| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1995: ¥300,000 (Direct Cost: ¥300,000)
Fiscal Year 1994: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1993: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Research Abstract |
Porphyromonas gingivalis (P.g.) is a gram-negative, anacrobic rod, which is believed to be a potential pathogen for an adult periodontitis. P.g. has a non-lectin-like hemagglutinin (HA) of which the activity is inhibited by arginine, protease inhibitors such as TLCK and leupeptin. The HA also has a cysteine protease activity (P) which cuts the bond next to an arginine residue. This bifunctional factor, called HA/P,was reported to be composed of a single kind of protein, the molecular weight of which is 44 kDa. HA/P has recently been thought to be one of the major tissue destructive agents. To help understand the structural relaitonship between HA and P and the mechanism for the regulation of these functions, we attempted to clone the structural gene of HA/P,named hap, recombine it into an expression vector, and express it in Escherichia coli. i) hap gene is more than 5 kbp long. We succeeded in cloning a 4 kbp-long DNA fragment. This fragment contained only 18 amino acid N-terminal por
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tion of HA/P.The downstream region of hap was further cloned by inverse PCR method. The orf continued more than 2 kbp and did not end. The nucleotide scquencing of the upstream region revealed the orf continuting about 2.3 kbp from the N-terminus of HA/P.The whole gene of hap was cloned by Pavloff et al. The sequence coincided with almost all of ours. ii) HA/P is a complex. iii) The subunits of the HA/P complex are encoded in one continuous orf. HA/P is thought to be matured by autocatalysis posttranslationally. iv) A cysteine protease called Arg-gingipain is encoded in the upstream region together with the sequences for its leader peptide and propeptide, that is, Arg-gingipain is translated in preproenzyme. v) HA is thought to be encoded in the downstream region, although there was no direct evidence. vi) We succeeded in expressing the N-terminal region of hap inserted in the pT7 expression vector in E.coli. The N-terminal amino acid sequence of the expressed peptide is QQTEL etc. Thus the initiation codon could not be determined experimentally. vii) Southern hybridization experiment with p ; arts of hap gene as a probe indicated that a material crossreacting with anti-HA/P (P.g.) antibody in P.endodontalis has a nucleotide sequence with low homology with that of hap in P.g. Less
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