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J. Biol. Chem., Vol. 283, Issue 34, 22942-22951, August 22, 2008

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The Catalytic and Lectin Domains of UDP-GalNAc:Polypeptide -N-Acetylgalactosaminyltransferase Function in Concert to Direct Glycosylation Site Selection^*

Jayalakshmi Raman, Timothy A. Fritz, Thomas A. Gerken^¶, Oliver Jamison, David Live^||, Mian Liu^||, and Lawrence A. Tabak¹

From the Section on Biological Chemistry, NIDDK, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland 20892, the W. A. Bernbaum Center for Cystic Fibrosis Research, ^¶Departments of Pediatrics and Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, and the ^||Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602

UDP-GalNAc:polypeptide -N-Acetylgalactosaminyltransferases (ppGalNAcTs), a family (EC 2.4.1.4 [EC] 1) of enzymes that initiate mucin-type O-glycosylation, are structurally composed of a catalytic domain and a lectin domain. Previous studies have suggested that the lectin domain modulates the glycosylation of glycopeptide substrates and may underlie the strict glycopeptide specificity of some isoforms (ppGalNAcT-7 and -10). Using a set of synthetic peptides and glycopeptides based upon the sequence of the mucin, MUC5AC, we have examined the activity and glycosylation site preference of lectin domain deletion and exchange constructs of the peptide/glycopeptide transferase ppGalNAcT-2 (hT2) and the glycopeptide transferase ppGalNAcT-10 (hT10). We demonstrate that the lectin domain of hT2 directs glycosylation site selection for glycopeptide substrates. Pre-steady-state kinetic measurements show that this effect is attributable to two mechanisms, either lectin domain-aided substrate binding or lectin domain-aided product release following glycosylation. We find that glycosylation of peptide substrates by hT10 requires binding of existing GalNAcs on the substrate to either its catalytic or lectin domain, thereby resulting in its apparent strict glycopeptide specificity. These results highlight the existence of two modes of site selection used by these ppGalNAcTs: local sequence recognition by the catalytic domain and the concerted recognition of distal sites of prior glycosylation together with local sequence binding mediated, respectively, by the lectin and catalytic domains. The latter mode may facilitate the glycosylation of serine or threonine residues, which occur in sequence contexts that would not be efficiently glycosylated by the catalytic domain alone. Local sequence recognition by the catalytic domain differs between hT2 and hT10 in that hT10 requires a pre-existing GalNAc residue while hT2 does not.

Received for publication, May 2, 2008 , and in revised form, June 17, 2008.

^* This work was supported, in whole or in part, by the National Institutes of Health, through funds of the intramural program of the NIDDK and Grants NCI-RO1 CA-78834 (to T. A. G.) and NIGMS-RO1 GM-066148 (to D. L.). 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.

¹ To whom correspondence should be addressed: Tel.: 301-496-3571; Fax: 301-402-2185; E-mail: tabakl{at}mail.nih.gov.

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