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Infection and Immunity, August 1999, p. 3816-3823, Vol. 67, No. 8
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Variable Carbohydrate Modifications to the Catalytic Chains of the RgpA and RgpB Proteases of Porphyromonas gingivalis W50

Michael A. Curtis,1,* Andrea Thickett,1 Jennifer M. Slaney,1 Minnie Rangarajan,1 Joseph Aduse-Opoku,2 Philip Shepherd,2 Nikolay Paramonov,1 and Elizabeth F. Hounsell3

MRC Molecular Pathogenesis Group, Department of Oral Microbiology, St. Bartholomew's and the Royal London School of Medicine and Dentistry, Queen Mary and Westfield College, London E1 2AA,1 Department of Immunology, United Medical and Dental Schools, London SE1 9RT,2 and Department of Biochemistry and Molecular Biology, University College London, London WC1E 6BT,3 United Kingdom

Received 7 December 1998/Returned for modification 9 March 1999/Accepted 4 May 1999

Proteases of Porphyromonas gingivalis are considered to be important virulence determinants of this periodontal bacterium. Several biochemical isoforms of arginine-specific proteases are derived from rgpA and rgpB. HRgpA is a heterodimer composed of the catalytic alpha  chain noncovalently associated with a beta  adhesin chain derived from the C terminus of the initial full-length translation product. The catalytic alpha  chain is also present as a monomer (RgpA) either free in solution or associated with membranes. rgpB lacks the coding region for the adhesin domain present in rgpA and yields only monomeric forms (RgpB) which again may be soluble or membrane associated. In this study, the catalytic chains of this unusual group of enzymes are shown to be differentially modified by the posttranslational addition of carbohydrate. A monoclonal antibody (MAb 1B5) raised to the monomeric RgpA did not react with the corresponding recombinant RgpA alpha  chain expressed in Escherichia coli but was immunoreactive with P. gingivalis lipopolysaccharide. MAb 1B5 also reacted with the membrane-associated forms of RgpA and RgpB but not with the heterodimeric HRgpA and the soluble form of RgpB. RgpA treated with denaturants was capable of binding to MAb 1B5 whereas treatment with periodate abolished this binding, suggesting the presence of carbohydrate residues within the epitope. Chemical deglycosylation abolished immunoreactivity with MAb 1B5 and caused a ~30% reduction in the size of the membrane-associated enzymes. Monosaccharide analysis of HRgpA and RgpA demonstrated 2.1 and 14.4%, respectively, carbohydrate by weight of protein. Furthermore, distinct differences were detected in their monosaccharide compositions, indicating that these protease isoforms are modified not only to different extents but also with different sugars. The variable nature of these additions may have a significant effect on the structure, stability, and immune recognition of these protease glycoproteins.

* Corresponding author. Mailing address: MRC Molecular Pathogenesis Group, Department of Oral Microbiology, St. Bartholomew's and the Royal London School of Medicine and Dentistry, Queen Mary and Westfield College, 32 Newark St., London E1 2AA, United Kingdom. Phone: 0171 377 0444. Fax: 0171 247 3428. E-mail: M.A.Curtis{at}mds.qmw.ac.uk.

Infection and Immunity, August 1999, p. 3816-3823, Vol. 67, No. 8
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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