Purification, cDNA cloning, and expression of UDP-N-acetylglucosamine: beta-D-mannoside beta-1,4N-acetylglucosaminyltransferase III from rat kidney.
UDP-N-acetylglucosamine: beta-D-mannoside beta-1,4N-acetylglucosaminyltransferase III (GnT-III: EC 2.4.1.144) catalyzes the addition of N-acetylglucosamine in beta 1-4 linkage to the beta-linked mannose of the trimannosyl core of N-linked sugar chains. The enzyme has been purified over 153,000-fold in 1.5% yield from a Triton X-100 extract of rat kidney by fractionation procedures utilizing QAE-Sepharose, Cu(2+)-chelating Sepharose, and affinity chromatography on UDP-hexanolamine and substrate-conjugated Sepharose. The purified protein migrates as one major and one minor band with apparent molecular masses of 62 kDa and 52 kDa, respectively. The purified enzyme was digested with trypsin, and the amino acid sequences of four peptides were determined. Oligonucleotide primers were designed according to those amino acid sequences and used in the polymerase chain reaction. Screening for the cDNA for GnT-III was carried out by plaque hybridization using a rat kidney cDNA library (lambda gt10) and a polymerase chain reaction product as the probe. Rat kidney GnT-III has 536 amino acids and three putative N-glycosylation sites. There is no sequence homology to other previously cloned glycosyltransferases, but the enzyme appears to be a type II transmembrane protein like the other glycosyltransferases. The GnT-III activity in transiently transfected COS-1 cells was found to be about 500-3600-fold as compared to that in non- or mock-transfected cells.
Nishikawa
A
A
Department of Biochemistry, Osaka University Medical School, Japan.
Ihara
Y
Y
Hatakeyama
M
M
Kangawa
K
K
Taniguchi
N
N
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002845:Chromatography; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D017886:Durapatite; D005964:Glucosyltransferases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D006882:Hydroxyapatites; D007668:Kidney / Q000201:enzymology*; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases; D010446:Peptide Fragments / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection
D006882:Hydroxyapatites; D010446:Peptide Fragments; D011994:Recombinant Proteins; D004247:DNA; D017886:Durapatite; D005964:Glucosyltransferases; D017351:N-Acetylglucosaminyltransferases; C054599:beta-1,4-mannosyl-glycoprotein beta-1,4-N-acetylglucosaminyltransferase