Molecular cloning and expression of two novel beta-N-acetylglucosaminidases from silkworm Bombyx mori.
Beta-N-acetylglucosaminidase is a major glycosidase involved in several physiological processes, such as fertilization, metamorphosis, glycoconjugate degradation, and glycoprotein biosynthesis in insects. A search using the Bombyx mori cDNA database revealed the existence of two putative beta-N-acetylglucosaminidase genes. Their full-length cDNAs were cloned by rapid amplification of cDNA ends and polymerase chain reaction using specific primers, and named BmGlcNAcase1 and BmGlcNAcase2. A BLAST search revealed that BmGlcNAcase1 and BmGlcNAcase2 are homologous to a beta-subunit homolog encoded by Drosophila melanogaster HEXO2 and the Spodoptera frugiperda beta-N-acetylglucosaminidase gene respectively. The recombinant proteins of BmGlcNAcase1 and BmGlcNAcase2 without putative transmembrane domains were expressed in the yeast Pichia pastoris. Both enzymes showed broad substrate specificity, and cleaved terminal N-acetylglucosamine residues from the alpha-3 and alpha-6 branches of a biantennary N-glycan substrate, and also hydrolyzed chitotriose to chitobiose.
Okada
Takahiro
T
The International Center for Biotechnology, Osaka University, Suita, Osaka, Japan.
Ishiyama
Seiji
S
Sezutsu
Hideki
H
Usami
Akihiro
A
Tamura
Toshiki
T
Mita
Kazuei
K
Fujiyama
Kazuhito
K
Seki
Tatsuji
T
D000118:Acetylglucosaminidase / Q000096:biosynthesis / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D012831:Bombyx / Q000201:enzymology*; D002240:Carbohydrate Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D007814:Larva / Q000235:genetics; D008969:Molecular Sequence Data
D018076:DNA, Complementary; D000118:Acetylglucosaminidase