cDNA cloning and expression of bovine UDP-N-acetylglucosamine: alpha1, 3-D-mannoside beta1,4-N-acetylglucosaminyltransferase IV.
UDP-N-acetylglucosamine:alpha1,3-D-mannoside beta1, 4-N-acetylglucosaminyltransferase (GnT-IV) is one of the essential enzymes in the production of tri- and tetra-antennary Asn-linked sugar chains. Recently, we have successfully purified GnT-IV from bovine small intestine. Based on the partial amino acid sequence of the purified bovine GnT-IV enzyme, its cDNA has been cloned from bovine small intestine. The open reading frame is 1,605 base pairs long, and this sequence produced GnT-IV activity on transient expression in COS-7 cells. Although the deduced amino acid sequence does not have any significant homology with other known N-acetylglucosaminyltransferases (GnTs), the hydrophobicity profile showed a typical type II transmembrane protein structure, which is common to many glycosyltransferases. N-terminal amino acid sequencing of the purified GnT-IV revealed that 92 amino acids, including a transmembrane region, were truncated during purification. Of the three potential N-glycosylation sites Asn-458 was actually glycosylated in the purified enzyme, although this N-glycosylation site could be abolished without any reduction in GnT-IV activity. Serial deletions at both the N and C termini proved that the catalytic domain of GnT-IV is located in the central region of the enzyme. The GnT-IV mRNA level correlated with enzymatic activity in the various bovine tissues tested.
Minowa
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MT
Central Laboratories for Key Technology, KIRIN Brewery Co., Ltd., 1-13-5 Fuku-ura, Kanazawa-ku, Yokohama, 236-0004, Japan.
Oguri
S
S
Yoshida
A
A
Hara
T
T
Iwamatsu
A
A
Ikenaga
H
H
Takeuchi
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002417:Cattle; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics*; D010449:Peptide Mapping; D012333:RNA, Messenger / Q000235:genetics; D015183:Restriction Mapping; D017384:Sequence Deletion; D012995:Solubility; D013329:Structure-Activity Relationship; D014162:Transfection
D018076:DNA, Complementary; D012333:RNA, Messenger; D017351:N-Acetylglucosaminyltransferases; C038111:alpha-1,3-mannosylglycoprotein beta-1,4-N-acetylglucosaminyltransferase