Porphyromonas gingivalis produces arginine-specific cysteine proteinase (Arg-gingipain, RGP) and lysinespecific cysteine proteinase (Lys-gingipain, KGP) in the extracellular and cell-associated forms. Two separate genes (rgpA and rgpB) and a single gene (kgp) have been found to encode RGP and KGP, respectively. We constructed rgpA rgpB kgp triple mutants by homologous recombination with cloned rgp and kgp DNA interrupted by drug resistance gene markers. The triple mutants showed no RGP or KGP activity in either cell extracts or culture supernatants. The culture supernatants of the triple mutants grown in a rich medium had no proteolytic activity toward bovine serum albumin or gelatin derived from human type I collagen. Moreover, the mutants did not grow in a defined medium containing bovine serum albumin as the sole carbon/energy source. These results indicate that the proteolytic activity of P. gingivalis toward bovine serum albumin and gelatin derived from human type I collagen appears to be attributable to RGP and KGP. The hemagglutinin gene hagA of P. gingivalis possesses the adhesin domain regions responsible for hemagglutination and hemoglobin binding that are also located in the Cterminal regions of rgpA and kgp. A rgpA kgp hagA triple mutant constructed in this study exhibited no hemagglutination using sheep erythrocytes or hemoglobin binding activity, as determined by a solid-phase binding assay with horseradish peroxidase-conjugated human hemoglobin, indicating that the adhesin domains seem to be particularly important for P. gingivalis cells to agglutinate erythrocytes and bind hemoglobin, leading to heme acquisition.

We recently reported that circular RNA is efficiently translated by a rolling circle amplification (RCA) mechanism in a cell-free Escherichia coli translation system. Recent studies have shown that circular RNAs composed of exonic sequences are abundant in human cells. However, whether these circular RNAs can be translated into proteins within cells remains unclear. In this study, we prepared circular RNAs with an infinite open reading frame and tested their translation in eukaryotic systems. Circular RNAs were translated into long proteins in rabbit reticulocyte lysate in the absence of any particular element for internal ribosome entry, a poly-A tail, or a cap structure. The translation systems in eukaryote can accept much simpler RNA as a template for protein synthesis by cyclisation. Here, we demonstrated that the circular RNA is efficiently translated in living human cells to produce abundant protein product by RCA mechanism. These findings suggest that translation of exonic circular RNAs present in human cells is more probable than previously thought.

Arg-gingipain (RGP) is an Arg-X-specific cysteine proteinase produced by the Gram-negative anaerobe Porphyromonas gingivalis and has been shown to be a potent virulence factor in progressive periodontal disease (Nakayama, K., Kadowaki, T., Okamoto, K., and Yamamoto, K. (1995) J. Biol. Chem. 270, 23619 -23626). In this study, we provide evidence that RGP acts as a major processing enzyme for various cell surface and secretory proteins in P. gingivalis. Fimbrilin, a major component of fimbriae, remained in the precursor form in the RGP-null mutant. Prefimbrilin expressed in Escherichia coli was converted to the mature fimbrilin in vitro when incubated with purified RGP, but its conversion was suppressed by potent RGP inhibitors. The results were consistent with the electron microscopic observation indicating little or no fimbriation in the RGP-null mutant. The immunogenic 75-kDa cell surface protein was also shown to retain its proform in the RGP-null mutant. In addition, Lys-gingipain (KGP) was found to be abnormally processed in the RGP-null mutant. In contrast, both prefimbrilin and the 75-kDa protein precursor were processed to their respective mature forms in the KGP-null mutant, suggesting that KGP is not involved in the normal processing mechanisms of these proteins. These results suggest that RGP not only acts as a direct virulence factor but also makes a significant contribution as a major processing enzyme to the virulence of P. gingivalis.Porphyromonas gingivalis has been frequently isolated from subgingival lesions of patients with progressive periodontal disease and is believed to be one of the major causative agents of the disease (1). This bacterium is known to produce a novel class of cysteine proteinases, now termed Arg-gingipain (RGP) 1 and Lys-gingipain (KGP) on the basis of the peptide cleavage specificity at arginine and lysine residues, respectively, which are responsible for the trypsin-like activity of P. gingivalis (2). Recent studies revealed that the proteolytic activities of both enzymes were closely associated with the virulence of the organism in terms of the proteolytic destruction of host connective-tissue proteins (3, 4) and the disruption of normal host defense mechanisms (3, 5-11).So far, several genes encoding Arg-X-(12-17) and Lys-Xspecific cysteine proteinases (18 -20) have been cloned and sequenced from various P. gingivalis strains. Our recent studies have revealed that two separate RGP-encoding genes (rgpA and rgpB) (21) and a single KGP-encoding gene (kgp) (18) are located on the chromosome of P. gingivalis. By analyses with the RGP-null (rgpA rgpB double) mutant and the KGP-null mutant, both RGP and KGP have been shown to act as major virulence factors of P. gingivalis in the progression of periodontal disease.The bacterial colonization on gingival tissues that precedes bacterial penetration and tissue destruction is critical in the pathogenic process of periodontal disease. A variety of cell surface structures, including fimbriae, hemagglutinins, and lipopolysaccharides,...

The oral anaerobic bacterium Porphyromonas gingivalis, a major pathogen of advanced adult periodontitis, produces a novel class of cysteine proteinases in both cell-associated and secretory forms. A lysine-specific cysteine proteinase (Lys-gingipain, KGP), as well as an arginine-specific cysteine proteinase (Arg-gingipain), is a major trypsin-like proteinase of the organism. Recent studies indicate that the secreted KGP is implicated in the destruction of periodontal tissue and the disruption of host defense mechanisms. In this study, we have constructed a KGP-deficient mutant to determine whether the cell-associated KGP is important for pathophysiology of the organism. Although the mutant retained the strong ability to disrupt the bactericidal activity of polymorphonuclear leukocytes, its hemagglutination activity was reduced to about one-half that observed with the wild-type strain. More important, the mutant did not form black-pigmented colonies on blood agar plates, indicating the defect of hemoglobin adsorption and heme accumulation. Immunoblot analysis showed that the expression of a 19-kDa hemoglobin receptor protein, which is thought to be responsible for hemoglobin binding by the organism, was greatly retarded in this mutant. The mutant also showed a marked decrease in the ability to degrade fibrinogen. These results suggest the possible involvement of KGP in the hemoglobin binding and heme accumulation of the organism and in the bleeding tendency in periodontal pockets.

Porphyromonas gingivalis, one of the major causative agents of periodontal diseases, produces large amounts of arginine- and lysine-specific cysteine proteinases in cell-associated and secretory forms, which are now referred to as Arg-gingipain (Rgp) and Lys-gingipain (Kgp), respectively. A number of studies have revealed that these proteinases are closely associated with the periodontopathogenesis of this bacterium: destruction of periodontal connective tissues, disruption of host defense mechanisms, and development and maintenance of inflammation in periodontal pockets. With respect to the physiology of the bacterium, Rgp and Kgp are indispensable for it to obtain nutrients from the environment, since it cannot utilize saccharides as carbon/energy sources for growth and totally depends on peptides and amino acids that are provided from environmental proteins by Rgp and Kgp. Furthermore, proteolytic activities of Rgp and Kgp contribute to processing/maturation of various cell-surface proteins of P. gingivalis, such as fimA fimbrilin (a subunit of major fimbriae), 75-kDa protein (a subunit of minor fimbriae), hemagglutinins, and the hemoglobin receptor protein, which are important for the bacterium to colonize and proliferate in the gingival crevice and to invade the periodontium. These findings strongly indicate critical roles of Rgp and Kgp in the virulence of P. gingivalis.

Arg-gingipain (Rgp) and Lys-gingipain (Kgp) are cysteine proteinases produced by Porphyromonas gingivalis, a major etiological bacterium of periodontal diseases. Here we show a series of small peptide analogs able to inhibit either Rgp or Kgp, which are synthesized on the basis of the cleavage site specificity of human salivary histatins by each enzyme. Among this series of compounds, carbobenzoxy-Lys-Arg-CO-Lys-N-(CH 3 ) 2 (KYT-1) and carbobenzoxy-Glu(NHN(CH 3 )Ph)-Lys-CO-NHCH 2 Ph (KYT-36) were found to be the most potent inhibitors of Rgp and Kgp, respectively, with K i values of 10 Ϫ11 to 10 Ϫ10 M order. Both inhibitors exhibited slight or no inhibition on mammalian proteinases such as trypsin and cathepsins B, L, and H. All of the virulence induced by the culture supernatant of P. gingivalis tested, including the degradation of various host proteins such as human type I collagen, immunoglobulins, fibronectin, and fibrinogen, disruption of the bactericidal activity of polymorphonuclear leukocytes, and enhancement of the vascular permeability, were strongly inhibited by a combined action of both inhibitors. The functions essential for the bacterium to grow and survive in the periodontal pocket, such as coaggregation and acquisition of amino acids, were also strongly inhibited by the combined action of both inhibitors. The disruption of the adhesion and viability of human fibroblasts and hemagglutination by the organism were strongly suppressed by a single use of KYT-1. These results thus indicate that the newly developed KYT-1 and KYT-36 both should provide a broader application in studies of this important class of enzymes and facilitate the development of new approaches to periodontal diseases.

RNA interference (RNAi) is a potent and highly specific genesilencing phenomenon that is initiated or triggered by doublestranded RNAs (dsRNAs). 1a Shortly after the development of RNAi, small interfering RNAs (siRNAs), which are 21 nucleotides in length with a 3′ nucleotide overhang, were shown to be very effective in mammalian cells. 1b Much effort has been dedicated to the application of siRNAs, both as biological tools and as therapeutic agents. 1c-e These approaches can be divided into two main classes. In the first, DNA vectors encoding the RNA polymerase II or III promoter are used to transcribe siRNAs or short hairpin RNAs (shRNAs) in mammalian cells. 1c-e In the second, chemically synthesized siRNA is introduced directly into cells. 1c-e Although the vector system is suitable for biological experiments, there are safety problems in clinical applications. 1c-e Currently, synthetic siRNA would be the method of choice for clinical purposes. 1e However, natural RNA strands are quickly degraded in biological fluids. 2a Chemically synthesized unnatural nucleotides have been developed and introduced into the siRNA strand. 1d For example, modification of the ribose moiety with a 2′-deoxy, 2′-O-methyl, or 2′-fluoro group or modification of the phosphate backbone has been examined. 1d Although these modifications improve the stability of siRNA in serum, they often cause a decrease in RNAi activity. 1d There is also concern that unnatural RNA derivatives are toxic in the human body. A method to stabilize nontoxic natural RNA strands should be very useful for applications in RNAi technology.DNA dumbbells, which consist of a double-helical stem closed by two hairpin loops, have been synthesized historically as physical models that can be used to analyze local structures in DNA. 3a,b They have more recently been applied successfully to transcriptional regulation, where they act as decoy systems because they exhibit increased stability against exonucleases and biological fluids without the need to introduce unnatural nucleotides. 3c-f A dumbbell-shaped RNA aptamer that contains two deoxynucleotides 4a and DNA/RNA chimera dumbbells for antisense applications 4b,c has been reported. Because 3′-exonuclease is a major enzyme involved in the degradation of nucleic acids in vivo, 2b RNA dumbbells are expected to be more stable than dsRNA. However, to our knowledge, this design has never been tested as an RNAi strategy. A key point to be determined is whether an RNA dumbbell is cleaved by the Dicer enzyme 5 to generate dsRNA, as shRNA is, 5c to achieve an RNAi effect in cells ( Figure 1).To test this idea, we designed and synthesized circular RNA dumbbells that contain a stem sequence encoding the firefly luciferase gene 1b and two 9-mer loops ( Figure 2a). The loop sequence is used widely in shRNA expression systems. 6 The stem sequences were designed according to a previous report, which showed that the expressed shRNA sequence worked well. 6b We added one more loop sequence on the other end of this shRNA to form dumbbel...

The oral anaerobic bacterium Porphyromonas gingivalis has been implicated as a major etiologic agent of progressive periodontal disease. A novel lysine-specific cysteine proteinase, termed "Lys-gingipain," was purified from the culture supernatant of the Arg-gingipain-deficient mutant of P. gingivalis (KDP112) by a simple method including immunoaffinity chromatography. The purified enzyme was found to be composed of a single polypeptide of Mr=51,000. Analysis of the enzymatic properties revealed several distinctive features of this enzyme. The proteolytic activity was remarkably activated by thiol-reducing agents and inhibited by idoacetamide, idoacetic acid, and leupeptin. The enzyme was also inhibited by the chloromethyl ketones of tosyl-L-lysine and tosyl-L-phenylalanine. However, internal protease inhibitors, such as cystatins and alpha1-antichymotrypsin, had no effect on the activity, suggesting its resistance to normal host defense systems in vivo. Despite its narrow specificity for synthetic substrates containing Lys in the P1 site, the enzyme extensively degraded human type I collagen and immunoglobulins G and A (both serum and secretory types). Most important, the enzyme was able to disrupt the functions of polymorphonuclear leukocytes, as shown by its inhibitory effect on the generation of active oxygen species from the activated cells. These results suggest that Lys-gingipain, like Arg-gingipain, plays a crucial role as a virulence factor from P. gingivalis in the development of periodontal disease via the direct destruction of periodontal tissue components and the disruption of normal host defense mechanisms.