Molecular cloning and expression of endo-beta-N-acetylglucosaminidase D, which acts on the core structure of complex type asparagine-linked oligosaccharides

Endo-beta -N-acetylglucosaminidase D (Endo D) produced by Streptococcus pne umoniae cleaves the di-N-acetylchitobiose structure in asparagine-linked ol igosaccharides. The enzyme generally acts on complex type oligosaccharides after removal of external sugars by neuraminidase, beta -galactosidase, and beta -N-acetylglucosaminidase. We cloned the gene encoding the enzyme and expressed it as a periplasm enzyme in Escherichia coli. The first 37 amino acids in the predicted sequence are removed in the mature enzyme, yielding a protein with a molecular mass of 178 kDa. The substrate specificity of th e recombinant enzyme is indistinguishable from the enzyme produced by S. pn eumoniae. Endo-beta -N-acetylglucosaminidase A (Endo A) hom Arthrobacter pr otophormiae, the molecular mass of which is 72 kDa, had 32% sequence identi ty to Endo D, starting from the N-terminal sides of both enzymes, although Endo A hydrolyzes high-mannose-type oligosaccharides and does not hydrolyze complex type ones. Endo D is not related to endo-beta -N-acetylglucosamini dases H, F-1, F-2, or F-3, which share common structural motifs. Therefore, there are two distinct groups of endo-beta -N-acetylglucosaminidases actin g on asparagine-linked oligosaccharides. The C-terminal region of Endo D sh ows homology to beta -galactosidase and beta -N-acetylglucosaminidase from S. pneumoniae and has an LPXTG motif typical of surface-associated proteins of Gram-positive bacteria, It is possible that Endo D is located on the su rface of the bacterium and, together with other glycosidases, is involved i n virulence.