Single nucleotide polymorphisms of pattern recognition receptors and chronic periodontitis.

BACKGROUND AND OBJECTIVE: Periodontitis is a multifactorial disease influenced partly by genetics. Activation of pattern recognition receptors (PRRs) can lead to the up-regulation of inflammatory pathways, resulting in periodontal tissue destruction. Hence, functional polymorphisms located in PRRs can explain differences in host susceptibility to periodontitis. This study investigated single nucleotide polymorphisms of PRRs including toll-like receptor (TLR)2 (G2408A), TLR4 (A896G), TLR9 (T1486C), TLR9 (T1237C) and CD14 (C260T) in patients with chronic periodontitis and in periodontally healthy subjects. METHODS: One-hundred and fourteen patients with chronic periodontitis and 77 periodontally healthy subjects were genotyped using TaqMan® allelic discrimination assays. Fisher's exact test and chi-square analyses were performed to compare genotype and allele frequencies. RESULTS: The frequency of subjects with the CC genotype of CD14 (C260T) (24.6% in the chronic periodontitis group vs. 13% in the periodontally healthy group) and those expressing the T allele of CD14 (C260T) (CT and TT) (75.4% in the chronic periodontitis group vs. 87% in the periodontally healthy group) was statistically different among groups (p = 0.04). Homozygocity for the C allele of the CD14 (C260T) polymorphism (CC) was associated with a two--fold increased susceptibility to periodontitis (p = 0.04; odds ratio, 2.49; 95% confidence interval, 1.06-6.26). Individuals with the CC genotype of TLR9 (T1486C) (14.9% in the chronic periodontitis group vs. 28.6% in the periodontally healthy group) and those expressing the T allele of TLR9 (T1486C) (CT and TT) (85.1% in the chronic periodontitis group vs. 71.4% in the periodontally healthy group) were also significantly differently distributed between groups without adjustment (p = 0.03). Further analysis of nonsmokers revealed a significant difference in the distribution of genotypes between groups for TLR9 (T1486C; p = 0.017) and CD14 (C260T; p = 0.03), polymorphisms again without adjustment. CONCLUSION: The CC genotype of CD14 (C260T) is related to susceptibility to chronic periodontitis in Caucasians. In addition, differences observed in the distribution of TLR9 (T1486C) genotypes between groups warrant further investigation.

Fibrinogen-neutrophil interactions in response to fMLP and Porphyromonas gingivalis fimbrial peptides.

Porphyromonas gingivalis (P.g) is the primary bacterial agent in many forms of chronic periodontitis. Since polymorphonuclear leukocytes (PMNs) are first-line responders to P.g.- induced inflammation, and fibrinogen is important for in vivo PMN in this disease, we have studied the effect of N-formyl-methionyl-leucyl-phenylalanine (fMLP) (an inflammatory stimulus), P.g. fimbriae and fimbrial peptides (based on FimA, the main structural protein of P.g. fimbriae) on PMN-fibrinogen interactions. Freshly isolated human PMNs were allowed to react with FITC-Fibrinogen and various fimbrial peptides (denoted as FimA followed by amino acid number within whole FimA protein), and FITC-Fibrinogen binding was measured using flow cytometry. Freshly isolated neutrophils were also challenged with Fibrinogen and/or fimbrial peptides to measure IL-8 secretion using ELISA. Our studies show that fibrinogen binding to PMNs is enhanced (p < 0.01) in response to fMLP as well as fimbrial peptides (FimA 61-80) containing the motif LTTE (p < 0.01) in a dose dependent manner but not in response to peptides without that motif. We also observed that fMLP and FimA 61-80 have an additive effect on fibrinogen binding to PMNs (p < 0.05), and fMLP and FimA 171-185 significantly inhibit fMLP-induced fibrinogen binding (p < 0.01). To determine of the role of inflammatory cytokines, we examined IL-8 release from PMNs in response to combinations of P. gingivalis fimbriae, fMLP and fibrinogen. In all cases, IL-8 release increased in a dose-dependent manner (p < 0.05). fMLP-fibrinogen effect on IL-8 release from PMNs was synergistic while fimbriae-fibrinogen effect was additive. In summary, PMN priming by fimbrial peptides facilitates fibrinogen-PMN interaction and may increase inflammation.

Periodontal infections contribute to elevated systemic C-reactive protein level.

BACKGROUND: Periodontitis is a local inflammatory process mediating destruction of periodontal tissues triggered by bacterial insult. However, this disease is also characterized by systemic inflammatory host responses that may contribute, in part, to the recently reported higher risk for cardiovascular disease (CVD) among patients with periodontitis. Moderate elevation of C-reactive protein (CRP) has been found to be a predictor of increased risk for CVD. Elevated CRP levels in periodontal patients have been reported by several groups. In this study, we examined whether CRP plasma levels are increased in periodontitis and if there is a relation to severity of periodontal disease and to the periodontal microflora. METHODS: CRP serum levels were assessed using radial immunodiffusion assay in 174 subjects, 59 with moderate mean clinical attachment loss (AL) (2.39+/-0.29 mm) and 50 with high AL (3.79+/-0.86 mm) as compared to 65 periodontally healthy controls (AL, 1.74+/-0.18 mm). Clinical attachment loss, probing depths, and percentage of periodontal pocket sites > or =5 mm were measured. The presence of periodontal pathogens Porphyromonas gingivalis (P.g.), Prevotella intermedia (P.i.), Campylobacter recta (C.r.), and Bacteroides forsythus (B.f.) in subgingival plaque samples was measured by immunofluorescence microscopy. RESULTS: Statistically significant increases in CRP levels were observed in subjects with periodontal disease when compared to healthy controls (P= 0.036). Subjects with high levels of mean clinical attachment loss had significantly higher mean CRP levels (4.06+/-5.55 mg/l) than controls (1.70+/-1.91 mg/l), P= 0.011. The CRP levels were adjusted for factors known to be associated with elevated CRP, including age, smoking, body mass index (BMI), triglycerides, and cholesterol. Age and BMI were found to be significant covariates. The reported range for CRP as a risk factor for CVD, peripheral vascular diseases, or stroke is 1.34 mg/l to 6.45 mg/l and the mean of this range is 3 mg/l. The percentage of subjects with elevated levels of CRP > or = 3 mm was significantly higher in the high clinical AL group (38%; 95% Cl: 26.7%, 49.3%) when compared to the control group (16.9%; 95% CI: 9.25%, 24.5%), P= 0.011. The presence of periodontal pathogens P.g., P.i., C.r., and B.f. in subgingival samples was positively associated with elevated CRP levels (P= 0.029). CONCLUSIONS: The extent of increase in CRP levels in periodontitis patients depends on the severity of the disease after adjusting for age, smoking, body mass index, triglycerides, and cholesterol. Also, there are elevated levels of CRP associated with infection with subgingival organisms often associated with periodontal disease, including P.g., P.i., C.r., and B.f. Recent investigations emphasized the role of moderate elevated CRP plasma levels as a risk factor for CVD. The positive correlation between CRP and periodontal disease might be a possible underlying pathway in the association between periodontal disease and the observed higher risk for CVD in these patients.

The role of inflammatory and immunological mediators in periodontitis and cardiovascular disease.

Epidemiological studies have implicated periodontitis (PD) as a risk factor for development of cardiovascular disease (CVD). Persistent infections such as periodontitis induce inflammatory and immune responses which may contribute to coronary atherogenesis, and, in conjunction with other risk factors, may lead to coronary heart disease (CHD). In this review, mechanisms are described that may help explain the association between periodontal infections and CHD. Periodontal diseases are bacterial infections associated with bacteremia, inflammation, and a strong immune response, all of which may represent significant risk factors for the development of atherogenesis, CHD, and myocardial infarction (MI). Several mechanisms may participate in this association, including those induced by oral organisms, and those associated with host response factors. This review will focus on host factors. Oral pathogens and inflammatory mediators (such as interleukin [IL]-1 and tumor necrosis factor [TNF]-alpha) from periodontal lesions intermittently reach the bloodstream inducing systemic inflammatory reactants such as acute-phase proteins, and immune effectors including systemic antibodies to periodontal bacteria. This review will describe the potential role of various inflammatory as well as immunologic factors that may play a role in periodontitis as a possible risk factor for CHD.

Interleukin 8, neutrophil-activating peptide-2 and GRO-alpha bind to and elicit cell activation via specific and different amino acid residues of CXCR2.

The objective of this investigation was to determine the amino acid residues of the human neutrophil CXC chemokine receptor-2 (CXCR2) that are critical for binding the ligands interleukin 8 (IL-8), neutrophil-activating peptide-2 (NAP-2), and growth-related protein alpha (GROalpha) and critical for receptor-mediated signal transduction. Charged residues of the amino terminus and the first extracellular loop of CXCR2 were targeted for point mutagenesis studies. Seven separate CXCR2 mutants (Glu7, Asp9, Glu12, Asp13, Lys108, Asn110, and Lys120, all to Ala) were generated. Based on the Scatchard analysis of radioligand binding studies, the following amino acids were deemed critical for ligand binding: (i) Asp9, Glu12, Lys108, and Lys120 for IL-8 and (ii) Glu7, Asp9, and Glu12 for GROalpha. Point mutations in the amino terminus domain (Asp9 and Glu12) and the first extracellular loop (Lys108, Asn110, and Lys120) of CXCR2 reduced cell activation to all three ligands as measured by changes in intracellular calcium concentration. In conclusion, high-affinity binding of IL-8, NAP-2, and GROalpha to CXCR2 involves interaction with specific and different amino acid residues of CXCR2. Furthermore, we propose that the CXCR2 amino acid residues required for cell activation are not necessarily the same residues required for ligand binding.

Pyk2 and Syk participate in functional activation of granulocytic HL-60 cells in a different manner.

The roles of the protein tyrosine kinases Pyk2 (also called RAFTK or CAK beta) and Syk in the process of functional activation of human myeloid cells were examined. During granulocytic differentiation of HL-60 cells with dimethyl sulfoxide (DMSO), the amounts of Pyk2 and beta2 integrin increased, whereas the amount of Syk was abundant before differentiation and did not change during differentiation. When the granulocytic cells were stimulated with N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), tyrosine phosphorylation of Pyk2 occurred promptly and subsequent association of Pyk2 with beta2 integrin was detected. In contrast, Syk was not tyrosine phosphorylated by fMLP stimulation but constitutively associated with beta2 integrin. Stimulation with fMLP also caused the alteration of beta2 integrin to an activated form, a finding that was confirmed by the observation of fMLP-induced cell attachment on fibrinogen-coated dishes and inhibition of this attachment by pretreatment with anti-beta2 integrin antibody. Cell attachment to fibrinogen caused the enhanced tyrosine phosphorylation of Pyk2 and the initial tyrosine phosphorylation of Syk, which was also inhibited by pretreatment with anti-beta2 integrin antibody. In vitro kinase assays revealed that Pyk2 and Syk represented kinase activities to induce tyrosine phosphorylation of several molecules in the anti-beta2 integrin immunoprecipitates of the attached cells. These results showed that Pyk2 is involved in the functional activation of granulocytic cells in 2 signaling pathways: an fMLP receptor-mediated "inside-out" signaling pathway that might cause beta2 integrin activation and a subsequent beta2 integrin-mediated "outside-in" signaling pathway. Syk was activated in relation to cell attachment to fibrinogen as a result of "outside-in" signaling, although it was already associated with beta2 integrin before fMLP stimulation. (Blood. 2000;96:1733-1739)

Chemotactic-like receptors and Abeta peptide induced responses in Alzheimer's disease.

Evidence suggests that beta-amyloid (Abeta) has chemokine-like properties and may act through formyl chemotactic receptors (FPR) to induce pathophysiologically important functional changes in Alzheimer's disease (AD) microglia. We have shown that Abeta 1-42, fibrillar Abeta 1-40, and Abeta 25-35 potentiate the release of interleukin-1beta (IL-1beta) from LPS activated human THP-1 monocytes [26] and LPS primed rat microglia. Moreover, Abeta-stimulated IL-1beta secretion seems to be receptor mediated because it is calcium dependent and requires activation of specific G-proteins [27]. Thus, we have evaluated the ability of Abeta 1-42 to mimic formyl chemotactic peptides in stimulating IL-1beta release from THP-1 monocytes. Several of the formyl chemotactic peptides and Abeta 1-42 significantly enhanced IL-1beta production in THP-1 monocytes. In contrast, a formyl chemotactic receptor antagonist inhibited Abeta 1-42-induced IL-1beta release from both human THP-1 monocytes and primary rat microglia. Further, primary rat microglia grown in culture expressed FPR as demonstrated by immunocytochemistry. Given the multiple pathophysiologic roles IL-1beta may play in AD, agents that block Abeta interactions with formyl chemotactic receptors on microglia might be important antiinflammatory therapeutic targets.

Isolation and characterization of a human neutrophil aggregation defective mutant of Fusobacterium nucleatum.

Fusobacterium nucleatum is known to adhere to human polymorphonuclear neutrophils (PMNs) and cause them to aggregate. In this study, we isolated a spontaneously occurring aggregation defective (AGG(-)) mutant and this mutant will be used for future study of the interactions between this bacterium and human PMN. Genomic DNA fingerprinting by random-primed polymerase chain reaction method revealed a difference between the parent strain and the AGG(-) mutant. This mutant also showed an altered phenotype in both microbicidal and phagocytic assays, suggesting that the bacterial factor involved in the aggregation may also be very important for the phagocytosis and, subsequently, the killing by human PMNs. Further study of this mutant may help to clarify the molecular mechanisms of the interaction between this pathogen and human PMNs.

Identification and characterization of human immunoglobulin G Fc receptors of Fusobacterium nucleatum.

Several human pathogens express components which can bind to the Fc portion of immunoglobulins. This study was undertaken to characterize the human immunoglobulin G (IgG) Fc-binding activity of Fusobacterium nucleatum, a suspected pathogen involved in periodontal diseases. Fc-binding activity was detected using whole-cell, cell envelope and outer membrane fractions, and it was found to be associated with polypeptides of 40 kDa and 42 kDa, respectively. Amino terminal sequencing of these components revealed them to be homologous to the bacterial porin encoded by fomA gene. Further sequencing of internal peptide fragments obtained by CNBr cleavage suggested that these two proteins are probably isoforms. In summary, we show that a porin-like protein on the surface of F. nucleatum can bind the Fc fragment of the human immunoglobulin G, and this protein may act as a virulence factor to facilitate this bacterium in evading host immune surveillance system.

Single nucleotide polymorphisms of the N-formyl peptide receptor in localized juvenile periodontitis.

BACKGROUND: Neutrophils from patients with localized juvenile periodontitis (LJP) exhibit decreased binding and responsiveness to various chemotactic agents, including N-formyl-1-methionyl-1-leucyl-1-phenylalanine (FMLP). This altered reaction of neutrophils is thought to account in part for the increased susceptibility of LJP patients to infections by periodontal organisms. Receptors for FMLP are involved in the activation and the subsequent response to certain chemotactic stimuli. METHODS: In order to determine if this decreased response is due to a genetic variation in the receptor, we directly compared DNA encoding the FMLP receptor from controls matched for gender and race and LJP patients by single-strand conformation polymorphism analysis (SSCP). RESULTS: Using this technique, we observed a characteristic SSCP pattern in 29 out of 30 patient samples in the FMLP receptor DNA. This pattern differed from those obtained from the 20 control subjects as well as 31 patients with adult periodontitis. DNA sequencing of 30 patients indicated single nucleotide polymorphisms (SNPs) in the FMLP receptor DNA from the LJP patients when compared to 20 controls (P = 0.0005). Two single nucleotide base alterations were consistently seen: either a thymine to cytosine substitution at base 329 in 17 LJP patients or a cytosine to guanine substitution at base 378 in 5 LJP patients. A combination of both alterations were seen in 7 LJP patients. Both alterations resulted in amino acid changes in the second intracellular loop of the receptor, specifically phenylalanine to serine at residue 110 and cysteine to tryptophan at residue 126. This region of the FMLP receptor has recently been shown to play a role in ligand binding and G-protein activation. CONCLUSIONS: This study suggests that a molecular alteration in the second intracellular loop of the FMLP receptor molecules in LP patients may play a role in the decreased chemotactic activity reported for some LJP patients.

Human formyl peptide receptor function role of conserved and nonconserved charged residues.

In this study, we investigated the role of charged residues in ligand binding interactions of f-Met-Leu-Phe receptors (FPR). Charged residues of FPR, both conserved and nonconserved, which are located close to the membrane interface were mutated to alanine to determine their role in ligand binding. The mutated residues belonged to specific domains of FPR which have previously been implicated in FPR ligand binding interactions. We demonstrate that nonconserved charged residues such as Arg84, Lys85, Arg205 and Asp284 and conserved charge residue Arg163 seem to play a role in ligand binding. However, alteration of nonconserved charged residue Asp106 did not have any effect. In conclusion, specific charged residues of FPR, both conserved nonconserved, may contribute to FPR function either directly or indirectly.

Expression and purification of recombinant human N-formyl-L-leucyl-L-phenylalanine (FMLP) receptor: generation of polyclonal antibody against FMLP receptor.

The recombinant formyl peptide receptor has been successfully expressed and purified, utilizing an Escherichia coli expression system. Purification of formyl peptide receptor was performed using gel filtration chromatography and affinity chromatography, and the purified protein migrated at an apparent molecular mass of 36,000 Da. The purified recombinant receptor retained functional activity as determined by a ligand binding assay. The yield of the recombinant purified receptor was approximately 1 mg/2 L of culture, and the binding activity was determined to be approximately 8 nM, which suggests the conclusion that glycosylation does not affect significantly ligand binding of the N-formyl-L-leucyl-L-phenylalanine (FMLP) receptor molecule. The recombinant receptor protein yield was found to be significantly higher than that obtained from neutrophils. The purified recombinant receptor was then utilized to generate antibody against the same. The reaction of the antibody against recombinant formylpeptide receptor and against native formylpeptide receptor on neutrophils was confirmed by western blot analysis and flow cytometric analysis, respectively. The antibody was also used successfully to detect recombinant formylpeptide receptor expression on transfected 293 cells. These results describe for the first time the expression, purification, and characterization of recombinant FMLP receptor with ligand binding activity and the generation of polyclonal antibody against the same. This work also provides a foundation for future biophysical studies of the FMLP receptor molecule, which have not been possible until now.

Mapping of the extracellular binding regions of the human interleukin-8 type B receptor.

This study was undertaken to define the regions of the human interleukin-8 type B receptor (IL8RB) which are critical for binding the ligands interleukin-8, NAP-2 and GRO alpha. Peptides corresponding to the N-terminus region and the first extracellular loop of the receptor demonstrated statistically significant (p = 0.001) inhibition of IL-8 control binding levels (inhibition levels of 73.0 +/- 5.1% and 89.9 +/- 2.2% respectively). In contrast, NAP-2 binding was inhibited only by the peptide representing the first extracellular loop (63.2 +/- 2.3%), while GRO alpha binding was inhibited by portions of the N-terminus (49.7 +/- 14.9% and 41.8 +/- 14.9%), but not the first extracellular loop. We suggest that: a) the chemokine receptor IL8RB, known to bind three related ligands with high affinity, seems to do so via distinct contact points and b.) the first extracellular loop is significant in the binding event.

Subtle differences between human and rabbit neutrophil receptors shown by the secretagogue activity of constrained formyl peptides.

Stereochemically constrained extended beta-antiparallel and folded beta-turn analogs of the chemotactic agent N-formyl-Met-Leu-Phe-OH were tested for their ability to induce the release of beta-glucuronidase from human and rabbit neutrophils. Selected biologically active peptides were further examined for their capacity to inhibit the binding of f-Met-Leu-[3H]Phe to whole human neutrophils at 4 degrees C. The results suggest that Dpg2 analogs with the extended backbone are significantly more potent in human peripheral blood neutrophils than the folded beta-turn analogs. Surprisingly, in rabbit peritoneal neutrophils, the extended Dpg2 analog appears to be marginally less active than the flexible parent peptide and the folded Ac6c2 analog. In human neutrophils, the secretagogue activity increases in the following order with alteration in the C-terminal functions: -CONH2 < -COOMe < -COOH << -COOBzl. However, this order of potency differs from that observed for the rabbit formyl peptide receptor (-COOH < -COOMe < -CONH2 << -COOBzl). In human neutrophils, the peptides' ability to compete for the receptor binding site of f-Met-Leu-[3H]Phe correlates well with their secretagogue potency. The results provide convincing evidence for the existence of subtle differences between human peripheral blood neutrophils and rabbit peritoneal neutrophils with regard to ligand-receptor interactions of constrained chemotactic peptides. What is new and novel in this report is that constrained peptides can distinguish between the rabbit and human chemotactic peptide receptors which have so far been believed to have similar response to secretagogue agents. The data emphasize that directly relating the secretagogue activity observed in rabbit neutrophils to that observed in human neutrophils may not be unequivocal.

Quantitative approach for detection and characterization of formyl peptide receptor in solution using a photoaffinity ligand.

In this paper we describe an assay method for the rapid detection and quantitation of human neutrophil FMLP (N-formyl-methionyl-leucyl-phenylalanine) receptor molecules. Although different assay methods are available to detect the receptor, none are rapid and at the same time allow quantitative detection of the binding affinity of the receptor molecule in solution. In our modified method, following a binding reaction using photoaffinity ligand, the amount of labeled ligand bound to the receptor is separated from the unbound one, thereby determining the binding affinity of the receptor protein. This simple procedure not only makes it possible to detect the recombinant FMLP receptor protein very rapidly, but also provides quantitative assessment of binding. This technique therefore allows a partial characterization (identification, determination of size and assessment of binding affinity) of receptor molecule in solution in less than 24 hours.

Serum cotinine levels, smoking, and periodontal attachment loss.

Cigarette smoking and tobacco use have been the subjects of numerous studies for many years. Smoking has also been associated with periodontal disease. However, no relationship between a reliable biochemical marker and increased severity of the periodontal condition has yet been described. It was thus the aim of this study to apply the measurement of cotinine, the major metabolite of nicotine, as a quantitative method to assess levels of smoking, and to correlate serum levels of cotinine with severity of periodontal disease. The degree of association between smoking and periodontal attachment loss was investigated in a study including 79 patients 25 to 64 years old suffering from periodontitis. Patients were examined and the following parameters recorded: Gingival Assessment (GA), Probing Pocket Depth (PPD), Clinical Attachment Level (CAL), and Bone Crest Height (BCH). In addition, self-reported histories of tobacco use as well as blood samples for quantitative analysis of serum levels of cotinine were taken. The serum samples were analyzed for cotinine content by means of a competitive-inhibition ELISA technique. The differences in mean cotinine levels were statistically significant (p = 0.0001) between smokers and non-smokers, showing no overlap between the groups. Severity of periodontal attachment loss was positively correlated with serum levels of cotinine for both measures of periodontal disease (CAL p = 0.005; BCH p = 0.008). Results from the present study indicate that serum cotinine levels used as a biochemical marker of smoking status are correlated with severity of periodontal attachment loss.

Identification of linear antigenic sites on the Porphyromonas gingivalis 43-kDa fimbrillin subunit.

The fimbrillin of Porphyromonas gingivalis is thought to be an important virulence factor that mediates adherence to host surfaces. The linear immunogenic and antigenic structure of P. gingivalis fimbrillin was investigated with synthetic peptides corresponding to the amino acid sequence predicted from the cloned fimbrillin gene for P. gingivalis 2561. A series of continuous and overlapping peptides corresponding to the entire sequence of P. gingivalis fimbrillin was used to immunize Wistar rats. The resulting polyclonal antibodies were used to test the antigenicity of the 43-kDa fimbrillin protein by enzyme-linked immunosorbent assay and Western blot analysis. All the peptides elicited specific antibodies directed to the corresponding peptides but differed in their ability to elicit antisera that cross-reacted with either native or denatured fimbrillin. Antisera to various C-terminal one-third peptides were more reactive to the denatured monomeric form of fimbrillin by Western blot analysis. Antisera to peptide 99-110 was by far the most reactive against the native form of the oligomeric fimbrillin as well as the partially denatured oligomeric form of fimbrillin. The results indicate that amino acid residues 99-110 on the native fimbrillin protein are accessible to antibody binding and that the immunogen 99-110, when conjugated to thyroglobulin, is able to mimic an epitope on the 43-kDa fimbrillin.

Structural and functional characterization of the human formyl peptide receptor ligand-binding region.

The formyl peptide (N-formyl-1-methionyl-1-leucyl-1-phenylalanine [FMLP]) receptor is involved in the activation of neutrophils and their subsequent response to chemotactic N-formylated peptides. Recently, we found that the first extracellular loop closest to the N-terminal end of the FMLP receptor exhibited the strongest ligand binding compared with that shown by other extracellular regions. By constructing amino acid substitutional variants of this domain, we have determined that residues Arg-84 and Lys-85 on this loop play major roles in ligand-binding activity. Furthermore, random rearrangement of the residues of this receptor region demonstrated that the position of these charged amino acids did not affect their involvement in ligand binding, although their presence was essential for this binding to occur. We propose that the portion of the first N-terminal extracellular loop of the FMLP receptor containing residues Arg-84 and Lys-85 contributes significantly to the active site in ligand-receptor binding. We further propose that this binding is not dependent on defined structure but rather that these charged moieties may function as important "contacts" in receptor-ligand interactions.

Porphyromonas gingivalis trypsin-like protease: a possible natural ligand for the neutrophil formyl peptide receptor.

Porphyromonous gingivalis is a periodontopathic Gram-negative anaerobe associated with chronic adult periodontitis. P. gingivalis proteases are considered important virulence factors in the pathogenesis of periodontal diseases. In addition, defective bactericidal activity of neutrophils has also been observed in periodontitis. In this report we describe the effects of trypsin-like protease(s) secreted from P. gingivalis cells on the ligand binding of FMLP receptor on neutrophils. It was observed that trypsin-like protease(s) from P. gingivalis stimulate neutrophils by means of superoxide anion production. Subsequently, the proteases were found to cleave the FMLP receptor protein as evident by direct labeling of the FMLP receptor molecule. These results suggest that trypsin-like protease(s) secreted from P. gingivalis cells contribute to attenuate the bactericidal activity of neutrophils by cleaving the polypeptide chain of the FMLP receptor molecule. The finding that neutrophils after the incubation with P. gingivalis released protease preparation fail to respond to further stimulation by FMLP suggests that P. gingivalis trypsin-like protease(s) may be a possible ligand for the FMLP receptor.

Recombinant expression and partial characterization of the human formyl peptide receptor.

FMLP-receptor DNA was expressed in Escherichia coli. The expressed product could specifically bind FMLP. This is the first-reported expression of a functional FMLP receptor in Escherichia coli. We confirm that receptor glycosylation is not essential for ligand binding. A deletion mutant did not bind FMLP, suggesting that the deleted portion plays a role in ligand binding.