The initiation and progression of adult-onset periodontitis has been associated with infection of the gingival sulcus byPorphyromonas gingivalis. This organism utilizes a multitude of virulence factors to evade host defenses as it establishes itself as one of the predominant pathogens in periodontal pockets. A feature common to many other oral pathogens is the production of ammonia due to its protective effect during acidic cleansing cycles in the mouth. Additionally, ammonia production byP. gingivalis has been proposed as a virulence factor due to its negative effects on neutrophil function. In this study, we describe the first purification of a peptidylarginine deiminase (PAD) from a prokaryote. PAD exhibits biochemical characteristics and properties that suggest that it may be a virulence agent. PAD deiminates the guanidino group of carboxyl-terminal arginine residues on a variety of peptides, including the vasoregulatory peptide-hormone bradykinin, to yield ammonia and a citrulline residue. The soluble protein has an apparent mass of 46 kDa, while the DNA sequence predicts a full-length protein of 61.7 kDa. PAD is optimally active at 55°C, stable at low pH, and shows the greatest activity above pH 9.0. Interestingly, in the presence of stabilizing factors, PAD is resistant to limited proteolysis and retains significant activity after short-term boiling. We propose that PAD, acting in concert with arginine-specific proteinases from P. gingivalis, promotes the growth of the pathogen in the periodontal pocket, initially by enhancing its survivability and then by assisting the organism in its circumvention of host humoral defenses.

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.

Antithrombin, the principal plasma inhibitor of coagulation proteinases, circulates in a form with low inhibitory activity due to partial insertion of its reactive site loop into the A-␤-sheet of the molecule. Recent crystallographic structures reveal the structural changes that occur when antithrombin is activated by the heparin pentasaccharide, with the exception of the final changes, which take place at the reactive center itself. Here we show that the side chain of the P 1 Arg of ␣-antithrombin is only accessible to modification by the enzyme peptidylarginine deiminase on addition of the heparin pentasaccharide, thereby inactivating the inhibitor, whereas the natural P 1 His variant, antithrombin Glasgow, is unaffected, indicating that only the P 1 Arg becomes accessible. Furthermore, the deimination of P 1 Arg converts antithrombin to a form with 4-fold higher affinity for the heparin pentasaccharide, similar to the affinity found for the P 1 His variant, due to a lowered dissociation rate constant for the antithrombinpentasaccharide complex. The results support the proposal that antithrombin circulates in a constrained conformation, which when released, in this study by perturbation of the bonding of P 1 Arg to the body of the molecule, allows the reactive site loop to take up the active inhibitory conformation with exposure of the P 1 Arg.The plasma serpin (1), antithrombin, is the major inhibitor of the serine proteinases of the coagulation network, especially thrombin and factor Xa (2). This inhibitory activity is stimulated by the complexing of antithrombin with the sulfated polysaccharide, heparin. In particular, the activity against factor Xa is mediated by a core pentasaccharide component of heparin. Antithrombin has an initial low affinity for heparin, but this immediately changes to a high affinity on initial binding, the change occurring concomitantly with the activation of inhibitory function (3).The interaction of the heparin pentasaccharide with antithrombin and the associated mechanism of conformational activation of inhibition has recently been revealed in the crystal structure of a dimer of antithrombin complexed with the pentasaccharide (4). Linkage between the two antithrombin molecules in the dimer directly involves the reactive site loop of the inhibitory component, with a consequent constraint in the movement at its reactive site. Hence, while showing the commencement of the movement of the reactive site loop that results in activation, the crystal structure does not show the change that occurs at the reactive center itself.There is, however, a good model of the likely unconstrained active conformation of the reactive site loop of antithrombin. This is provided by the structure of the closely related serpin, ␣ 1 -antitrypsin (5), in which the loop is fixed in the optimal canonical inhibitory conformation present in all other families of serine proteinase inhibitors. The transition of antithrombin to this conformation would require a shift of the side chain of the P 1 arginine from an i...

We have investigated the functional relationship between metalloendopeptidase EC 3.4.24.15 (MP24.15) and the amyloid precursor protein involved in Alzheimer's disease (AD) and discovered that the enzyme promotes A␤ degradation. We show here that conditioned medium (CM) of MP24.15 antisense-transfected SKNMC neuroblastoma has significantly higher levels of A␤. Furthermore, synthetic-A␤ degradation was increased or decreased following incubation with CM of sense or antisense-transfected cells, respectively. Soluble A␤1-42 was degraded more slowly than soluble A␤1-40, while aggregated A␤1-42 showed almost no degradation. Alzheimer's disease (AD)1 is a progressive neurodegenerative disorder and the most common form of dementia in the elderly (1). Evidence indicates that accumulation of amyloid-␤ (A␤) deposits in senile plaques and in cerebrovasculature is associated with the pathophysiology of AD (2). The A␤ peptide is composed of 40 -42 amino acids (3). The events leading to its formation from the transmembrane amyloid precursor protein (APP) involve proteolytic cleavage by enzymes that have been termed: 1) ␤-secretase, which cleaves at the amino terminus of A␤, and 2) ␥-secretase, which cleaves at the carboxyl terminus. In the senile plaques A␤ is associated with a number of proteins, including ␣ 1 -antichymotrypsin (ACT) (4), which is a serine proteinase inhibitor of the serpin family as well as an acute-phase protein (5). Thus, we hypothesized 10 years ago that ACT may play a role in APP processing (4, 6).Soluble A␤ peptide can be detected in the conditioned media of a variety of cultured mammalian cells in vitro (7-9), as well as in serum and cerebrospinal fluid in vivo (10). The majority of secreted A␤ is 40 amino acids in length (A␤1-40), but approximately 10% of all A␤ is 42 amino acids in length (A␤1-42) (11). Little is known about how secreted A␤ is degraded and cleared from the extracellular milieu. The excessive cerebral accumulation of A␤ that occurs in AD could be explained in part by a decreased ability of the brain to degrade and clear A␤. If neural cells can be shown to release specific A␤-degrading proteinases, changes in the activity of such proteinases and/or their upregulation could represent a therapeutic approach to AD.We have explored the role of the metalloendopeptidase MP24.15 in the degradation of A␤. MP24.15 is a thiol-dependent enzyme that was purified to homogeneity from AD brain as a candidate ␤-secretase (12). McDermot et al. (13) also identified a partially purified MP24.15 as a potential ␤-secretase using synthetic peptide substrates. In vitro, MP24.15 has been shown to be involved in the inactivation of a number of neuropeptides, including somatostatin, bradykinin, substance P, and neurotensin (14 -16). The cDNA coding for MP24.15 was subsequently cloned from a human brain library (17,18). In an attempt to further examine the ␤-secretase properties of MP24.15 under physiologic conditions, we transfected human neuroblastoma cells with MP24.15 cDNA in the sense and antisense directi...

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