Cloning of a human UDP-N-acetyl-alpha-D-Galactosamine:polypeptide N-acetylgalactosaminyltransferase that complements other GalNAc-transferases in complete O-glycosylation of the MUC1 tandem repeat.
A fourth human UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase, designated GalNAc-T4, was cloned and expressed. The genomic organization of GalNAc-T4 is distinct from GalNAc-T1, -T2, and -T3, which contain multiple coding exons, in that the coding region is contained in a single exon. GalNAc-T4 was placed at human chromosome 12q21.3-q22 by in situ hybridization and linkage analysis. GalNAc-T4 expressed in Sf9 cells or in a stably transfected Chinese hamster ovary cell line exhibited a unique acceptor substrate specificity. GalNAc-T4 transferred GalNAc to two sites in the MUC1 tandem repeat sequence (Ser in GVTSA and Thr in PDTR) using a 24-mer glycopeptide with GalNAc residues attached at sites utilized by GalNAc-T1, -T2, and -T3 (TAPPAHGVTSAPDTRPAPGSTAPPA, GalNAc attachment sites underlined). Furthermore, GalNAc-T4 showed the best kinetic properties with an O-glycosylation site in the P-selectin glycoprotein ligand-1 molecule. Northern analysis of human organs revealed a wide expression pattern. Immunohistology with a monoclonal antibody showed the expected Golgi-like localization in salivary glands. A single base polymorphism, G1516A (Val to Ile), was identified (allele frequency 34%). The function of GalNAc-T4 complements other GalNAc-transferases in O-glycosylation of MUC1 showing that glycosylation of MUC1 is a highly ordered process and changes in the repertoire or topology of GalNAc-transferases will result in altered pattern of O-glycan attachments.
Bennett
E P
EP
Faculty of Health Sciences, School of Dentistry, Copenhagen, Denmark.
Hassan
H
H
Mandel
U
U
Mirgorodskaya
E
E
Roepstorff
P
P
Burchell
J
J
Taylor-Papadimitriou
J
J
Hollingsworth
M A
MA
Merkx
G
G
van Kessel
A G
AG
Eiberg
H
H
Steffensen
R
R
Clausen
H
H
D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary / Q000737:chemistry; D008040:Genetic Linkage; D006031:Glycosylation; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D018396:Mucin-1 / Q000378:metabolism*; D017350:N-Acetylgalactosaminyltransferases / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D018411:Spodoptera; D013363:Submandibular Gland / Q000201:enzymology; D013379:Substrate Specificity; D020080:Tandem Repeat Sequences; D013912:Threonine / Q000378:metabolism; D000097763:Polypeptide N-acetylgalactosaminyltransferase
D018076:DNA, Complementary; D018396:Mucin-1; D017350:N-Acetylgalactosaminyltransferases; D013912:Threonine; D000097763:Polypeptide N-acetylgalactosaminyltransferase