Purification of a chitooligosaccharidolytic beta-N-acetylglucosaminidase from Bombyx mori larvae during metamorphosis and the nucleotide sequence of its cDNA.
Three beta-N-acetylglucosaminidase (catalyzing hydrolysis of p-nitrophenyl-beta-D-GlcNAc) were purified from the integument tissue of Bombyx mori larvae during metamorphosis into pupae. The largest enzyme (66 kDa by SDS-PAGE, 126 kDa by gel-filtration chromatography) reacted with chitooligosaccharides to produce GlcNAc. A full-length cDNA encoding this chitooligosaccharidolytic beta-GlcNAcase was isolated. Based on the amino acid sequence deduced from the nucleotide sequence, the pre-beta-GlcNAcase was found to consist of 596 amino acid residues including a characteristic signal peptide of 23 residues and have an M(r) of 68,212. Homoloyg search and limited proteolytic digestion showed that the enzyme has a C-terminal 58-kDa catalytic domain very similar to that of human lysosomal beta-hexosaminidase that is responsible for hydrolyzing gangliosides. Two other enzymes (composed of 58-kDa and 48-kDa polypeptides, respectively) did not hydrolyze chitooligosaccharides, and were not proteolytic fragments from the largest enzyme judged by amino acid sequencing analyses. Natural substrates for the beta-GlcNAcases are unknown.
Nagamatsu
Y
Y
Department of Applied Biochemistry, Faculty of Applied Biological Science, Hiroshima University, Japan.
Yanagisawa
I
I
Kimoto
M
M
Okamoto
E
E
Koga
D
D
D000117:Acetylglucosamine / Q000378:metabolism; D000118:Acetylglucosaminidase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D012831:Bombyx / Q000201:enzymology* / Q000235:genetics; D002686:Chitin / Q000378:metabolism*; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006596:Hexosaminidases / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007814:Larva / Q000201:enzymology / Q000235:genetics; D008247:Lysosomes / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009844:Oligosaccharides / Q000378:metabolism*; D021382:Protein Sorting Signals; D017386:Sequence Homology, Amino Acid; D048828:beta-N-Acetyl-Galactosaminidase; D001619:beta-N-Acetylhexosaminidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D009844:Oligosaccharides; D021382:Protein Sorting Signals; D002686:Chitin; D006596:Hexosaminidases; D000118:Acetylglucosaminidase; D001619:beta-N-Acetylhexosaminidases; D048828:beta-N-Acetyl-Galactosaminidase; D000117:Acetylglucosamine