Molecular cloning of a human cDNA encoding beta-1,4-galactosyltransferase with 37% identity to mammalian UDP-Gal:GlcNAc beta-1,4-galactosyltransferase.
A cDNA encoding a beta-1,4-galactosyltransferase named beta-1,4-GalT II was cloned from a cDNA library of the human breast tumor cell line, MRK-nu-1. Initially, a 860-bp PCR fragment was obtained from MRK-nu-1 mRNA by 3'-rapid amplification of cDNA ends by using two nested degenerate oligonucleotide primers based on a highly conserved amino acid sequence found in the catalytic domain of mammalian beta-1,4-galactosyltransferases and Lymnaea stagnalis beta-1,4-N-acetylglucosaminyltransferase (beta-1,4-GlcNAcT), both of which utilize the same sugar acceptor. This subsequently was used as a probe to isolate a 4.7-kb cDNA that contained an ORF of 1,164 bp predicting a polypeptide of 388 aa. Its deduced amino acid sequence shows an identity of 37% with that of the previously characterized human beta-1,4-galactosyltransferase (referred to as beta-1,4-GalT I) and of 28% with that of L. stagnalis beta-1,4-GlcNAcT. Study of the properties of the beta-1,4-GalT II fused to protein A expressed as a soluble form in COS-7 cells revealed that it is a genuine beta-1,4-GalT but has no lactose synthetase activity in the presence of alpha-lactalbumin. Northern blot analysis of 24 human tissues showed that they all express the beta-1,4-GalT II transcript, although the levels varied. These results indicate that human cells contain another beta-1,4-GalT.
Sato
T
T
Department of Biosignal Research, Tokyo Metropolitan Institute of Gerontology, Japan.
Furukawa
K
K
Bakker
H
H
Van den Eijnden
D H
DH
Van Die
I
I
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D008969:Molecular Sequence Data; D012689:Sequence Homology, Nucleic Acid; D006022:beta-N-Acetylglucosaminylglycopeptide beta-1,4-Galactosyltransferase / Q000235:genetics*
D018076:DNA, Complementary; D006022:beta-N-Acetylglucosaminylglycopeptide beta-1,4-Galactosyltransferase