Identification of a Novel Protein Binding Motif within the T-synthase for the Molecular Chaperone Cosmc

• Type of Material: Article
• Authors: Rajindra P. Aryal, Emory University; Tongzhong Ju, Emory University; Richard Cummings, Emory University
• Language: English
• Date: 2014-04-25
• Publisher: American Society for Biochemistry and Molecular Biology
• Publication Version: Final Published Version
• Copyright Statement: © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.
• Final Published Version (URL): http://dx.doi.org/10.1074/jbc.M114.555870
• Title of Journal or Parent Work: Journal of Biological Chemistry
• ISSN: 0021-9258
• Volume: 289
• Issue: 17
• Start Page: 11630
• End Page: 11641
• Grant/Funding Information: This work was supported, in whole or in part, by National Institutes of Health Grants DK0808760 (to T. J.) and GM068559 (to R. D. C.).
• Keywords: LEUKOCYTE RECRUITMENT; T-synthase; COLLAGEN TRIPLE-HELIX; IN-VITRO; O-GLYCOSYLATION; Glycosylation; MUCIN; CORE-1 BETA-1,3-GALACTOSYLTRANSFERASE; Glycoprotein; Science & Technology; TOLL-LIKE RECEPTORS; Protein Folding; Molecular Chaperone; Endoplasmic Reticulum (ER); UDP-GLCNAC; CAENORHABDITIS-ELEGANS; Cosmc; Glycobiology; Biochemistry & Molecular Biology; PSGL-1 BINDING; Glycoprotein Biosynthesis; Life Sciences & Biomedicine
• Research Categories: Chemistry, Biochemistry

Abstract

Prior studies suggested that the core 1 β3-galactosyltransferase (T-synthase) is a specific client of the endoplasmic reticulum chaperone Cosmc, whose function is required for T-synthase folding, activity, and consequent synthesis of normal O-glycans in all vertebrate cells. To explore whether the T-synthase encodes a specific recognition motif for Cosmc, we used deletion mutagenesis to identify a cryptic linear and relatively hydrophobic peptide in the N-terminal stem region of the T-synthase that is essential for binding to Cosmc (Cosmc binding region within T-synthase, or CBRT). Using this sequence information, we synthesized a peptide containing CBRT and found that it directly interacts with Cosmc and also inhibits Cosmc-assisted in vitro refolding of denatured T-synthase. Moreover, engineered T-synthase carrying mutations within CBRT exhibited diminished binding to Cosmc that resulted in the formation of inactive T-synthase. To confirm the general recognition of CBRT by Cosmc, we performed a domain swap experiment in which we inserted the stem region of the T-synthase into the human β4GalT1 and found that the CBRT element can confer Cosmc binding onto the β4GalT1 chimera. Thus, CBRT is a unique recognition motif for Cosmc to promote its regulation and formation of active T-synthase and represents the first sequence-specific chaperone recognition system in the ER/Golgi required for normal protein O-glycosylation.