K21-Antigen: A Molecule Shared by the Microenvironments of the Human Thymus and Germinal Centers

The mouse IgG1 monoclonal antibody (mAb) K21 recognizes a 230-kD molecule (K21-Ag) on Hassall's corpuscles in the human thymus. This mAb also stains cultured thymic epithelial cells as well as other epithelial cell lines, revealing a predominant intracellular localization. Further analysis with mAb K21 on other lymphoid tissues showed that it also stains cells within the germinal centers of human tonsils, both lymphoid (B) cells and some with the appearance of follicular dendritic cells. Double immunostaining of tonsil sections shows that K21-Ag is not expressed by T cells, whereas staining with anti-CD22 and -CD23 mAb revealed some doublepositive cells. A subpopulation of the lymphoid cells express the K21-Ag much more strongly. This K21++/CD23++ subpopulation of cells is localized in the apical light zone of germinal centers, suggesting that K21-Ag may be an important marker for the selected centrocytes within germinal centers and may play a role in B-cell selection and/or development of B-cell memory. Flow cytometric analysis showed that K21-Ag is expressed on the surface of a very low percentage of thymocytes, tonsillar lymphocytes, and peripheral blood mononuclear cells. Analysis of purified/separated tonsillar T and B lymphocytes showed that T cells do not express the K21-Ag; in contrast, B cells express low levels of the K21-Ag, and this together with CD23 is upregulated after mitogenic stimulation. Our data therefore raise the possibility that the K2l- Ag may play a role in B-lymphocyte activation/selection

Down-regulation of human extracellular cysteine protease inhibitors by the secreted staphylococcal cysteine proteases, staphopain A and B

Of seven human cystatins investigated, none inhibited the cysteine proteases staphopain A and B secreted by the human pathogen Staphylococcus aureus. Rather, the extracellular cystatins C, D and E/M were hydrolyzed by both staphopains. Based on MALDI-TOF time-course experiments, staphopain A cleavage of cystatin C and D should be physiologically relevant and occur upon S. aureus infection. Staphopain A hydrolyzed the Glyl 1 bond of cystatin C and the Ala10 bond of cystatin D with similar K-m values of approximately 33 and 32 mu m, respectively. Such N-terminal truncation of cystatin C caused > 300-fold lower inhibition of papain, cathepsin B, L and K, whereas the cathepsin H activity was compromised by a factor of ca. 10. Similarly, truncation of cystatin D caused alleviated inhibition of all endogenous target enzymes investigated. The normal activity of the cystatins is thus down-regulated, indicating that the bacterial enzymes can cause disturbance of the host protease-inhibitor balance. To illustrate the in vivo consequences, a mixed cystatin C assay showed release of cathepsin B activity in the presence of staphopain A. Results presented for the specificity of staphopains when interacting with cystatins as natural protein substrates could aid in the development of therapeutic agents directed toward these proteolytic virulence factors

The Human Protease Inhibitor Cystatin C Is an Activating Cofactor for the Streptococcal Cysteine Protease IdeS

SummaryHuman cystatin C is considered the physiologically most important inhibitor of endogenous papain-like cysteine proteases. We present here an unexpected function of cystatin C. Instead of acting as an inhibitor, cystatin C acts as a facultative, endogenous cofactor for the papain-like IgG-cleaving enzyme IdeS of the human pathogen Streptococcus pyogenes. IdeS activity is not dependent on cystatin C, but is significantly enhanced in the presence of cystatin C. We report a protease inhibitor that accelerates the activity of its putative target protease and a unique example of how a host protease inhibitor is “hijacked” by a bacterial protease to increase its activity. This finding has important implications for the view on protease-inhibitor interactions, and is relevant to consider in the therapeutic use of protease inhibitors

Cleavage of IgG1 and IgG3 by gingipain K from Porphyromonas gingivalis may compromise host defense in progressive periodontitis

Degradation of immunoglobulins is an effective strategy of bacteria to evade the immune system. We have tested whether human IgG is a substrate for gingipain K of Porphyromonas gingivalis and found that the enzyme can hydrolyze subclass 1 and 3 of human IgG. The heavy chain of IgG(1) was cleaved at a single site within the hinge region, generating Fab and Fc fragments. IgG(3) was also cleaved within the heavy chain, but at several sites around the CH2 region. Investigation of the enzyme kinetics of IgG proteolysis by gingipain K, using FPLC- and isothermal titration calorimetry-based assays followed by Hill plots, revealed non-Michaelis-Menten kinetics involving a mechanism of positive cooperativity. In ex vivo studies, it was shown that gingipain K retained its IgG hydrolyzing activity in human plasma despite the high content of natural protease inhibitors; that IgG(1) cleavage products were detected in gingival crevicular fluid samples from patients with severe periodontitis; and that gingipain K treatment of serum samples from patients with high antibody titers against P. gingivalis significantly hindered opsonin-dependent phagocytosis of clinical isolates of P. gingivalis by neutrophils. Altogether, these findings underline a biological function of gingipain K as an IgG protease of pathophysiological importance