title: Design and synthesis of sensitive fluorogenic substrates specific for Lys-gingipain.
abstract: Lys-gingipain (Kgp) is a major cysteine proteinase produced by the oral anaerobic bacterium Porphyromonas gingivalis, and has been implicated as a major pathogen in the development and progression of advanced adult periodontitis. This enzyme is believed to act as a major virulence factor of the disease, yet there exist no convenient and sensitive substrates for analyzing its biological activity. For a better understanding of the importance of this enzyme in the organism, there is an urgent need for specific substrates. Here we designed and synthesized two peptide 4-methyl-coumaryl-7-amides (MCA), carbobenzoxy (Z)-His-Glu-Lys-MCA, and Z-Glu-Lys-MCA, and tested their possible use as sensitive substrates for Kgp with limited specificity. Both substrates exhibited greater k(cat)/K(m) values than the best known Kgp substrates described so far. Both substrates were resistant to Arg-gingipain, another pathogenic cysteine proteinase from P. gingivalis, as well as trypsin and cathepsins B, L, and H. The levels of Kgp in various microorganisms and human cells were determined with Z-His-Glu-Lys-MCA. Little or no Kgp-like activity was detected in either other microorganisms or human cells tested. These results indicate that the present substrates are a valuable and fast tool for routine assays and for mechanistic studies on Kgp.
authors.affiliation: Department of Pharmacology, Graduate School of Dental Science, Kyushu University, Higashi-ku, Fukuoka 812-8582, Japan.
authors.forename: N
authors.initials: N
authors.lastname: Abe
authors.forename: A
authors.initials: A
authors.lastname: Baba
authors.forename: T
authors.initials: T
authors.lastname: Kadowaki
authors.forename: K
authors.initials: K
authors.lastname: Okamoto
authors.forename: S
authors.initials: S
authors.lastname: Okazaki
authors.forename: T
authors.initials: T
authors.lastname: Asao
authors.forename: K
authors.initials: K
authors.lastname: Yamamoto
mesh_terms: D018829:Adhesins, Bacterial / Q000378:metabolism*; D000328:Adult; D000595:Amino Acid Sequence; D002384:Catalysis; D003374:Coumarins / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000378:metabolism; D000080867:Gingipain Cysteine Endopeptidases; D006388:Hemagglutinins / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis* / Q000378:metabolism; D016966:Porphyromonas gingivalis
chemical_list: D018829:Adhesins, Bacterial; D003374:Coumarins; D005456:Fluorescent Dyes; D000080867:Gingipain Cysteine Endopeptidases; D006388:Hemagglutinins; D009842:Oligopeptides; C419355:carbobenzoxy-glutamyl-lysyl-4-methyl-coumaryl-7-amide; C419356:carbobenzoxy-histidyl-glutamyl-lysyl-4-methyl-coumaryl-7-amide; C074332:tertiary butyloxycarbonylvalyl-leucyl-lysinyl-4-methylcoumarin-7-amide; D003546:Cysteine Endopeptidases