title: Identification of a novel protein binding motif within the T-synthase for the molecular chaperone Cosmc.
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.
authors.affiliation: Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322.
authors.forename: Rajindra P
authors.initials: RP
authors.lastname: Aryal
authors.affiliation: Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322. Electronic address: tju@emory.edu.
authors.forename: Tongzhong
authors.initials: T
authors.lastname: Ju
authors.affiliation: Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322. Electronic address: rdcummi@emory.edu.
authors.forename: Richard D
authors.initials: RD
authors.lastname: Cummings
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D005700:Galactosyltransferases / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid
chemical_list: C505369:C1GALT1C1 protein, human; D017931:DNA Primers; D018832:Molecular Chaperones; C505293:C1GALT1 protein, human; D005700:Galactosyltransferases
keywords: Cosmc; Endoplasmic Reticulum (ER); Glycobiology; Glycoprotein; Glycoprotein Biosynthesis; Glycosylation; Molecular Chaperone; Protein Folding; T-synthase