Cloning of cDNA encoding the membrane-bound form of bovine beta 1,4-galactosyltransferase.
UDPgalactose: N-acetyl-D-glucosamine 4-beta-D-galactosyltransferase (EC 2.4.1.38) (GalT) is a Golgi-membrane-bound enzyme that participates in the biosynthesis of the oligosaccharide structures of glycoproteins and glycolipids. Synthetic DNA oligomers representing segments of the published partial cDNA sequence for bovine GalT were used as molecular probes to isolate from bovine-liver cDNA libraries overlapping cDNA clones that span 1728 nucleotides and potentially code for the entire polypeptide chain of bovine galactosyltransferase. The cDNA sequence for bovine GalT reveals a 1206-base-pair open reading frame that codes for 402 amino acids, including a presumptive N-terminal membrane anchoring domain of 20 hydrophobic amino acids. The colinearity between the cDNA sequence and 29 non-overlapping amino acid residues which were positively identified by N-terminal sequencing of two polypeptides isolated from the soluble form of the enzyme was consistent with the translation frame and confirmed the authenticity of the cDNA clones. The finding of an N-terminal hydrophobic segment which serves as the membrane anchor and signal sequence suggests that the C-terminal region of the GalT polypeptide is oriented within the lumen of the Golgi membranes. This conclusion is in agreement with previous biochemical studies which indicated that the 51-kDa and 42-kDa soluble forms of the enzyme which encompass the C-terminal 324 and 297 amino acid residues of the entire GalT polypeptide, respectively, include the catalytic site.
D'Agostaro
G
G
Department of Medical Genetics, University of Toronto.
Bendiak
B
B
Tropak
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001482:Base Composition; D001665:Binding Sites; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D003001:Cloning, Molecular; D004247:DNA / Q000302:isolation & purification*; D015342:DNA Probes; D005700:Galactosyltransferases / Q000235:genetics*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D010455:Peptides / Q000302:isolation & purification; D012689:Sequence Homology, Nucleic Acid; D006022:beta-N-Acetylglucosaminylglycopeptide beta-1,4-Galactosyltransferase / Q000235:genetics*
D015342:DNA Probes; D010446:Peptide Fragments; D010455:Peptides; D004247:DNA; D005700:Galactosyltransferases; D006022:beta-N-Acetylglucosaminylglycopeptide beta-1,4-Galactosyltransferase