Porphyromonas gingivalis fimbriae carbohydrate specificity assessment by glycomics

Porphyromonas gingivalis is a major pathogen in adult periodontitis. Fimbriae play an important role in the initial interaction between the bacteria and the host. Our earlier studies suggested that the oligosaccharide moiety of lactoferrin is involved in the interaction with fimbriae. Porphyromonas gingivalis fimbriae bound strongly to albumin-fucosylamide (albumin-1-amido-1-deoxy-l-fucose) and to a lesser extent to albumin-N-acetyl-d-galactosamine (albumin-p-aminophenyl-N-acetyl-β-d-galactosaminide, but failed to bind bovine serum albumin. In this study we explored the glycan array to determine the carbohydrate-binding specificity of P.?gingivalis fimbriae. Purified fimbriae bind to glycans which have a Lewis(x) , Galβ1-4(Fucα1-3) GlcNAcβ structure in common. Interestingly, all glycans have a terminal fucose moiety and if fucose is removed, the fimbriae fail to bind. This is the first study that suggests that fucose is important for P.?gingivalis fimbriae binding.     

Adherence of Porphyromonas gingivalis to matrix proteins via a fimbrial cryptic receptor exposed by its own arginine-specific protease

Porphyromonas gingivalis , a Gram-negative anaerobe, is known to be involved in the pathogenesis of periodontitis. P. gingivalis fimbriae, which are proteinaceous appendages extending from the cell surface, may contribute to the adherence of the organism to the host cell surface. We previously suggested that arginine-specific protease produced by P. gingivalis enhanced the adherence of purified fimbriae to fibroblasts or matrix proteins. In this study, we have revealed the mechanism of the enhanced binding of fimbriae by the protease in more detail. Arg-specific protease and fimbriae were obtained from P. gingivalis 381 cells and purified. We then analysed the interaction of fimbriae and immobilized fibronectins (intact or partially degraded fibronectin by the purified protease) by using the real-time biomolecular interaction analysis (BIAcore) system with an optical biosensor based on the principles of surface plasmon resonance. BIAcore profiles demonstrated an enhanced interaction between fimbriae and protease-degraded fibronectin. We also showed specific binding of fimbriae to the degraded fibronectin by means of BIAcore analysis. The binding of biotinylated fimbriae to immobilized fibronectin was examined by enzyme-linked biotin–avidin assay. The purified protease enhanced the fimbrial binding to the immobilized fibronectin. The enhancement was inhibited by the addition of l -Arg, or oligopeptides containing the Arg residue at the C-terminus in the fimbrial binding reaction, suggesting that the P. gingivalis fimbriae may potentially have an ability to bind tightly to the Arg residue at C-terminus. Taken together, these studies indicate that P. gingivalis arginine-specific protease can expose a cryptitope in the matrix protein molecules, i.e. the C-terminal Arg residue of the host matrix proteins, so that the organism can adhere to the surface layer in the oral cavity through fimbriae–Arg interaction (a novel host–parasite relationship).     

Evidence that Porphyromonas (Bacteroides) gingivalis fimbriae function in adhesion to Actinomyces viscosus.

Porphyromonas (Bacteroides) gingivalis adheres to gram-positive bacteria, such as Actinomyces viscosus, when colonizing the tooth surface. However, little is known of the adhesins responsible for this interaction. A series of experiments were performed to determine whether P. gingivalis fimbriae function in its coadhesion with A. viscosus. Fimbriae typical of P. gingivalis were isolated from strain 2561 (ATCC 33277) by the method of Yoshimura et al. (F. Yoshimura, K. Takahashi, Y. Nodasaka, and T. Suzuki, J. Bacteriol. 160:949-957, 1984) in fractions enriched with a 40-kDa subunit, the fimbrillin monomer, P. gingivalis-A. viscosus coaggregation was inhibited by purified rabbit antifimbrial immunoglobulin G (IgG) at dilutions eightfold higher than those of preimmune IgG, providing indirect evidence implicating P. gingivalis fimbriae in coadhesion. Three types of direct binding assays further supported this observation. (i) Mixtures of isolated P. gingivalis fimbriae and A. viscosus WVU627 cells were incubated for 1 h, washed vigorously with phosphate-buffered saline (pH 7.2), and subjected to electrophoresis. Transblots onto nitrocellulose were probed with antifimbrial antiserum. Fimbrillin labeled positively on these blots. No reaction occurred with the control protein, porcine serum albumin, when blots were exposed to anti-porcine serum albumin, (ii) A. viscosus cells incubated with P. gingivalis fimbriae were agglutinated only after the addition of antifimbrial antibodies. (iii) Binding curves generated from an enzyme immunoassay demonstrated concentration-dependent binding of P. gingivalis fimbriae to A. viscosus cells. From these lines of evidence, P. gingivalis fimbriae appear to be capable of binding to A. viscosus and mediating the coadhesion of these species.     

Adhesion of cellulose to cell walls to Trichoderma reesei

Carbohydrate-binding components were shown to be present at the surface of Listeria monocytogenes by means of a panel of neoglycoproteins using direct agglutination. These lectin-like components bind on neoglycoproteins bearing D-glucosamine, L-fucosylamine, or para-amino-phenyl-alpha-D-mannopyrannoside residues. The interactions were inhibited by the carbohydrate moieties specific to the neoglycoproteins. The protein nature of the lectin-like components of L. monocytogenes was ascertained by the loss of carbohydrate-binding capacity following protease treatment.     

Surface Listeria monocytogenes carbohydrate-binding components revealed by agglutination with neoglycoproteins

Carbohydrate-binding components were shown to be present at the surface of Listeria monocytogenes by means of a panel of neoglycoproteins using direct agglutination. These lectin-like components bind on neoglycoproteins bearing d -glucosamine, l -fucosylamine, or para-amino-phenyl-α- d -mannopyrannoside residues. The interactions were inhibited by the carbohydrate moieties specific to the neoglycoproteins. The protein nature of the lectin-like components of L. monocytogenes was ascertained by the loss of carbohydratebinding capacity following protease treatment.     

Human lactoferrin binds and removes the hemoglobin receptor protein of the periodontopathogen Porphyromonas gingivalis

Porphyromonas gingivalis possesses a hemoglobin receptor (HbR) protein on the cell surface as one of the major components of the hemoglobin utilization system in this periodontopathogenic bacterium. HbR is intragenically encoded by the genes of an arginine-specific cysteine proteinase (rgpA), lysine-specific cysteine proteinase (kgp), and a hemagglutinin (hagA). Here, we have demonstrated that human lactoferrin as well as hemoglobin have the abilities to bind purified HbR and the cell surface of P. gingivalis through HbR. The interaction of lactoferrin with HbR led to the release of HbR from the cell surface of P. gingivalis. This lactoferrin-mediated HbR release was inhibited by the cysteine proteinase inhibitors effective to the cysteine proteinases of P. gingivalis. P. gingivalis could not utilize lactoferrin for its growth as an iron source and, in contrast, lactoferrin inhibited the growth of the bacterium in a rich medium containing hemoglobin as the sole iron source. Lactoferricin B, a 25-amino acid-long peptide located at the N-lobe of bovine lactoferrin, caused the same effects on P. gingivalis cells as human lactoferrin, indicating that the effects of lactoferrin might be attributable to the lactoferricin region. These results suggest that lactoferrin has a bacteriostatic action on P. gingivalis by binding HbR, removing it from the cell surface, and consequently disrupting the iron uptake system from hemoglobin.     

Identification of glyceraldehyde-3-phosphate dehydrogenase of epithelial cells as a second molecule that binds to Porphyromonas gingivalis fimbriae

Binding of Porphyromonas gingivalis to the host cells is an essential step in the pathogenesis of periodontal disease. P. gingivalis binds to and invades epithelial cells, and fimbriae are thought to be involved in this process. In our earlier studies, two major epithelial cell components of 40 and 50 kDa were identified as potential fimbrial receptors. Sequencing of a cyanogen bromide digestion fragment of the 50-kDa component resulted in an internal sequence identical to keratin I molecules, and hence this cytokeratin represents one of the epithelial cell receptors for P. gingivalis fimbriae. In this study, the 40-kDa component of KB cells was isolated and its amino-terminal sequence determined. The N-terminal amino sequence was found to be GKVKVGVNGF and showed perfect homology with human glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Furthermore, purified P. gingivalis fimbriae were found to bind to rabbit muscle GAPDH. Antibodies directed against internal peptide 49-68 and 69-90 of fimbrillin were shown to inhibit the binding of P. gingivalis and of fimbriae to epithelial cells. Antibodies against these peptides also inhibited the binding of fimbriae to GAPDH. Our results confirmed that the amino-terminal domain corresponding to amino residues 49-68 of the fimbrillin protein is the major GAPDH binding domain. These studies point to GAPDH as a major receptor for P. gingivalis major fimbriae and, as such, GAPDH likely plays a role in P. gingivalis adherence and colonization of the oral cavity, as well as triggering host cell processes involved in the pathogenesis of P. gingivalis infections.     

Fimbria-mediated coaggregation between human oral anaerobes Treponema medium and Porphyromonas gingivalis.

Bacterial binding phenomena among different bacterial genera or species play an important role in bacterial colonization in a mixed microbiota such as in the human oral cavity. The coaggregation reaction between two gram-negative anaerobes, Treponema medium and Porphyromonas gingivalis, was characterized using fimbria-deficient mutants of P. gingivalis and specific antisera against purified fimbriae and bacterial whole cells. T. medium ATCC 700273 strongly coaggregated with fimbriate P. gingivalis strains ATCC 33277 and 381, but not with afimbriate strains including transposon-induced fimbria-deficient mutants and KDP98 as a fimA-disrupted mutant of P. gingivalis ATCC 33277. In the P. gingivalis-T. medium coaggregation assay, the presence of rabbit antiserum against the purified fimbriae or the whole cells of P. gingivalis ATCC 33277 produced different "aggregates" consisting predominantly of P. gingivalis cells with few spirochetes, but both preimmune serum and the antiserum against the afimbriate KDP98 cells did not inhibit the coaggregation reaction. Heated P. gingivalis cells lost their ability to bind both heated and unheated T. medium cells. This T. medium-P. gingivalis coaggregation reaction was inhibited by a cysteine proteinase inhibitor, leupeptin, and also by arginine and lysine, but not by EDTA or sugars including lactose. A binding assay on nitrocellulose membranes and immunoelectron microscopy demonstrated that a heat-stable 37 kDa surface protein on the T. medium cell attached to the P. gingivalis fimbriae.     

Inhibitory effects of Porphyromonas gingivalis fimbriae on interactions between extracellular matrix proteins and cellular integrins

Porphyromonas gingivalis is a predominant periodontal pathogen, whose fimbriae are considered to be a major virulence factor, especially for bacterial adherence and invasion of host cells. In the present study, we investigated the influence of fimbriae on the interactions between alphavbeta3- and alpha5beta1-integrins and their ligand extracellular matrix (ECM) proteins (vitronectin and fibronectin), using human alphavbeta3- and alpha5beta1-integrin-overexpressing CHO cell lines (CHOalphavbeta3 and CHOalpha5beta1, respectively). P. gingivalis was found to have significantly greater binding to CHOalphavbeta3 and CHOalpha5beta1 than to control cells, whereas a fimbria-deficient mutant showed negligible binding to any of the tested cell lines. CHOalphavbeta3 and CHOalpha5beta1 cells attached to the polystyrene culture dishes in the presence of their ligand ECM proteins, while fimbriae markedly inhibited those attachments in a dose-dependent manner, with the highest dose of fimbriae achieving complete inhibition. In addition, the binding of vitronectin and fibronectin to CHOalphavbeta3 and CHOalpha5beta1 was inhibited by P. gingivalis cells. These results suggest that P. gingivalis fimbriae compete with ECM proteins for alphavbeta3- and alpha5beta1-integrins, and inhibit integrin/ECM protein-related cellular functions.     

Oral streptococcal glyceraldehyde-3-phosphate dehydrogenase mediates interaction with Porphyromonas gingivalis fimbriae

Interaction of Porphyromonas gingivalis with plaque-forming bacteria is necessary for its colonization in periodontal pockets. Participation of Streptococcus oralis glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and P. gingivalis fimbriae in this interaction has been reported. In this investigation, the contribution of various oral streptococcal GAPDHs to interaction with P. gingivalis fimbriae was examined. Streptococcal cell surface GAPDH activity was measured by incubation of a constant number of streptococci with glyceraldehyde-3-phosphate and analysis for the conversion of NAD+ to NADH based on the absorbance at 340 nm. Coaggregation activity was measured by a turbidimetric assay. Cell surface GAPDH activity was correlated with coaggregation activity (r = 0.854, P < 0.01) with Spearman's rank correlation coefficient. S. oralis ATCC 9811 and ATCC 10557, Streptococcus gordonii G9B, Streptococcus sanguinis ATCC 10556, and Streptococcus parasanguinis ATCC 15909 exhibited high cell surface GAPDH activity and coaggregation activity; consequently, their cell surface GAPDHs were extracted with mutanolysin and purified on a Cibacron Blue Sepharose column. Subsequently, their DNA sequences were elucidated. Purified GAPDHs bound P. gingivalis recombinant fimbrillin by Western blot assay, furthermore, their DNA sequences displayed a high degree of homology with one another. Moreover, S. oralis recombinant GAPDH inhibited coaggregation between P. gingivalis and the aforementioned five streptococcal strains in a dose-dependent manner. These results suggest that GAPDHs of various plaque-forming streptococci may be involved in their attachment to P. gingivalis fimbriae and that they may contribute to P. gingivalis colonization.     

Bacterial fimbriae stimulate proinflammatory activation in the endothelium through distinct TLRs

The major and minor fimbriae proteins produced by the human pathogen Porphyromonas gingivalis are required for invasion of human aortic endothelial cells and for the stimulation of potent inflammatory responses. In this study, we report that native forms of both the major and minor fimbriae proteins bind to and signal through TLR2 for this response. Major and minor fimbriae bound to a human TLR2:Fc chimeric protein with an observed K(d) of 28.9 nM and 61.7 nM, respectively. Direct binding of the major and minor fimbriae to a human chimeric CD14-Fc protein also established specific binding of the major and minor fimbriae to CD14 with classic saturation kinetics. Using a P. gingivalis major and minor fimbriae mutant, we confirmed that TLR2 binding in whole cells is dependent on the expression of the major and minor fimbriae. Although we did not observe binding with the major or minor fimbriae to the TLR4-Fc chimeric protein, signaling through TLR4 for both proteins was demonstrated in human embryonic kidney 293 cells transfected with TLR4 and only in the presence MD-2. Transient transfection of dominant-negative forms of TLR2 or TLR4 reduced IL-8 production by human aortic endothelial cells following stimulation with major or minor fimbriae. The ability of two well-defined microbe-associated molecular patterns to select for innate immune recognition receptors based on accessory proteins may provide a novel way for a pathogen to sense and signal in appropriate host environments.     

Roles of porphyrins and host iron transport proteins in regulation of growth of Porphyromonas gingivalis W50.

Porphyromonas gingivalis (Bacteroides gingivalis) requires iron in the form of hemin for growth and virulence in vitro, but the contributions of the porphyrin ring structure, porphyrin-associated iron, host hemin-sequestering molecules, and host iron-withholding proteins to its survival are unknown. Therefore, the effects of various porphyrins, host iron transport proteins, and inorganic iron sources on the growth of P. gingivalis W50 were examined to delineate the various types of iron molecules used for cellular metabolism. Cell envelope-associated hemin and iron stores contributed to the growth of P. gingivalis in hemin-free culture, and depletion of these endogenous reserves required eight serial transfers into hemin-free medium for total suppression of growth. Comparable growth of P. gingivalis was observed with 7.7 microM equivalents of hemin as hemoglobin (HGB), methemoglobin, myoglobin, hemin-saturated serum albumin, lactoperoxidase, cytochrome c, and catalase. Unrestricted growth was recorded in the presence of haptoglobin-HGB and hemopexin-hemin complexes, indicating that these host defense proteins do not sequester HGB and hemin from P. gingivalis. The iron chelator 2,2'-bipyridyl functionally chelated hemin-associated iron, resulting in dose-dependent inhibition of growth in hemin-restricted cultures at 1 to 25 microM 2,2'-bipyridyl concentrations. In the absence of an exogenous iron source, protoporphyrin IX did not support P. gingivalis growth. These findings suggest that the iron atom in the hemin molecule is the critical constituent for growth and that the tetrapyrrole porphyrin ring structure may represent an important vehicle for delivery of iron into the P. gingivalis cell. P. gingivalis does not have a strict requirement for porphyrins, since growth occurred with nonhemin iron sources, including high concentrations (200 muM) of ferric, ferrous, and nitrogenous inorganic iron, and P. gingivalis exhibited unrestricted growth in the presence of host transferrin, lactoferrin, and serum albumin. The diversity of iron substrates utilized by P. gingivalis and the observation that growth was not affected by the bacteriostatic effects of host iron-withholding proteins, which it may encounter in the periodontal pocket, may explain why P. gingivalis is such a formidable pathogen in the periodontal disease process.     

TLR2 transmodulates monocyte adhesion and transmigration via Rac1- and PI3K-mediated inside-out signaling in response to Porphyromonas gingivalis fimbriae

We present evidence for a novel TLR2 function in transmodulating the adhesive activities of human monocytes in response to the fimbriae of Porphyromonas gingivalis, a pathogen implicated in chronic periodontitis and atherosclerosis. Monocyte recruitment into the subendothelium is a crucial step in atherosclerosis, and we investigated the role of P. gingivalis fimbriae in stimulating monocyte adhesion to endothelial cells and transendothelial migration. Fimbriae induced CD11b/CD18-dependent adhesion of human monocytes or mouse macrophages to endothelial receptor ICAM-1; these activities were inhibited by TLR2 blockade or deficiency or by pharmacological inhibitors of PI3K. Moreover, this inducible adhesive activity was sensitive to the action of Clostridium difficile toxin B, but was not affected by Clostridium botulinum C3 exoenzyme, pertussis toxin, or cholera toxin. Accordingly, we subsequently showed through the use of dominant negative signaling mutants of small GTPases, that Rac1 mediates the ability of fimbria-stimulated monocytes to bind ICAM-1. A dominant negative mutant of Rac1 also inhibited the lipid kinase activity of PI3K suggesting that Rac1 acts upstream of PI3K in this proadhesive pathway. Furthermore, fimbriae stimulated monocyte adhesion to HUVEC and transmigration across HUVEC monolayers; both activities required TLR2 and Rac1 signaling and were dependent upon ICAM-1 and the high-affinity state of CD11b/CD18. P. gingivalis-stimulated monocytes displayed enhanced transendothelial migration compared with monocytes stimulated with nonfimbriated isogenic mutants. Thus, P. gingivalis fimbriae activate a novel proadhesive pathway in human monocytes, involving TLR2, Rac1, PI3K, and CD11b/CD18, which may constitute a mechanistic basis linking P. gingivalis to inflammatory atherosclerotic processes.     

Inactivation of the Porphyromonas gingivalis fimA gene blocks periodontal damage in gnotobiotic rats.

Fimbrial production by Porphyromonas gingivalis was inactivated by insertion-duplication mutagenesis, using the cloned gene for the P. gingivalis major fimbrial subunit protein, fimA. by several criteria, this insertion mutation rendered P. gingivalis unable to produce fimbrilin or an intact fimbrial structure. A nonfimbriated mutant, DPG3, hemagglutinated sheep erythrocytes normally and was unimpaired in the ability to coaggregate with Streptococcus gordonii G9B. The cell surface hydrophobicity of DPG3 was also unaffected by the loss of fimbriae. However, DPG3 was significantly less able to bind to saliva-coated hydroxyapatite than wild-type P. gingivalis 381. This suggested that P. gingivalis fimbriae are important for adherence of the organism to saliva-coated oral surfaces. Further, DPG3 was significantly less able to cause periodontal bone loss in a gnotobiotic rat model of periodontal disease. These observations are consistent with other data suggesting that P. gingivalis fimbriae play an important role in the pathogenesis of human periodontal disease.     

Fimbrial proteins of porphyromonas gingivalis mediate in vivo virulence and exploit TLR2 and complement receptor 3 to persist in macrophages

Porphyromonas gingivalis is an oral/systemic pathogen implicated in chronic conditions, although the mechanism(s) whereby it resists immune defenses and persists in the host is poorly understood. The virulence of this pathogen partially depends upon expression of fimbriae comprising polymerized fimbrillin (FimA) associated with quantitatively minor proteins (FimCDE). In this study, we show that isogenic mutants lacking FimCDE are dramatically less persistent and virulent in a mouse periodontitis model and express shorter fimbriae than the wild type. Strikingly, native fimbriae allowed P. gingivalis to exploit the TLR2/complement receptor 3 pathway for intracellular entry, inhibition of IL-12p70, and persistence in macrophages. This virulence mechanism also required FimCDE; indeed, mutant strains exhibited significantly reduced ability to inhibit IL-12p70, invade, and persist intracellularly, attributable to failure to interact with complement receptor 3, although not with TLR2. These results highlight a hitherto unknown mechanism of immune evasion by P. gingivalis that is surprisingly dependent upon minor constituents of its fimbriae, and support the concept that pathogens evolved to manipulate innate immunity for promoting adaptive fitness and thus their capacity to cause disease.     

An endogenous carbohydrate-binding agglutinin of BHK cells. Purification, specificity and interaction with normal and ricin-resistant cell lines

Several lectin-like activities were detected on the surface of unfixed, viable BHK cells by reaction with FITC-labelled glycosylated albumin derivatives. A prominent surface staining was obtained with the beta-lactosyl, beta-N-acetylgalactosaminyl, alpha-mannosyl and beta-N-acetylglucosaminyl derivatives. Endogenous lectin-like activities were also detected in BHK cell homogenates by haemagglutination of glutaraldehyde-fixed rabbit erythrocytes. Haemagglutinating activity was purified by chromatography of sodium deoxycholate-extracts of BHK cell microsomal fractions on Sepharose 4B and asialofetuin-Sepharose 4B. A purified agglutinin was eluted from the latter column with 0.2 M thiodigalactoside. The haemagglutination mediated by the purified factor was inhibited by thiodigalactoside, N-acetylgalactosamine, galactosyl-beta 1-4-N-acetylglucosamine and several glycoproteins. The purified agglutinin agglutinated trypsinised, fixed normal BHK cells more readily than several ricin-resistant cell lines. By contrast, a mannose-binding lectin from rabbit serum reacted equally well with normal and mutant cells. These results are in general agreement with models of cell-cell adhesion involving the interaction of surface located lectins with carbohydrate sequences of normal BHK cell surface glycoproteins.     

A novel lectin-independent interaction of P fimbriae of Escherichia coli with immobilized fibronectin

Binding of P fimbriae of uropathogenic Escherichia coli to purified human fibronectin and human placental type IV collagen was studied. In an enzyme immunoassay, purified P fimbriae bound strongly to immobilized intact fibronectin and to the aminoterminal 30-kDa fragment and the 120-140-kDa carboxyterminal fragments of fibronectin. Binding to the gelatin-binding 40-kDa fragment of fibronectin was considerably weaker. No binding to immobilized type IV collagen was seen. The interaction between P fimbriae and immobilized fibronectin was not inhibited by alpha-D-Gal-(1-4)-beta-D-Gal-1-O-Me, a receptor analog of P fimbriae. Moreover, a mutated P fimbria lacking the lectin activity behaved similarly in the adherence assays. Recombinant strains expressing the corresponding cloned fimbriae genes bound to immobilized fibronectin, but no binding to soluble 125I-labelled fibronectin was found. The results suggest that P fimbriae interact with immobilized fibronectin and that the binding mechanism does not involve the lectin activity of the fimbriae.     

Purification and characterization of a novel type of fimbriae from the oral anaerobe Bacteroides gingivalis.

Fimbriae and their constituent protein (fimbrilin) were purified to homogeneity from the bacterial wash fluid and cell lysate fraction, respectively, of Bacteroides gingivalis 381. Fimbriae, observed by negative staining, were curly, single-stranded filaments with a diameter of ca. 5 nm. The apparent molecular weight of the fimbrilin was 43,000. Fimbriae were resistant to sodium dodecyl sulfate denaturation at 70 degrees C. Heating at 100 degrees C in sodium dodecyl sulfate was needed to completely dissociate them to monomers of fimbrilin. Different sets of antigenic determinants seemed to be exposed on the surfaces of fimbriae and sodium dodecyl sulfate-denatured fimbrilin. Purified fimbriae did not show either hemagglutinating activity or hemagglutination inhibitory activity, although it has been inferred on the basis of circumstantial evidence that fimbriae are correlated to hemagglutinating activity of the organism. Hemagglutinin activity, however, was detected in culture supernatant, and this observation suggests that fimbriae of a different type or a lectin-like protein may be acting as hemagglutinin in B. gingivalis.     

Characterization of the rabbit sperm membrane autoantigen, RSA, as a lectin-like zona binding protein

Adhesion between spermatozoa and the egg's extracellular coat, the zona pellucida, involves the sperm's zona binding proteins (ZBP) and their interaction with the carbohydrate residues of the zona. To investigate this interaction in more detail, a purified nonenzymatic ZBP, the rabbit sperm membrane autoantigen, RSA, was used. RSA-zona binding was demonstrated on nitrocellulose blots and by using the Denny-Jaffe crosslinking reagent which identified an 87,000 molecular weight zona component as the ligand for RSA. The RSA-zona binding was of high affinity with a dissociation constant of 5.6 X 10(-13) M. Furthermore, the binding of capacitated spermatozoa to intact zona was inhibited in the presence of RSA. Characterization of the RSA-zona interaction with a variety of simple and complex carbohydrates indicated that the sulfated, complex carbohydrates fucoidin, dextran sulfate, chondroitin sulfate B, and heparin strongly inhibited RSA-zona binding while chondroitin sulfates A and C, cholesterol-3-sulfate, and monosaccharides such as galactose inhibited RSA-zona binding only weakly. It is concluded that RSA functions as a sperm lectin-like molecule to bind the spermatozoon to the zona pellucida.