Binding of the Golgi sorting receptor muclin to pancreatic zymogens through sulfated O-linked oligosaccharides.
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.
Department of Anatomy , University of Kansas School of Medicine, Kansas City, Kansas 66160, USA.
Igor
I
Boulatnikov
Robert C
RC
De Lisle
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D015153:Blotting, Western; D002135:Calcium-Binding Proteins; D004268:DNA-Binding Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors / Q000378:metabolism*; D006020:Glycopeptides / Q000737:chemistry; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000378:metabolism; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D009077:Mucins / Q000737:chemistry* / Q000378:metabolism; D009844:Oligosaccharides / Q000737:chemistry*; D010179:Pancreas / Q000166:cytology / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry; D011402:Pronase / Q000378:metabolism; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D025521:Tumor Suppressor Proteins; D021601:trans-Golgi Network
D000596:Amino Acids; D002135:Calcium-Binding Proteins; D004268:DNA-Binding Proteins; C114202:Dmbt1 protein, mouse; D004792:Enzyme Precursors; D006020:Glycopeptides; D009077:Mucins; D009844:Oligosaccharides; D010455:Peptides; D025521:Tumor Suppressor Proteins; D011402:Pronase