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.
Fritz
Timothy A
TA
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.
Hurley
James H
JH
Trinh
Loc-Ba
LB
Shiloach
Joseph
J
Tabak
Lawrence A
LA
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