Identification and Characterization of aβ1,3-Glucosyltransferase That Synthesizes the Glc-β1,3-Fuc Disaccharide on Thrombospondin Type 1 Repeats

• Published in: The Journal of biological chemistry 2006-12, Vol.281 (48), p.36742
• Main Authors: Krisztina Kozma, Jeremy J. Keusch, Björn Hegemann, Kelvin B. Luther, Dominique Klein, Daniel Hess, Robert S. Haltiwanger, Jan Hofsteenge
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M605912200

Thrombospondin type 1 repeats (TSRs) are biologically important domains of extracellular proteins. They are modified with a unique Glcβ1,3Fucα1- O -linked disaccharide on either serine or threonine residues. Here we identify the putative glycosyltransferase, B3GTL, as the β1,3-glucosyltransferase involved in the biosynthesis of this disaccharide. This enzyme is conserved from Caenorhabditis elegans to man and shares 28% sequence identity with Fringe, the β1,3- N -acetylglucosaminyltransferase that modifies O- linked fucosyl residues in proteins containing epidermal growth factor-like domains, such as Notch. β1,3-Glucosyltransferase glucosylates properly folded TSR-fucose but not fucosylated epidermal growth factor-like domain or the non-fucosylated modules. Specifically, the glucose is added in a β1,3-linkage to the fucose in TSR. The activity profiles of β1,3-glucosyltransferase and protein O- fucosyltransferase 2, the enzyme that carries out the first step in TSR O- fucosylation, superimpose in endoplasmic reticulum subfractions obtained by density gradient centrifugation. Both enzymes are soluble proteins that efficiently modify properly folded TSR modules. The identification of the β1,3-glucosyltransferase gene allows us to manipulate the formation of the rare Glcβ1,3Fucα1 structure to investigate its biological function.