Isolation and expression of a cDNA clone encoding a bovine UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase.
NH2-terminal amino acid sequence obtained from a UDP-GalNAc:polypeptide N-acetylgalactosaminyl-transferase (GalNAc-transferase) isolated from bovine colostrum was used for the construction of synthetic oligonucleotide primers. Subsequent polymerase chain reaction and library screenings of a bovine intestine cDNA library produced seven positive clones. The largest clone had a 2294-base pair insert that contained an open reading frame coding for a protein composed of 559 amino acids with a predicted polypeptide molecular mass of 64,173 Da. The cloned molecule has no significant sequence homology to previously reported cloned glycosyltransferases, but appears to have a similar domain structure. It is a type II membrane protein with a 23-amino acid putative transmembrane region starting 8 amino acids from the NH2 terminus. The transmembrane segment of the molecule is immediately followed by a sequence rich in proline residues. The molecule contains three consensus sequences for N-linked glycosylation and five predicted sites for O-glycosylation. Northern blot analysis of poly(A+) mRNA isolated from Madin-Darby bovine kidney cells, bovine mammary tissue, and eight human tissues demonstrated the expression of two transcripts differing in size by approximately 1 kilobase. The cloned DNA was expressed in insect cells using a baculovirus vector. This resulted in an almost 100-fold increase in GalNAc-transferase activity in lysates prepared from cells infected with virus containing the GalNAc-transferase gene compared to cells infected with virus containing DNA coding for an unrelated molecule or uninfected cells. Immunoprecipitation from lysates prepared from infected cells labeled in vivo with [35S] methionine showed a large increase in the recovery of an approximately 67-kDa protein.
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Molecular Biology Research Unit, Upjohn Company, Kalamazoo, Michigan 49001.
Hollander
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Lehman
D J
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Thomsen
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Elhammer
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular; D003126:Colostrum / Q000201:enzymology*; D004247:DNA / Q000302:isolation & purification; D005260:Female; D015870:Gene Expression; D015723:Gene Library; D006031:Glycosylation; D007421:Intestine, Small / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D009036:Moths; D017350:N-Acetylgalactosaminyltransferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D009838:Oligodeoxyribonucleotides; D011061:Poly A / Q000302:isolation & purification / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D011499:Protein Processing, Post-Translational; D012313:RNA / Q000302:isolation & purification / Q000378:metabolism; D012333:RNA, Messenger / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D014162:Transfection; D000097763:Polypeptide N-acetylgalactosaminyltransferase
D004247:DNA; D017350:N-Acetylgalactosaminyltransferases; D009838:Oligodeoxyribonucleotides; D011061:Poly A; D012313:RNA; D012333:RNA, Messenger; D011994:Recombinant Proteins; D000097763:Polypeptide N-acetylgalactosaminyltransferase