Studies on gonococcus infection. XVI. Purification of Neisseria gonorrhoeae immunoglobulin A1 protease

Abstract

A protease which cleaves human immunoglobulin A1 (IgAj) has been purified from broth cultures of Neisseria gonorrhoeae. This IgAj protease is produced by pilated and nonpilated gonococci throughout their growth cycles. A combination of ammonium sulfate precipitation, column chromatography, and either isoelectric focusing or affinity chromatography was utilized to obtain an enzyme preparation that showed approximately 3,800-fold purification and exhibited two bands (65,000 and 70,000 daltons) by analytical polyacrylamide electrophoresis in the presence of sodium dodecyl sulfate and reducing conditions. IgAj protease activity is dependent on divalent cations and is heat labile. Detection and quantitation of IgA protease activity utilized an assay in which [251I]IgA, is incubated with protease preparations and the cleavage products are analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.In 1971 Mueller described a substance in culture supematants of Neisseria gonorrhoeae and several other bacterial species which, when reacted with serum proteins, caused immunoglobulin A (IgA) to migrate more negatively in immunoelectrophoresis (14). This phenomenon was explained when Plaut et al. described a protease in gonococcal culture broth which cleaves IgA1 in the hinge region to form Fab and Fc fragments (16).A role of this enzyme in gonococcal infections or in modifying the host immune response has not been established. Antibodies to gonococcal antigens can be found in sera of patients who have had gonorrhea (1-3,8), but because uncomplicated, acute gonorrhea is largely confined to mucosal surfaces, the local immune response is of greater theoretical importance in this infection. O'Reilly and co-workers demonstrated the presence of secretory immunoglobulins specific for gonococci in exudates from both male and female patients with gonorrheal infections (7, 8).These results plus our findings that IgAj protease can be detected in vaginal washings of women with gonorrhea (M. S. Blake et al., J. Infect. Dis., in press) suggest that there could be interactions between secretory IgA (sIgA) and gonococcal protease in the course of gonorrheal infections. This paper describes methods to purify IgAj protease from culture supernatants of N. gonorrhoeae. t Present address: The Rockefeller University, New York, NY 10021. (This manuscript was submitted to the University of Utah by M.S.B. in partial fulfillment of the requirements for the Ph.D. degree in experimental pathology.) MATERIALS AND METHODS Assay system. IgA, was isolated from human myeloma serum by the method of Putnam et al. ( 18) and labeled with '25I (New England Nuclear Corp., Boston, Mass.) by using a modification of the method described by Marchalonis (13). This method was modified only by using lactoperoxidase (Sigma Chemical Co., St. Louis, Mo.) which had been coupled to Sepharose 4B (Pharmacia Fine Chemicals, Inc., Piscataway, N.J.) (4). Unbound '25I was removed by filtering the solution over a Sephadex G 25 column (0.9 by 15 cm) equilibrated with 0.05...

Purification and characterization of an immunoglobulin A1 protease from Bacteroides melaninogenicus

“…It did not attach other human immunoglobulin isotypes, and IgAs from a variety of animal species were resistant to proteolysis. The molecular weight determined for the enzyme (62K) is close to the molecular weight of IgAl protease purified from N. gonorrhoeae (2). Furthermore, the Bacteroides protease resembles other IgAl proteases in cleaving IgAl over a broad pH range (pH 4.5 to 7.5), although maximal activity occurs at a pH somewhat lower (pH 5.0) than that of several other IgAl proteases (2, 13, 23, Lineweaver-Burke double-reciprocal plot of the B. melaninogenicus IgAl protease activity at pH 5.5.…”

“…Previous studies of IgAl proteases from N. meningitidis, H. influenzae, S. pneumoniae, S. sanguis, and N. gonorrhoeae have shown that enzymes from different species may differ with respect to the exact site of cleavage in the ot1 chain (11,17,22), susceptibility to EDTA (7, 8, 11), immunochemical properties (10,11), and isoelectric point and molecular weight (2,13,23). However, common to all IgAl proteases that have been examined in detail is that they belong to the group of metal-dependent proteases.…”

Antigenic heterogeneity of immunoglobulin A1 proteases from encapsulated and non-encapsulated Haemophilus influenzae

“…Despite many common characteristics, detailed studies have shown that IgAl proteases from different species may differ with respect to the site of attack in the al chain (14,18,25,26,32,33), susceptibility to EDTA (12,14,18), immunochemical properties (17,18), isoelectric point, and molecular weight (6,19,34). Furthermore, strain differences within some of the species have recently been demonstrated.…”

Subversion of Phagocytosis for Pathogen Survival

“…There are also more specific strategies to avoid (Kilian et al, 1979), Neisseria spp. (Blake and Swanson, 1978), and Staphylococcus aureus (Ryan et al, 2008) produce immunoglobulin (Ig) proteases, which cleave Ig in the hinge region. This results in separation of the Fab (fragment, antibody-binding) and Fc regions of the antibody, precluding capture of the immune complex by Fc receptors.…”