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J. Biol. Chem., Vol. 279, Issue 39, 40918-40926, September 24, 2004

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Binding of the Golgi Sorting Receptor Muclin to Pancreatic Zymogens through Sulfated O-linked Oligosaccharides^*

Igor Boulatnikov and Robert C. De Lisle

From the Departments of Anatomy and Cell Biology, University of Kansas School of Medicine, Kansas City, Kansas 66160

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.

Received for publication, June 6, 2004 , and in revised form, July 22, 2004.

^* This work was supported by National Institutes of Health Grant DK55998. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 913-588-2742; Fax: 913-588-2710; E-mail: rdelisle{at}kumc.edu.

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