Adhesins from oral bacteria perform an important function in colonizing target tissues within the dentogingival cavity. In Porphyromonas gingivalis certain of these adhesion proteins exist as a complex with either of two major proteinases referred to as gingipain R (arginine-specific gingipain) and gingipain K (lysine-specific gingipain) (R. N. Pike, W. T. McGraw, J. Potempa, and J. Travis, J. Biol. Chem. 269:406-411, 1994). With specific proteinase inhibitors, it was shown that hemagglutination by either proteinase-adhesin complex could occur independently of proteinase activity. Significantly, low concentrations of fibrinogen, fibronectin, and laminin inhibited hemagglutination, indicating that adherence to these proteins and not the hemagglutination activity was a primary property of the adhesin activity component of complexes. Binding studies with gingipain K and gingipain R suggest that interaction with fibrinogen is a major function of the adhesin domain, with dissociation constants for binding to fibrinogen being 4 and 8.5 nM, respectively. Specific association with fibronectin and laminin was also found. All bound proteins were degraded by the functional proteinase domain, with gingipain R being more active on laminin and fibronectin and gingipain K being more effective in the digestion of fibrinogen. Cumulatively, these data suggest that gingipain R and gingipain K, acting as proteinase-adhesin complexes, progressively attach to, degrade, and detach from target proteins. Since such complexes appear to be present on the surfaces of both vesicles and membranes of P. gingivalis, they may play an important role in the attachment of this bacterium to host cell surfaces.

MENT (Myeloid and Erythroid NuclearTermination stage-specific protein) is a developmentally regulated chromosomal serpin that condenses chromatin in terminally differentiated avian blood cells. We show that MENT is an effective inhibitor of the papain-like cysteine proteinases cathepsins L and V. In addition, ectopic expression of MENT in mammalian cells is apparently sufficient to inhibit a nuclear papain-like cysteine proteinase and prevent degradation of the retinoblastoma protein, a major regulator of cell proliferation. MENT also accumulates in the nucleus, causes a strong block in proliferation, and promotes condensation of chromatin. Variants of MENT with mutations or deletions within the M-loop, which contains a nuclear localization signal and an AT-hook motif, reveal that this region mediates nuclear transport and morphological changes associated with chromatin condensation. Noninhibitory mutants of MENT were constructed to determine whether its inhibitory activity has a role in blocking proliferation. These mutations changed the mode of association with chromatin and relieved the block in proliferation, without preventing transport to the nucleus. We conclude that the repressive effect of MENT on chromatin is mediated by its direct interaction with a nuclear protein that has a papain-like cysteine proteinase active site.MENT (Myeloid and Erythroid Nuclear Termination stagespecific protein), a developmentally regulated nuclear protein, is present in three main avian blood cell types (erythrocytes, lymphocytes, and granulocytes) where it is the predominant non-histone protein associated with compact heterochromatin (1). In vitro, MENT brings about a dramatic remodeling and condensation of chromatin higher order structure by forming protein "bridges" connecting separate nucleosomes in nucleosome arrays (for review see Ref.2). MENT has no homology with other known chromatin proteins but belongs to the intracellular branch of the serpin superfamily (3). Serpins were originally characterized as serine proteinase inhibitors; however, more recently certain members have been shown to be capable of inhibiting other proteinase classes such as caspases (the viral serpin crmA (4)) and papain-like cysteine proteinases (SCCA-1 (5)). The inhibitory members of the serpin family are notable for their ability to undergo a large scale conformational transition (for review see Ref. 6) that is critical for inhibition of target proteinases (7, 8) and for self-association or polymerization (9 -11). Multiple sequence alignments and comparison with known serpin structures reveal that MENT contains a large insertion, the "M-loop," between the C-and D-helices. This loop contains two critical functional motifs as follows: a classical nuclear localization signal (NLS) 1 that is required for nuclear import, and an AT-hook motif that is involved in chromatin and DNA binding (3). Like other serpins, MENT possesses a reactive center loop (RCL) 2 through which interaction with a cognate proteinase occurs. The presence of an inhibitory...

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Trypanosoma brucei contains a soluble serine oligopeptidase (OP-Tb) that is released into the host bloodstream during infection, where it has been postulated to participate in the pathogenesis of African trypanosomiasis. Here, we report the identification of a single copy gene encoding the T. brucei oligopeptidase and a homologue from the related trypanosomatid pathogen Leishmania major. The enzymes encoded by these genes belong to an emerging subgroup of the prolyl oligopeptidase family of serine hydrolases, referred to as oligopeptidase B. The trypanosomatid oligopeptidases share 70% amino acid sequence identity with oligopeptidase B from the intracellular pathogen Trypanosoma cruzi, which has a demonstrated role in mammalian host cell signaling and invasion. OP-Tb exhibited no activity toward the prolyl oligopeptidase substrate H-Gly-Pro-7-amido-4-methylcoumarin. Instead, it had activity toward substrates of trypsin-like enzymes, particularly those that have basic amino acids in both P 1 and P 2 (e.g. benzyloxycarbonyl-Arg-Arg-7-amido-4-methylcoumarin k cat /K m ‫؍‬ 529 s ؊1 M ؊1). The activity of OP-Tb was enhanced by reducing agents and by polyamines, suggesting that these agents may act as in vivo regulators of OP-Tb activity. This study provides the basis of the characterization of a novel subgroup of serine oligopeptidases from kinetoplastid protozoa with potential roles in pathogenesis.

African trypanosomes contain proteases that may be released into the bloodstream of their infected hosts. This paper describes a novel, combined isolation of a cysteine proteinase (called trypanopain-Tb) and a serine oligopeptidase (which we call oligopeptidase-Tb) from Trypanosoma brucei brucei, as well as a comparison of the activities of these two enzymes against several host regulatory molecules.The enzymes differed in various respects. Firstly, purified trypanopain-Tb was shown to readily cleave proteins such as gelatin maximally at acidic pH. In contrast, oligopeptidase-Tb, which is optimally active at alkaline pH, did not hydrolyse proteins larger than 4 kDa. However, it readily hydrolysed various polypeptides, including neurotensin and atrial natriuretic factor.The interaction of the two enzymes with mammalian protease inhibitors also differed. Cystatins and a,-macroglobulin effectively inhibited trypanopain-Tb, with the K, values for cystatin C and low-molecular-mass kininogen (-1O-~" M) predicting that trypanopain-Tb is likely to be effectively controlled by these inhibitors if released into the host bloodstream. In contrast, oligopeptidase-Tb was not inhibited by serpins or a,-macroglobulin, suggesting that it may remain active if released into the host bloodstream. In support of these in v i t m results, the blood of trypanosome-infected rats displayed no trypanopain-Tblike activity, but exhibited high oligopeptidase-Tb-like activity. Thus, while trypanopain-Tb seems likely to be confined to an intracellular role within the parasite, oligopeptidase-Tb has the potential to remain active in the host bloodstream and so contribute directly to pathogenesis.