Cloning and expression in Escherichia coli of the nahA gene from Porphyromonas gingivalis indicates that beta-N-acetylhexosaminidase is an outer-membrane-associated lipoprotein.
Porphyromonas gingivalis has been implicated in human periodontal diseases. It expresses a number of exoglycosidase enzymes capable of hydrolysing host proteoglycan residues. As a first stage to explore the role of these enzymes in periodontal tissue damage, the nahA gene of P. gingivalis W83, which encodes beta-N-acetylhexosaminidase (beta-Nahase), was cloned. The gene was expressed poorly in Escherichia coli, but increased expression was achieved by cloning the nahA gene downstream of the tac promoter. Southern blot analysis revealed that nahA was present as a single copy, and it was found in all the other P. gingivalis strains tested. In contrast, sequences homologous to nahA were not detected in either P. endodontalis or P. asaccharolytica. The nahA gene was 2331 bp long and encoded a beta-Nahase enzyme of 777 amino acids with a predicted molecular mass of 87 kDa. A characteristic signal peptide for an acylated lipoprotein was present at the amino-terminus, suggesting that the mature beta-Nahase is a lipoprotein. The predicted amino acid sequence of the P. gingivalis beta-Nahase shared homology with the catalytic domains of the human beta-Nahase enzyme and the chitinase of Vibrio harveyi, suggesting a common catalytic mechanism.
Lovatt
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Department of Microbiology and Immunology, University of Leicester, UK.
Roberts
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D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial; D006801:Humans; D008074:Lipoproteins / Q000235:genetics; D008969:Molecular Sequence Data; D010510:Periodontal Diseases / Q000209:etiology; D016966:Porphyromonas gingivalis / Q000201:enzymology* / Q000235:genetics* / Q000472:pathogenicity; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D001619:beta-N-Acetylhexosaminidases / Q000235:genetics*
D001425:Bacterial Outer Membrane Proteins; D004269:DNA, Bacterial; D008074:Lipoproteins; D001619:beta-N-Acetylhexosaminidases