Kgp DNA Vaccine Prevents Experimental Periodontitis.

PURPOSE: To investigate the prophylactic effect of lysine-specific protease (Kgp) vaccine on experimental periodontitis in mice. MATERIALS AND METHODS: We constructed the eukaryotic expression plasmid pVAX1-kgp and immunised mice with the recombinant plasmid. Mice were divided into two groups and immunised with pVAX1-kgp or pVAX1 three times at 2-week intervals. Immunoglobulin (Ig)G, IgG1 and IgG2a antibodies were detected by enzyme-linked immunosorbent assay (ELISA) before and after immunisation. At the last immunisation, a silk ligature infiltrated with Porphyromonas gingivalis (P. gingivalis) was tied at the neck of the maxillary second molar to induce experimental periodontitis. Each group was euthanised after 10 days, and microcomputed tomography (micro-CT) and hematoxylin-eosin (HE) staining were used to detect the loss of alveolar bone. RESULTS: Comparison with the pVAX1 group indicated that mice immunised with Kgp had higher levels of IgG (P < 0.05); the levels of the IgG1 were statistically significantly different (p < 0.05), and the levels of the IgG2a subtype were not significantly different. The results of micro-CT and HE staining showed that the alveolar bone loss in the pVAX1-kgp group was statistically significantly less than that in the pVAX1 group (p < 0.05). The expression of the related inflammatory factors, including interleukin-1ß (IL-ß), tumour necrosis factor (TNF-α) and interleukin-6 (IL-6), was lower in the pVAX1-kgp group than in the pVAX1 group. CONCLUSION: The Kgp DNA vaccine can enhance IgG levels in a model of experimental periodontitis, effectively activate immunity, and mitigate alveolar bone loss.

An efficient and concise synthesis of a selective small molecule non-peptide inhibitor of cathepsin L: KGP94.

KGP94 is a potent, selective, and competitive inhibitor of the lysosomal endopeptidase enzyme (Cathepsin L) currently in preclinical trials for the treatment of metastatic cancer, which is a leading cause of cancer-associated death. Herein, we report two new synthetic routes for synthesizing the target compound through four consecutive steps, using a Weinreb amide approach starting from a common 3-bromobenzoyl chloride. A key step in the approach is a coupling reaction of a readily available Grignard reagent with amide 4 to produce 6, a previously unreported coupling pattern. These new strategies offer an efficient and alternative approach to synthesis of target compound with an excellent overall yield.

Expanded CAG Repeats in ATXN1, ATXN2, ATXN3, and HTT in the 1000 Genomes Project.

BACKGROUND: Spinocerebellar ataxia types 1, 2, 3 and Huntington disease are neurodegenerative disorders caused by expanded CAG repeats. METHODS: We performed an in-silico analysis of CAG repeats in ATXN1, ATXN2, ATXN3, and HTT using 30× whole-=genome sequencing data of 2504 samples from the 1000 Genomes Project. RESULTS: Seven HTT-positive, 3 ATXN2-positive, 1 ATXN3-positive, and 6 possibly ATXN1-positive samples were identified. No correlation was found between the repeat sizes of the different genes. The distribution of CAG alleles varied by ethnicity. CONCLUSION: Our results suggest that there may be asymptomatic small expanded repeats in almost 0.5% of these populations. © 2020 International Parkinson and Movement Disorder Society.

Enzymatic Characteristics and Activities of Gingipains from Porphyromonas gingivalis.

Porphyromonas gingivalis is a gram-negative, rod-shaped, nonmotile bacterium belonging to the phylum Bacteroidetes. It produces abundant amounts of proteases in both cell-associated and secretory forms, including a group of cysteine proteases referred to as gingipains, which have attracted much attention due to their high proteolytic activity associated with pathogenicity. Gingipains are grouped into arginine (R)-specific (RgpA and RgpB) and lysine (K)-specific (Kgp) types. Both Rgp (collective term for RgpA and RgpB) and Kgp gingipains play crucial roles in the virulence of P. gingivalis, including the degradation of host periodontal tissues, disruption of host defense mechanisms, and loss of viability in host cells, such as fibroblasts and endothelial cells. In addition to their function in virulence, gingipains are also essential for the growth and survival of P. gingivalis in periodontal pockets through the acquisition of amino acids and heme groups. Furthermore, Rgp and Kgp gingipains are critical in processing fimbriae and several bacterial proteins that contribute to hemagglutination, coaggregation, and hemoglobin binding. This chapter describes the methods used to analyze gingipains.

Proteoform-Resolved FcɤRIIIa Binding Assay for Fab Glycosylated Monoclonal Antibodies Achieved by Affinity Chromatography Mass Spectrometry of Fc Moieties.

Fcɤ receptors (FcɤR) mediate key functions in immunological responses. For instance, FcɤRIIIa is involved in antibody-dependent cell-mediated cytotoxicity (ADCC). FcɤRIIIa interacts with the fragment crystallizable (Fc) of immunoglobulin G (IgG). This interaction is known to be highly dependent on IgG Fc glycosylation. Thus, the impact of glycosylation features on this interaction has been investigated in several studies by numerous analytical and biochemical techniques. FcɤRIIIa affinity chromatography (AC) hyphenated to mass spectrometry (MS) is a powerful tool to address co-occurring Fc glycosylation heterogeneity of monoclonal antibodies (mAbs). However, MS analysis of mAbs at the intact level may provide limited proteoform resolution, for example, when additional heterogeneity is present, such as antigen-binding fragment (Fab) glycosylation. Therefore, we investigated middle-up approaches to remove the Fab and performed AC-MS on the IgG Fc to evaluate its utility for FcɤRIIIa affinity assessment compared to intact IgG analysis. We found the protease Kgp to be particularly suitable for a middle-up FcɤRIIIa AC-MS workflow as demonstrated for the Fab glycosylated cetuximab. The complexity of the mass spectra of Kgp digested cetuximab was significantly reduced compared to the intact level while affinity was fully retained. This enabled a reliable assignment and relative quantitation of Fc glycoforms in FcɤRIIIa AC-MS. In conclusion, our workflow allows a functional separation of differentially glycosylated IgG Fc. Consequently, applicability of FcɤRIIIa AC-MS is extended to Fab glycosylated IgG, i.e., cetuximab, by significantly reducing ambiguities in glycoform assignment vs. intact analysis.

Spinocerebellar ataxia type 10 (SCA10): Mutation analysis and common haplotype based inference suggest its rarity in Indian population.

Spinocerebellar ataxia type 10 (SCA10) is a rare autosomal dominant cerebellar ataxia caused by nucleotide ATTCT expansion in ATXN10 gene. SCA10 has been reported in patients of cerebellar ataxia from Amerindian/Latin America and in East Asian ancestry. A common founder has been ascribed to the origin of ATTCT repeat expansion mutation in both the population. Here we present our investigation of the SCA10 pentanucleotide repeat expansion in 461 SCA patients of the Indian population. The analysis of multi-ethnic at-risk haplotype C-(ATTCT)n-GGC was performed using genotype data of various ethnic population included in the 1000 Genomes Project (KGP) to infer the prevalence of at-risk haplotype in the Indian populations. Unsurprisingly, none of the patient's DNA samples with (ATTCT)n expansion was observed in pathological range, however, the observed normal range of (ATTCT)n was 8-22 repeats, suggesting very rare or absence of the occurrence of SCA10 in Indian SCA patients. The at-risk haplotype, CGGC was found to be the most prevalent haplotype across different populations and no segregation of CGGC haplotype with large normal or small normal ATTCT repeats length was observed. However, on extended haplotype analysis, some lineage of CGGC with a flanking divergence at 5' end was observed specifically in the American or East Asian population but not in other population in KGP dataset. Together, these evidence points towards the absence of SCA10 in Indian population and haplotype-based analysis also suggests its occurrence to be rare in South Asian, European and African population. Further investigations are required to establish the present finding. SIGNIFICANCE: The implications of the findings of this study are 1.) For the diagnostic work-up of SCAs in the Indian population and to decide upon inclusion of SCA10 in panel based genetic investigations even for Indians living abroad. 2.) The haplotype based inference of its presumptive prevalence through the estimation of at-risk haplotype using population genetics approach (South-Asians as the background) allowed us to estimate the possible absence of SCA10 in Indian population. SCA10 is a rare autosomal dominant cerebellar ataxia mostly reported among SCA patients from Latin America and recently described in East Asia population. The genetic study of SCA10 performed in the unrelated Indian spinocerebellar ataxia patients with heterogeneous ethnicity confirmed its absence from the Indian population and that conforms to population genetic based inference of its rarity or absence. 3.) This approach may be adopted for the screening of other subtypes of SCAs, i.e. other rare SCAs e.g. SCA31, SCA36, and SCA37.

Generating and Purifying Fab Fragments from Human and Mouse IgG Using the Bacterial Enzymes IdeS, SpeB and Kgp.

Fab fragments are valuable research tools in various areas of science including applications in imaging, binding studies, removal of Fc-mediated effector functions, mass spectrometry, infection biology, and many others. The enzymatic tools for the generation of Fab fragments have been discovered through basic research within the field of molecular bacterial pathogenesis. Today, these enzymes are widely applied as research tools and in this chapter, we describe methodologies based on bacterial enzymes to generate Fab fragments from both human and mouse IgG. For all human IgG subclasses, the IdeS enzyme from Streptococcus pyogenes has been applied to generate F(ab')2 fragments that subsequently can be reduced under mild conditions to generate a homogenous pool of Fab' fragments. The enzyme Kgp from Porphyromonas gingivalis has been applied to generate intact Fab fragments from human IgG1 and the Fab fragments can be purified using a CH1-specific affinity resin. The SpeB protease, also from S. pyogenes, is able to digest mouse IgGs and has been applied to digest antibodies and Fab fragments can be purified on light chain affinity resins. In this chapter, we describe methodologies that can be used to obtain Fab fragments from human and mouse IgG using bacterial proteases.

Cathepsin L in tumor angiogenesis and its therapeutic intervention by the small molecule inhibitor KGP94.

A significant proportion of breast cancer patients harbor clinically undetectable micrometastases at the time of diagnosis. If left untreated, these micro-metastases may lead to disease relapse and possibly death. Hence, there is significant interest in the development of novel anti-metastatic agents that could also curb the growth of pre-established micrometastases. Like primary tumor, the growth of metastases also is driven by angiogenesis. Although the role of cysteine protease Cathepsin L (CTSL) in metastasis associated tumor cell functions such as migration and invasion is well recognized, its role in tumor angiogenesis remains less explored. The present study examines the contribution of CTSL to breast cancer angiogenesis and evaluates the anti-angiogenic efficacy of CTSL inhibitor KGP94. CTSL semi-quantitative RT-PCR analysis on breast tissue panels revealed significant upregulation of CTSL in breast cancer patients which strongly correlated with increased relapse and metastatic incidence and poor overall survival. Preclinically, CTSL ablation using shRNA or KGP94 treatment led to a significant reduction in MDA-MB-231 tumor cell induced angiogenesis in vivo. In-vitro assessments demonstrated a significant decrease in various angiogenic properties such as endothelial cell sprouting, migration, invasion, tube formation and proliferation in the presence of KGP94. Microarray analyses revealed a significant upregulation of cell cycle related genes by CTSL. Western blot analyses further confirmed upregulation of members of the cyclin family by CTSL. Collectively, these data indicate that CTSL is an important contributor to tumor angiogenesis and that the CTSL inhibition may have therapeutic utility in the treatment of breast cancer patients.

Cathepsin L inactivation leads to multimodal inhibition of prostate cancer cell dissemination in a preclinical bone metastasis model.

It is estimated that approximately 90% of patients with advanced prostate cancer develop bone metastases; an occurrence that results in a substantial reduction in the quality of life and a drastic worsening of prognosis. The development of novel therapeutic strategies that impair the metastatic process and associated skeletal adversities is therefore critical to improving prostate cancer patient survival. Recognition of the importance of Cathepsin L (CTSL) to metastatic dissemination of cancer cells has led to the development of several CTSL inhibition strategies. The present investigation employed intra-cardiac injection of human PC-3ML prostate cancer cells into nude mice to examine tumor cell dissemination in a preclinical bone metastasis model. CTSL knockdown confirmed the validity of targeting this protease and subsequent intervention studies with the small molecule CTSL inhibitor KGP94 resulted in a significant reduction in metastatic tumor burden in the bone and an improvement in overall survival. CTSL inhibition by KGP94 also led to a significant impairment of tumor initiated angiogenesis. Furthermore, KGP94 treatment decreased osteoclast formation and bone resorptive function, thus, perturbing the reciprocal interactions between tumor cells and osteoclasts within the bone microenvironment which typically result in bone loss and aggressive growth of metastases. These functional effects were accompanied by a significant downregulation of NFκB signaling activity and expression of osteoclastogenesis related NFκB target genes. Collectively, these data indicate that the CTSL inhibitor KGP94 has the potential to alleviate metastatic disease progression and associated skeletal morbidities and hence may have utility in the treatment of advanced prostate cancer patients.

Exopeptidases and gingipains in Porphyromonas gingivalis as prerequisites for its amino acid metabolism.

Porphyromonas gingivalis, an asaccharolytic bacterium, utilizes amino acids as energy and carbon sources. Since amino acids are incorporated into the bacterial cells mainly as di- and tri-peptides, exopeptidases including dipeptidyl-peptidase (DPP) and tripeptidyl-peptidase are considered to be prerequisite components for their metabolism. We recently discovered DPP11, DPP5, and acylpeptidyl oligopeptidase in addition to previously reported DPP4, DPP7, and prolyl tripeptidyl peptidase A. DPP11 is a novel enzyme specific for acidic P1 residues (Asp and Glu) and distributed ubiquitously in eubacteria, while DPP5 is preferential for the hydrophobic P1 residue and the first entity identified in prokaryotes. Recently, acylpeptidyl oligopeptidase with a preference for hydrophobic P1 residues was found to release N-terminally blocked di- and tri-peptides. Furthermore, we also demonstrated that gingipains R and K contribute to P1-basic dipeptide production. These observations implicate that most, if not all, combinations of di- and tri-peptides are produced from extracellular oligopeptides even with an N-terminal modification. Here, we review P. gingivalis exopeptidases mainly in regard to their enzymatic characteristics. These exopeptidases with various substrate specificities benefit P. gingivalis for obtaining energy and carbon sources from the nutritionally limited subgingival environment.

Structure of the lysine specific protease Kgp from Porphyromonas gingivalis, a target for improved oral health.

The oral pathogen Porphyromonas gingivalis is a keystone pathogen in the development of chronic periodontitis. Gingipains, the principle virulence factors of P. gingivalis are multidomain, cell-surface proteins containing a cysteine protease domain. The lysine specific gingipain, Kgp, is a critical virulence factor of P. gingivalis. We have determined the X-ray crystal structure of the lysine-specific protease domain of Kgp to 1.6 Å resolution. The structure provides insights into the mechanism of substrate specificity and catalysis.

The profile of Porphyromonas gingivalis kgp biotype and fimA genotype mosaic in subgingival plaque samples.

Combined analysis of allelic variation of the virulence-associated, strain-specific lys-gingipain gene (kgp) and major fimbrial gene (fimA) of Porphyromonas gingivalis was undertaken in 116 subgingival plaque samples to understand the kgp biotype and fimA genotype profile in a subject-specific manner. Allelic variation in the polyadhesin domain of kgp from P. gingivalis strains 381 (ATCC 33277), HG66 and W83 generated four isoforms corresponding to four biotypes of P. gingivalis. Similarly, variation in the fimA subunit of the fimA gene cluster of P. gingivalis resulted in six fimA genotypes. Strain-specific differential PCR was performed for kgp and fimA using DNA isolated from subgingival plaque samples. Our findings demonstrate that all of the P. gingivalis kgp biotypes detected in this study were predominantly associated with the fimA II genotype. Dominance of kgp biotypes 381 or HG66 combined with fimA II fimbriae could imply an adaptive strategy by P. gingivalis to generate the fittest strains for survival in the host environment.

kgp, rgpA, and rgpB DNA vaccines induce antibody responses in experimental peri-implantitis.

BACKGROUND: Peri-implantitis is the key factor for implant failure. This study aims to evaluate kgp, rgpA, and rgpB DNA vaccines to induce an immune response and prevent peri-implantitis. METHODS: The kgp, rgpA, and rgpB genes were amplified by polymerase chain reaction (PCR) from Porphyromonas gingivalis (Pg) ATCC 33277 and cloned into the pVAX1 vector. Titanium implants were placed into the mandibular bone of dogs. Three months later, the animals were divided into four groups, immunized with pVAX1-kgp, pVAX1-rgpA, pVAX1-rgpB, or pVAX1. Cotton ligatures infiltrated with Pg were tied around the neck of the implants. Immunoglobulin (Ig)G and IgA antibodies were detected by enzyme-linked immunosorbent assay before and after immunization. RESULTS: The kgp, rgpA, and rgpB genes were successfully cloned into the pVAX1 plasmid. Animals immunized with pVAX1-kgp and pVAX1-rgpA showed higher titers of IgG and IgA antibodies compared to those before immunization (P <0.05) and compared to those that were immunized with pVAX1 and pVAX1-rgpB, whereas there were no significant differences in the animals treated with pVAX1 and pVAX1-rgpB. Furthermore, among these, the kgp DNA vaccine was more effective. The bone losses of the groups with pVAX1-kgp and pVAX1-rgpA were significantly attenuated. CONCLUSION: pVAX1-kgp and pVAX1-rgpA DNA vaccines enhanced immunity responses and significantly retarded bone loss in experimental peri-implantitis animal models, whereas pVAX1-rgpB was ineffective.

Genotipificación de los genes rgpA y kgp que codifican para las gingipaínas de Porphyromonas gingivalis / Genotyping of rgpA and kgp genes encoding Pophyromonas gingivalis gingipains

Porphyromonas gingivalis es un microorganismo fuertemente asociado con la etiología de la periodontitis. Esta bacteria posee varios factores de virulencia, dentro de los que destacan las gingipaínas, debido a sus múltiples acciones relacionadas con la destrucción de la matriz extracelular del tejido conectivo periodontal, la modulación del sistema inmune del hospedero y la estimulación de la expresión de citoquinas pro-inflamatorias. Estas proteinasas tienen afinidades específicas siendo Arg-gingipaínas (RgpA y RgpB, codificadas por los genes rgpA y rgpB, respectivamente) y Lys-gingipaínas (Kgp, codificada por el gen kgp). Se ha descrito que existen polimorfismos en los genes que codifican para esta proteinasas. El objetivo del presente estudio fue describir la frecuencia de los genotipos identificados para los genes rgpA y kgp en aislados clínicos de P. gingivalis, obtenidos desde pacientes con periodontitis. Para ello se utilizó amplificación por PCR de los genes rgpA y kgp, seguido de análisis de restricción. De un total de 47 aislados provenientes de 4 individuos con periodontitis crónica y 2 con periodontitis agresiva, se genotipificaron 38 aislados para el gen rgpA, exhibiendo la totalidad de éstos el patrón electroforético A (100 por ciento). Para el gen kgp se genotipificaron 43 aislados, presentando 28 de ellos (65.2 por ciento) el perfil electroforético kgp-I y 15 aislados (34.8 por ciento) el perfil kgp-II. En los aislados provenientes de un individuo fue posible apreciar ambos genotipos descritos para el gen kgp. Los resultados indican un predominio del patrón electroforético A (rgpA) y que el genotipo kgp-I fue el más frecuentemente encontrado de los genotipos kgp.

Porphyromonas gingivalis is a microorganism strongly associated with the etiology of periodontitis. This periodontal bacterium possesses an array of virulence factors, among which gingipains have a key importance, being involved with extracellular matrix destruction of periodontal tissues, modulation of host immune response and stimulation in the production of pro-inflammatory cytokines by different types of cells. These proteinases have specific affinities, being Arg-gingipains (RgpA and RgpB, encoded by rgpA and rgpB genes, respectively) and Lys-gingipains (Kgp, encoded by the kgp gene). It has been described that there are polymorphisms in the genes encoding for gingipains. Therefore, the aim of the present study was to describe the frequency of rgpA and kgp genotypes in clinical isolates of P. gingivalis obtained from periodontitis patients. For determining the rgpA and kgp genotypes, we used PCR amplification and restriction analysis. From 47 isolates obtained from 4 individuals with chronic periodontitis and 2 subjects with aggressive periodontitis, 38 were typified for rgpA gene and all exhibited the electrophoretic pattern A (100 percent). For kgp gene, we characterized 43 isolates, 28 of them (65.2 percent) with the kgp-I electrophoretic profile and 15 isolates (34.8 percent) with the kgp-II profile. In the isolates belonging to one individual, we found both genotypes of kgp gene. The results indicate a clear predominance of the electrophoretic pattern A (for rgpA gene) and kgp-I genotype was the most frequently found of the kgp genotypes.

Expression of gingipain K in Porphyromonas gingivalis in the subgingival plaque of puberty gingivitis / 实用口腔医学杂志

Objective:To detect and compare the intensity of gingipain K(Kgp)in culture medium and cell extract of Porphyromonas gingivalis(P.gingivalis)isolates in puberty gingivitis,and then to reveal the possible relationship between Kgp and puberty gingivitis.Methods:36 patients with puberty gingivitis aged from 14 to 17 years were enrolled.Clinical parameters including GI,SBI and PD were evaluated before subgingival plaque samples collection.Subgingival plaque samples were collected and then P.gingivalis isolates were obtained.16S rRNA PCR was used to confirm the presence of P.gingivalis in clinical isolates.Bacteria were cultured in BHI agar base and harvested at the end of log-phase growth.Culture fractions of P.gingivalis(culture medium and cell extracts)were performed with SDS-PAGE and Western blot technique using primary antibody against specific anti-Kgp N-terminal IgG subdomain.The data were statistically analyzed using SPSS 11.5 software.The relationship between the Kgp intensity and the clinical parameters was statistically analyzed using sum rank test.Results:There was positive correlation between the intensity of Kgp N-terminal IgG subdomain and the clinical parameters(P