Biomedical subjects

C Collyer

Publications and source records attributed to C Collyer.

6 recordsLinked to original sources

PubMed2003

Enhancement of Th2 pathways and direct activation of B cells by the gingipains of Porphyromonas gingivalis.

Porphyromonas gingivalis cysteine proteinases (gingipains) have been associated with virulence in destructive periodontitis, a disease process that has been linked with Th2 pathways. Critical in maintaining Th2 activity is the response of B lymphocytes to environmental interleukin (IL)-4, a cytokine that also counteracts Th1-cell differentiation. Here we demonstrate that while the gingipains effectively degrade interleukin (IL)-4 under serum-free conditions, limited hydrolysis was observed in the presence of serum even after prolonged incubation. Gingipains up-regulated CD69 expression directly in purified peripheral blood B cell preparations. Further, the induction of IL-4 receptor expression on B cells by gingipains correlates with B cell activation, which is also manifested by a mitogenic response. These results suggest that the gingipains of P. gingivalis act during the early stage of B-cell growth as a competence signal, whereby sensitized B cells might become more responsive to further challenge in the disease-susceptible individual.

Adhesins, Bacterial↗

PubMed2001

Hydrolysis of interleukin-12 by Porphyromonas gingivalis major cysteine proteinases may affect local gamma interferon accumulation and the Th1 or Th2 T-cell phenotype in periodontitis.

Porphyromonas gingivalis cysteine proteinases (gingipains) have been associated with virulence in destructive periodontitis, a disease process variously considered to represent an unregulated stimulation of either T helper type 1 (Th1)- or Th2-type cells. Critical in maintaining Th1 activity is the response of T lymphocytes to environmental interleukin 12 (IL-12) in the form of up-regulation of gamma interferon (IFN-gamma) production. Here we demonstrate that in the presence or absence of serum, gingipains were able to hydrolyze IL-12 and reduce the IL-12-induced IFN-gamma production from CD4+ T cells. However, the induction of IL-12 receptors on T cells by gingipains did not correlate with the enhancement of IFN-gamma production. The gingipains cleaved IL-12 within the COOH-terminal region of the p40 and p35 subunit chains, which leads to IL-12 inactivity, whereas IL-2 in these assays was not affected. Inactivation of IL-12 by the gingipains could disrupt the cytokine balance or favor Th2 activities in the progression of periodontitis.

Adhesins, Bacterial↗

PubMed1999

Porphyrin-mediated binding to hemoglobin by the HA2 domain of cysteine proteinases (gingipains) and hemagglutinins from the periodontal pathogen Porphyromonas gingivalis.

Heme binding and uptake are considered fundamental to the growth and virulence of the gram-negative periodontal pathogen Porphyromonas gingivalis. We therefore examined the potential role of the dominant P. gingivalis cysteine proteinases (gingipains) in the acquisition of heme from the environment. A recombinant hemoglobin-binding domain that is conserved between two predominant gingipains (domain HA2) demonstrated tight binding to hemin (Kd = 16 nM), and binding was inhibited by iron-free protoporphyrin IX (Ki = 2.5 microM). Hemoglobin binding to the gingipains and the recombinant HA2 (rHA2) domain (Kd = 2.1 nM) was also inhibited by protoporphyrin IX (Ki = 10 microM), demonstrating an essential interaction between the HA2 domain and the heme moiety in hemoglobin binding. Binding of rHA2 with either hemin, protoporphyrin IX, or hematoporphyrin was abolished by establishing covalent linkage of the protoporphyrin propionic acid side chains to fixed amines, demonstrating specific and directed binding of rHA2 to these protoporphyrins. A monoclonal antibody which recognizes a peptide epitope within the HA2 domain was employed to demonstrate that HA2-associated hemoglobin-binding activity was expressed and released by P. gingivalis cells in a batch culture, in parallel with proteinase activity. Cysteine proteinases from P. gingivalis appear to be multidomain proteins with functions for hemagglutination, erythrocyte lysis, proteolysis, and heme binding, as demonstrated here. Detailed understanding of the biochemical pathways for heme acquisition in P. gingivalis may allow precise targeting of this critical metabolic aspect for periodontal disease prevention.

Adhesins, Bacterial↗

PubMed1995

Inhibition of bovine beta-trypsin, human alpha-thrombin and porcine pancreatic beta-kallikrein-B by 4',6-diamidino-2-phenylindole, 6-amidinoindole and benzamidine: a comparative thermodynamic and X-ray structural study.

The inhibitory effect of 4',6-diamidino-2-phenylindole (DAPI) and 6-amidinoindole on the catalytic properties of bovine beta-trypsin (trypsin), human alpha-thrombin (thrombin) and porcine pancreatic beta-kallikrein-B (kallikrein) was investigated (between pH 3.0 and 7.0, I = 0.1 M; T = 30.0 +/- 0.5 degrees C), and analyzed in parallel with that of benzamidine, commonly taken as a molecular inhibitor model of serine proteinases. Next, the X-ray crystal structure of the trypsin:DAPI complex was solved at 1.9 A resolution (R = 0.161). Over the whole pH range explored, values of the association inhibition constant (Ki) for DAPI and 6-amidinoindole binding to trypsin, thrombin and kallikrein are higher than those found for benzamidine association, suggesting a binding mode of DAPI to the enzyme primary specificity pocket-based on the indole moiety of the inhibitor. On lowering the pH from 5.5 to 3.0, the decrease in affinity for DAPI, 6-amidinoindole and benzamidine binding to trypsin, thrombin and kallikrein reflects the acidic pK shift of the Asp189 invariant residue, present at the bottom of the primary specificity subsite of the serine proteinases considered, from 4.5, in the free enzyme, to 3.7, in the proteinase:inhibitor complexes. Inspection of the refined crystal structure of the trypsin:DAPI complex, however, does not allow a unique interpretation of the inhibitor binding mode. The present data were analysed in parallel with those reported for related serine (pro)enzyme/inhibitor systems.

Animals↗

PubMed1993

Crystallisation and preliminary crystallographic analysis of recombinant Xenopus laevis Cu,Zn superoxide dismutase b.

The recombinant Cu,Zn superoxide dismutase from the South African frog Xenopus laevis, expressed in E. coli, has been crystallized in a form suitable for high resolution crystallographic investigations. The crystals grow from polyethylene glycol solutions, at pH 6.0, 28 degrees C, and belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell edges a = 73.33, b = 68.86, c = 59.73 A, one protein dimer (32,000 M(r)) per asymmetric unit. Diffraction data have been collected to 3.0 A resolution, and a molecular replacement solution found for Xenopus laevis superoxide dismutase using the bovine enzyme as search model. The crystallographic R-factor corresponding to this solution is 0.412, in the 15.0-3.0 A resolution range.

Animals↗

PubMed1993

Arthrobacter D-xylose isomerase: protein-engineered subunit interfaces.

Mutants of Arthrobacter D-xylose isomerase were constructed in which one or two disulphide bridges or additional salt bridges were introduced at the A-A* subunit interfaces. These showed no change in enzyme activity or stability compared with the wild-type enzyme. However, a Tyr253 mutant in which a disulphide bridge was introduced at the A-B* subunit interface showed reduced thermostability that was identical in both oxidized and reduced forms, and also reduced stability in urea. X-ray-crystallographic analysis of the Mn(2+)-xylitol form of oxidized Y253C (the Tyr253-->Cys mutant) showed a changed conformation of Glu185 and also alternative conformations for Asp254, which is a ligand to the Site-[2] metal ion. With fructose, Mg(2+)-Y253C has a similar Km to that of the wild-type, and its Vmax. is also similar below pH 6.4, but declined thereafter. In the presence of Co2+, Y253C has lower activity than wild-type at all pH values, but its activity also declines at alkaline pH. These results suggest that electrostatic repulsion from the new position of Glu185 causes Asp254 to move when His219 is unprotonated, thereby preventing M2+ binding at Site [2]. These results also suggest that subunit dissociation does not lie on the pathway of thermal inactivation of D-xylose isomerase, but that movements of active-site groups are a trigger for conformational changes that initiate the unfolding process.

Aldose-Ketose Isomerases↗