Disruption of gingipain oligomerization into non-covalent cell-surface attached complexes

Abstract RgpA and Kgp gingipains are non-covalent complexes of endoprotease catalytic and hemagglutinin-adhesin domains on the surface of Porphyromonas gingivalis. A motif conserved in each domain has been suggested to function as an oligomerization motif. We tested this hypothesis by mutating motif residues to hexahistidine or insertion of hexahistidine tag to disrupt the motif within the Kgp catalytic domain. All modifications led to the secretion of entire Kgp activity into the growth media, predominantly in a form without functional His-tag. This confirmed the role of the conserved motif in correct posttranslational proteolytic processing and assembly of the multidomain complexes.

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Role of vimA in cell surface biogenesis in Porphyromonas gingivalis

Microbiology ◽

10.1099/mic.0.038331-0 ◽

2010 ◽

Vol 156 (7) ◽

pp. 2180-2193 ◽

Cited By ~ 13

Author(s):

Devon O. Osbourne ◽

Wilson Aruni ◽

Francis Roy ◽

Christopher Perry ◽

Lawrence Sandberg ◽

...

Keyword(s):

Cell Surface ◽

Outer Membrane ◽

Porphyromonas Gingivalis ◽

Type Strain ◽

Wild Type Strain ◽

Phenotypic Expression ◽

Surface Proteins ◽

Wild Type ◽

In The Wild

The Porphyromonas gingivalis vimA gene has been previously shown to play a significant role in the biogenesis of gingipains. Further, in P. gingivalis FLL92, a vimA-defective mutant, there was increased auto-aggregation, suggesting alteration in membrane surface proteins. In order to determine the role of the VimA protein in cell surface biogenesis, the surface morphology of P. gingivalis FLL92 was further characterized. Transmission electron microscopy demonstrated abundant fimbrial appendages and a less well defined and irregular capsule in FLL92 compared with the wild-type. In addition, atomic force microscopy showed that the wild-type had a smoother surface compared with FLL92. Western blot analysis using anti-FimA antibodies showed a 41 kDa immunoreactive protein band in P. gingivalis FLL92 which was missing in the wild-type P. gingivalis W83 strain. There was increased sensitivity to globomycin and vancomycin in FLL92 compared with the wild-type. Outer membrane fractions from FLL92 had a modified lectin-binding profile. Furthermore, in contrast with the wild-type strain, nine proteins were missing from the outer membrane fraction of FLL92, while 20 proteins present in that fraction from FLL92 were missing in the wild-type strain. Taken together, these results suggest that the VimA protein affects capsular synthesis and fimbrial phenotypic expression, and plays a role in the glycosylation and anchorage of several surface proteins.

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Cell-Surface-Anchoring Role of N-Terminal Surface Layer Homology Domains of Clostridium cellulovorans EngE

Journal of Bacteriology ◽

10.1128/jb.184.4.884-888.2002 ◽

2002 ◽

Vol 184 (4) ◽

pp. 884-888 ◽

Cited By ~ 41

Author(s):

Akihiko Kosugi ◽

Koichiro Murashima ◽

Yutaka Tamaru ◽

Roy H. Doi

Keyword(s):

Cell Wall ◽

Surface Layer ◽

Cell Surface ◽

Catalytic Domain ◽

Glycosyl Hydrolase ◽

Sodium Dodecyl ◽

Scaffolding Protein ◽

Clostridium Cellulovorans ◽

Surface Anchoring

ABSTRACT engE, coding for endoglucanase E, one of the three major subunits of the Clostridium cellulovorans cellulosome, has been cloned and sequenced (Y. Tamaru and R. H. Doi, J. Bacteriol. 181:3270-3276, 1999). The N-terminal-half region of EngE possesses three repeated surface layer homology (SLH) domains, which are homologous to those of some bacterial S-layer proteins. Also, the C-terminal-half region consists of a catalytic domain of glycosyl hydrolase family 5 and a duplicated sequence (dockerin) for binding EngE to scaffolding protein CbpA. Our hypothesis is that the SLH domains serve in the role of anchoring to the cell surface. This model was investigated by using recombinant EngEs (rEngE) with and without SLH domains that were synthesized in Escherichia coli and cell wall preparations from C. cellulovorans. When rEngE and SLH polypeptides of EngE were incubated with cell wall fragments prepared by sodium dodecyl sulfate treatment, these proteins bound strongly to the cell wall. However, rEngEs without SLH domains lost their ability to bind to cell walls. When rEngE was incubated with mini-CbpA, consisting of two cohesin domains, and cell wall fragments, the mini-CbpA was able to bind to the cell wall with rEngE. However, the binding of mini-CbpA was dramatically inhibited by addition of a chelating reagent, such as EDTA, which prevents cohesin-dockerin interactions. These results suggest not only that the SLH domains of EngE can bind to the cell surface but also that EngE plays an anchoring role for cellulosomes through the interaction of its dockerin domain with a CbpA cohesin.

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Proteolytic processing of the serine protease matriptase-2: identification of the cleavage sites required for its autocatalytic release from the cell surface

Biochemical Journal ◽

10.1042/bj20091565 ◽

2010 ◽

Vol 430 (1) ◽

pp. 87-95 ◽

Cited By ~ 46

Author(s):

Marit Stirnberg ◽

Eva Maurer ◽

Angelika Horstmeyer ◽

Sonja Kolp ◽

Stefan Frank ◽

...

Keyword(s):

Cell Surface ◽

Disulfide Bonds ◽

Serine Proteases ◽

Catalytic Domain ◽

Transmembrane Domain ◽

Mutational Analysis ◽

Bond Cleavage ◽

Proteolytic Processing ◽

Cleavage Sites ◽

Single Chain

Matriptase-2 is a member of the TTSPs (type II transmembrane serine proteases), an emerging class of cell surface proteases involved in tissue homoeostasis and several human disorders. Matriptase-2 exhibits a domain organization similar to other TTSPs, with a cytoplasmic N-terminus, a transmembrane domain and an extracellular C-terminus containing the non-catalytic stem region and the protease domain. To gain further insight into the biochemical functions of matriptase-2, we characterized the subcellular localization of the monomeric and multimeric form and identified cell surface shedding as a defining point in its proteolytic processing. Using HEK (human embryonic kidney)-293 cells, stably transfected with cDNA encoding human matriptase-2, we demonstrate a cell membrane localization for the inactive single-chain zymogen. Membrane-associated matriptase-2 is highly N-glycosylated and occurs in monomeric, as well as multimeric, forms covalently linked by disulfide bonds. Furthermore, matriptase-2 undergoes shedding into the conditioned medium as an activated two-chain form containing the catalytic domain, which is cleaved at the canonical activation motif, but is linked to a released portion of the stem region via a conserved disulfide bond. Cleavage sites were identified by MS, sequencing and mutational analysis. Interestingly, cell surface shedding and activation of a matriptase-2 variant bearing a mutation at the active-site serine residue is dependent on the catalytic activity of co-expressed or co-incubated wild-type matriptase-2, indicating a transactivation and trans-shedding mechanism.

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Porphyromonas gingivalis proteinases in periodontitis, a review.

Acta Biochimica Polonica ◽

10.18388/abp.1996_4477 ◽

1996 ◽

Vol 43 (3) ◽

pp. 455-465 ◽

Cited By ~ 35

Author(s):

J Potempa ◽

J Travis

Keyword(s):

Porphyromonas Gingivalis ◽

Catalytic Domain ◽

Gingival Crevicular Fluid ◽

Proteolytic Enzymes ◽

Periodontal Diseases ◽

Proteolytic Processing ◽

Tissue Destruction ◽

Single Chain ◽

Neutrophil Accumulation ◽

Crevicular Fluid

Porphyromonas gingivalis has been closely associated with the initiation and progression of some forms of periodontal diseases and its proteolytic enzymes have been implicated in invasion, tissue destruction and evasion of host antibacterial defenses. Recently, the primary focus of research has been on cysteine proteinases, referred to as gingipain R and gingipain K which are produced in large quantities and are directly involved in pathological events during development and progression of periodontitis, contributing to clinical hallmarks of the disease including: flow of gingival crevicular fluid, neutrophil accumulation and bleeding on probing. Gingipain R exists as 110-, 95-, 70- to 90- and 50-kDa proteins, the first two being a complex of the 50-kDa catalytic subunit with hemagglutinin/adhesins, with or without an added membrane anchorage peptide. The other forms are single-chain enzymes. The predominant form of gingipain K in P. gingivalis strains is a complex of a 60-kDa catalytic protein with hemagglutinin/adhesins. Molecular cloning and structural characterization of the gingipain R and gingipain K genes has shown that they code for 1704 and 1722 amino-acid residue preproenzymes, respectively. Although both structures show no similarity within the preprofragment and only limited identity within the catalytic domain (27%) they are essentially identical within the putative hemagglutinin/adhesin domain. Furthermore, on the basis of gene structure it is now apparent that various soluble and membrane bound forms of gingipains are derived through proteolytic processing of the preproenzymes, and it can be assumed that the Arg-X-specific enzyme is responsible for this processing.

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The Role of the Interaction between Fe(III) and Cell Surface in the Accumulation of 67Ga by Tumor Cells

Nuklearmedizin ◽

10.1055/s-0038-1629590 ◽

1991 ◽

Vol 30 (06) ◽

pp. 290-293 ◽

Cited By ~ 1

Author(s):

P. Maleki ◽

A. Martinezi ◽

M. C. Crone-Escanye ◽

J. Robert ◽

L. J. Anghileri

Keyword(s):

Cell Surface ◽

Tumor Cells ◽

Ionic Composition ◽

Iron Complex ◽

Iron Binding ◽

Complex Time ◽

Interaction Product ◽

Thermodynamic Constant ◽

Number Of Cells

The study of the interaction between complexed iron and tumor cells in the presence of 67Ga-citrate indicates that a phenomenon of iron-binding related to the thermodynamic constant of stability of the iron complex, and a hydrolysis (or anion penetration) of the interaction product determine the uptake of 67Ga. The effects of various parameters such as ionic composition of the medium, nature of the iron complex, time of incubation and number of cells are discussed.

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