Agnieszka Banbula

Crystal structure of gingipain R: an Arg-specific bacterial cysteine proteinase with a caspase-like fold

Gingipains are cysteine proteinases acting as key virulence factors of the bacterium Porphyromonas gingivalis, the major pathogen in periodontal disease. The 1.5 and 2.0 Å crystal structures of free and D‐Phe‐Phe‐Arg‐chloromethylketone‐inhibited gingipain R reveal a 435‐residue, single‐polypeptide chain organized into a catalytic and an immunoglobulin‐like domain. The catalytic domain is subdivided into two subdomains comprising four‐ and six‐stranded β‐sheets sandwiched by α‐helices. Each subdomain bears topological similarities to the p20‐p10 heterodimer of caspase‐1. The second subdomain harbours the Cys‐His catalytic diad and a nearby Glu arranged around the S1 specificity pocket, which carries an Asp residue to enforce preference for Arg‐P1 residues. This gingipain R structure is an excellent template for the rational design of drugs with a potential to cure and prevent periodontitis. Here we show the binding mode of an arginine‐containing inhibitor in the active‐site, thus identifying major interaction sites defining a suitable pharmacophor.

Prolyl Tripeptidyl Peptidase from Porphyromonas gingivalis A NOVEL ENZYME WITH POSSIBLE PATHOLOGICAL IMPLICATIONS FOR THE DEVELOPMENT OF PERIODONTITIS

Porphyromonas gingivalis possesses a complex proteolytic system, which is essential for both its growth and evasion of host defense mechanisms. In this report we characterized, both at a protein and genomic level, a novel peptidase of this system with prolyl tripeptidyl peptidase activity. The enzyme was purified to homogeneity, and its enzymatic activity and biochemical properties were investigated. The amino acid sequence at the amino terminus and of internal peptide fragments enabled identification of the gene encoding this enzyme, which we refer to as PtpA for prolyl tripeptidyl peptidase A. The gene encodes an 82-kDa protein, which contains a GWSYGG motif, characteristic for members of the S9 prolyl oligopeptidase family of serine proteases. However, it does not share any structural similarity to other tripeptidyl peptidases, which belong to the subtilisin family. The production of prolyl tripeptidyl peptidase may contribute to the pathogenesis of periodontal tissue destruction through the mutual interaction of this enzyme, host and bacterial collagenases, and dipeptidyl peptidases in the degradation of collagen during the course of infection.

Emerging Family of Proline-Specific Peptidases of Porphyromonas gingivalis: Purification and Characterization of Serine Dipeptidyl Peptidase, a Structural and Functional Homologue of Mammalian Prolyl Dipeptidyl Peptidase IV

ABSTRACT Porphyromonas gingivalis is an asaccharolytic and anaerobic bacterium that possesses a complex proteolytic system which is essential for its growth and evasion of host defense mechanisms. In this report, we show the purification and characterization of prolyl dipeptidyl peptidase IV (DPPIV) produced by this organism. The enzyme was purified to homogeneity, and its enzymatic activity and biochemical properties were investigated. P. gingivalis DPPIV, like its human counterpart, is able to cleave the N terminus of synthetic oligopeptides with sequences analogous to those of interleukins 1β and 2. Additionally, this protease hydrolyzes biologically active peptides including substance P, fibrin inhibitory peptide, and β-casomorphin. Southern blot analysis of genomic DNA isolated from several P. gingivalis strains reveal that a single copy of the DPPIV gene was present in all strains tested.

Isolation and Properties of Stachyrase A, a Chymotrypsin-Like Serine Proteinase from Stachybotrys chartarum

ABSTRACT A strain of the common mold Stachybotrys chartarum has been isolated from the lung of a child with pulmonary hemorrhage. We report the purification of stachyrase A, a new serine chymotrypsin-like proteinase from S. chartarum. This enzyme cleaves major protease inhibitors, several biologically active peptides, and collagen, all of which are found in the lung.

Isolation and characterization of a highly specific serine endopeptidase from an oral strain of Staphylococcus epidermidis.

Abstract Infection by Staphylococcus epidermidis, an opportunistic pathogen, has become a major problem due to the increased use of implanted medical devices and the growing number of patients who are therapeutically or infectiously immunosuppressed. These infections appear to proceed via modulation of the coagulation and complement systems. In this communication we describe the purification and characterization of a novel extracellular proteinase from an oral strain of S. epidermidis that can degrade fibrinogen, complement protein C5, and several other proteins. This proteinase has a strong preference for cleavage after glutamic acid residues, but not after aspartic acid. The S. epidermidis enzyme may be a multifunctional protein which not only provides this organism with both the ability to evade the complement defense system and to dysregulate the coagulation cascade, but also supplies nutrients for its growth through the degradation of Glurich proteins.

The C‐terminal domains of the gingipain K polyprotein are necessary for assembly of the active enzyme and expression of associated activities

The Porphyromonas gingivalis lysine‐specific cysteine protease (gingipain K, Kgp) is expressed as a large precursor protein consisting of a leader sequence, a pro‐fragment, a catalytic domain with a C‐terminal IgG‐like subdomain (IgSF) and a large haemagglutinin/adhesion (HA) domain. In order to directly study the role of these non‐catalytic domains in pro‐Kgp processing and maturation in P. gingivalis, the wild‐type form of the gene was replaced with deletion variants encoding C‐terminally truncated proteins, including KgpΔHA3/4 (Δ1292–1732 aa), KgpΔHA2‐4 (Δ1157–1732 aa), KgpΔHA1‐4 (Δ738–1732 aa), KgpΔC‐term/HA (Δ681–1732 aa) and KgpΔIg/C‐term/HA (602–1732 aa). Northern blot and reverse transcription polymerase chain reaction (RT‐PCR) analysis revealed that all truncated variants of the kgp gene were transcribed in P. gingivalis. Despite high levels of kgpΔC‐term/HA and kgpΔIg/C‐term/HA transcripts, no Kgp‐specific antigen was detected in cultures of these mutants as determined by Western blot analysis with monoclonal antibodies specific for the Kgp catalytic domain. Furthermore, only barely measurable amounts of Kgp‐specific activity were detected in these two mutants. The remaining mutants expressed significant Kgp activity, however, at lower levels when compared with the parental strain. The decreased activity most probably resulted from altered folding and/or hindered secretion of the protein. The kgp gene truncation was also demonstrated to alter the distribution of the gingipain protein between membrane‐associated and ‐secreted forms. While both gingipain K activity and the protein were cell membrane‐associated in the parental strain, the mutants released significant amounts of both protein and activity into the media. Taken together, these results suggest that the C‐terminal HA domains of Kgp are not only essential for full expression of gingipain activity, but also for proper processing of the multiprotein complex assembly on the P. gingivalis outer membrane. Moreover, our results indicate that the immunoglobulin‐like subdomain is indispensable for proper folding and expression of the gingipains.

Novel extracellular x-prolyl dipeptidyl-peptidase (DPP) from Streptococcus gordonii FSS2: an emerging subfamily of viridans Streptococcal x-prolyl DPPs.

ABSTRACT Streptococcus gordonii is generally considered a benign inhabitant of the oral microflora, and yet it is a primary etiological agent in the development of subacute bacterial endocarditis (SBE), an inflammatory state that propagates thrombus formation and tissue damage on the surface of heart valves. Strain FSS2 produced several extracellular aminopeptidase and fibrinogen-degrading activities during growth in culture. In this report we describe the purification, characterization, and cloning of a serine class dipeptidyl-aminopeptidase, an x-prolyl dipeptidyl-peptidase (Sg-xPDPP, for S. gordonii x-prolyl dipeptidyl-peptidase), produced in a pH-controlled batch culture. Purification of this enzyme by anion exchange, gel filtration, and hydrophobic interaction chromatography yielded a protein monomer of approximately 85 kDa, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) under denaturing conditions. However, under native conditions, the protein appeared to be a homodimer on the basis of gel filtration and PAGE. Kinetic studies indicated that purified enzyme had a unique and stringent x-prolyl specificity that is comparable to both the dipeptidyl-peptidase IV/CD26 and lactococcal x-prolyl dipeptidyl-peptidase families. Nested PCR cloning from an S. gordonii library enabled the isolation and sequence analysis of the full-length gene. A 759-amino-acid polypeptide with a theoretical molecular mass of 87,115 Da and a calculated pI of 5.6 was encoded by this open reading frame. Significant homology was found with the PepX gene family fromLactobacillus and Lactococcus spp. and putative x-prolyl dipeptidyl-peptidases from other streptococcal species. Sg-xPDPP may serve as a critical factor for the sustained bacterial growth in vivo and furthermore may aid in the proteolysis of host tissue that is commonly observed during SBE pathology.

The Role of Bacterial and Host Proteinases in Periodontal Disease

It is abundantly obvious that the uncontrolled degradation and/or activation of host defense pathways is the major pathway by which the periodontal pathogen P. gingivalis promotes its growth and proliferation. By being able to shed host receptors, degrade cytokines, and activate coagulation, complement, and kallikrein/kinin pathways it is clear that this organism has found a mechanism(s) to evade host defense and at the same time develop a system for cannibalizing host proteins for its own nutritional usage (Fig 2). Thus, it seems only logical that the development of inhibitors against these bacterial proteinases would be a useful method for negating their activities and making such pathogens more susceptible to attack by host phagocyte cells. In this respect, the structure of the truncated form of RGP has just been elucidated. Thus, it should only be a question of time before inhibitors to this enzyme will be developed and, hopefully, be used to reduce the pathologies associated with the development of periodontitis and/or eliminate the disease altogether.