Abstract
Three β-N-acetylglucosaminidase (catalyzing hydrolysis of p-nitrophenyl-β-D-GlcNAc) were purified from the integument tissue of Bombyx mori larvae during metamorphosis into pupae. The largest enzyme (66kDa by SDS-PAGE, 126kDa by gel-filtration chromatography) reacted with chitooligosaccharides to produce GlcNAc. A full-length cDNA encoding this chitooligosaccharidolytic β-GlcNAcase was isolated. Based on the amino acid sequence deduced from the nucleotide sequence, the pre-β-GlcNAcase was found to consist of 596 amino acid residues including a characteristic signal peptide of 23 residues and have an Mr of 68, 212. Homology search and limited proteolytic digestion showed that the enzyme has a C-terminal 58-kDa catalytic domain very similar to that of human lysosomal β-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 β-GlcNAcases are unknown.
