S]sulfate biosynthetically labeled Muclin bound directly at mildly acidic pH to the zymogen granule content proteins amylase, prolipase, pro-carboxypeptidase A1, pro-elastase II, chymotrypsinogen B, and Reg1. Denaturation of Muclin with reducing agents to break the numerous intrachain disulfide bonds in Muclin's scavenger receptor cysteine-rich and CUB domains did not interfere with binding. Non-sulfated [35S]Met/Cys-labeled Muclin showed decreased binding in the overlay assay. Extensive Pronase E digestion of unlabeled Muclin was used to produce glycopeptides, which competed for binding of [35S]sulfate-labeled Muclin to zymogens. The results 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.
Copyright 2004 American Society for Biochemistry and Molecular Biology, Inc.