Purification and cDNA cloning of a human UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase.
A UDP-GalNAc:polypeptide N-acetylgalactosaminyl-transferase (GalNAc-transferase) from human placenta was purified to apparent homogeneity using a synthetic acceptor peptide as affinity ligand. The purified GalNAc-transferase migrated as a single band with an approximate molecular weight of 52,000 by reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Based on a partial amino acid sequence, the cDNA encoding the transferase was cloned and sequenced from a cDNA library of a human cancer cell line. The cDNA sequence has a 571-amino acid coding region indicating a protein of 64.7 kDa with a type II domain structure. The deduced protein sequence showed significant similarity to a recently cloned bovine polypeptide GalNAc-transferase (Homa, F.L., Hollanders, T., Lehman, D.J., Thomsen, D.R., and Elhammer, A.P. (1993) J. Biol. Chem. 268, 12609-12616). A polymerase chain reaction construct was expressed in insect cells using a baculovirus vector. Northern analysis of eight human tissues differed clearly from that of the bovine GalNAc-transferase. Polymerase chain reaction cloning and sequencing of the human version of the bovine transferase are presented, and 98% similarity at the amino acid level was found. The data suggest that the purified human GalNAc-transferase is a novel member of a family of polypeptide GalNAc-transferases, and a nomenclature GalNAc-T1 and GalNAc-T2 is introduced to distinguish the members.
White
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Faculty of Health Sciences, School of Dentistry, University of Copenhagen, Denmark.
Bennett
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EP
Takio
K
K
Sørensen
T
T
Bonding
N
N
Clausen
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002417:Cattle; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D018076:DNA, Complementary; D005260:Female; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D017350:N-Acetylgalactosaminyltransferases / Q000096:biosynthesis* / Q000302:isolation & purification* / Q000378:metabolism; D015345:Oligonucleotide Probes; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010920:Placenta / Q000201:enzymology*; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D000097763:Polypeptide N-acetylgalactosaminyltransferase
D018076:DNA, Complementary; D017350:N-Acetylgalactosaminyltransferases; D015345:Oligonucleotide Probes; D010446:Peptide Fragments; D012333:RNA, Messenger; D011994:Recombinant Proteins; D000097763:Polypeptide N-acetylgalactosaminyltransferase