The beginnings of mucin biosynthesis: the crystal structure of UDP-GalNAc:polypeptide alpha-N-acetylgalactosaminyltransferase-T1.
UDP-GalNAc:polypeptide alpha-N-acetylgalactosaminyltransferases (ppGaNTases) initiate the formation of mucin-type, O-linked glycans by catalyzing the transfer of alpha-N-acetylgalactosamine from UDP-GalNAc to Ser or Thr residues of core proteins to form the Tn antigen (GalNAc-alpha-1-O-Ser/Thr). ppGaNTases are unique among glycosyltransferases in containing a C-terminal lectin domain. We present the x-ray crystal structure of a ppGaNTase, murine ppGaNTase-T1, and show that it folds to form distinct catalytic and lectin domains. The association of the two domains forms a large cleft in the surface of the enzyme that contains a Mn2+ ion complexed by invariant D209 and H211 of the "DXH" motif and by invariant H344. Each of the three potential lectin domain carbohydrate-binding sites (alpha, beta, and gamma) is located on the active-site face of the enzyme, suggesting a mechanism by which the transferase may accommodate multiple conformations of glycosylated acceptor substrates. A model of a mucin 1 glycopeptide substrate bound to the enzyme shows that the spatial separation between the lectin alpha site and a modeled active site UDP-GalNAc is consistent with the in vitro pattern of glycosylation observed for this peptide catalyzed by ppGaNTase-T1. The structure also provides a template for the larger ppGaNTase family, and homology models of several ppGaNTase isoforms predict dramatically different surface chemistries consistent with isoform-selective acceptor substrate recognition.
Section on Biological Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Department of Health and Human Services, Bethesda, MD 20892, USA.
Timothy A
TA
Fritz
James H
JH
Hurley
Loc-Ba
LB
Trinh
Joseph
J
Shiloach
Lawrence A
LA
Tabak
D000818:Animals; D001665:Binding Sites; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D037102:Lectins / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D009077:Mucins / Q000096:biosynthesis*; D017350:N-Acetylgalactosaminyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D000097763:Polypeptide N-acetylgalactosaminyltransferase
D037102:Lectins; D009077:Mucins; D017350:N-Acetylgalactosaminyltransferases; D000097763:Polypeptide N-acetylgalactosaminyltransferase