Purification, cloning, and expression of a bovine UDP-GalNAc: polypeptide N-acetyl-galactosaminyltransferase.
Partial amino acid sequence was obtained from UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (polypeptide GalNAc transferase) purified from bovine colostrum. Oligonucleotide primers designed from these sequences were used to amplify and clone a polypeptide GalNAc transferase cDNA from bovine placental mRNA. The cDNA encodes an open reading frame, which is 519 amino acids in length and contains the predicted N-terminal and internal amino acid sequence derived from three Lys-C peptides obtained from the purified protein. There was no sequence homology with the UDP-GalNAc: Fuc alpha 1,2Gal alpha 1,3GalNAc transferase. To verify the authenticity of the clone, the cDNA was cloned in frame with an insulin secretion sequence and was expressed transiently in COS-7 cells. Polypeptide GalNAc transferase activity was detected in the culture medium; no activity was detected in the media of mock-transfected cells. Previous studies have shown that the polypeptide GalNAc transferase from bovine colostrum glycosylates threonine residues more efficiently than serine residues in the same peptide context (O'Connell, B. C., Hagen, F. K., and Tabak, L. A. (1992) J. Biol. Chem. 267, 25010-25018). We found that the cloned polypeptide GalNAc transferase glycosylates the threonine-containing peptide, PPDAATAAPLR, at a 58-fold greater rate than the serine-containing homologue, PPDAASAAPLR. The ratio of the in vitro threonine and serine glycosylation rate is identical for the cloned placental and purified colostral enzymes. It is not known if the preference for threonine over serine is merely context-dependent on the specific amino acids that flank the glycosylation site or if there are discrete threonine- and serine-specific isoforms of this transferase. Alternatively, there may be additional factors required to enhance the glycosylation of serine residues in vivo.
Hagen
F K
FK
Department of Dental Research, School of Medicine and Dentistry, University of Rochester, New York 14642.
Van Wuyckhuyse
B
B
Tabak
L A
LA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular; D003126:Colostrum / Q000201:enzymology; D004247:DNA / Q000737:chemistry / Q000235:genetics; D005260:Female; D006031:Glycosylation; D008969:Molecular Sequence Data; D017350:N-Acetylgalactosaminyltransferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D010920:Placenta / Q000201:enzymology; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D013912:Threonine / Q000378:metabolism; D014162:Transfection; D000097763:Polypeptide N-acetylgalactosaminyltransferase
D004247:DNA; D017350:N-Acetylgalactosaminyltransferases; D012333:RNA, Messenger; D011994:Recombinant Proteins; D012694:Serine; D013912:Threonine; D000097763:Polypeptide N-acetylgalactosaminyltransferase