| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2000: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1999: ¥2,200,000 (Direct Cost: ¥2,200,000)
|
|---|
| Research Abstract |
Helicobacter pylori, a Gram negative gastric bacterium, secretes VacA, a cytotoxin that causes vacuolar degeneration of susceptible cells. Exposure of VacA to acid or alkali stimulated its binding to AZ-521 cells. Immunoprecipitates of AZ-521 cells previously exposed to acid- and alkali-treated VacA with polyclonal antibodies against VacA include a 250-kDa glycoprotein, containing galactose-_(1-3)-N- acetylgalactosamine and galactose-_ (1-4)-N-acetylglucosamine. p250 purified by chromatography on peanut agglutinin (PNA) affinity- and Superose 6 columns contained N-terminal and internal amino acid sequences of YRQQRKLVEEIGWSYT and LIIQDEILEATQDDY, respectively. These sequences are identical to those of receptor protein tyrosine phosphatase (RPTPβ/PTPζ), and p250 reacted with antihuman RPTPβ monoclonal antibody. Immunoprecipitation studies with anti-human RPTPβ_antibody of solubilized membrane preparations incubated with VacA or heat-inactivated VacA indicated that RPTPβ bound native Vac
… More
A but not denatured VacA.These data suggest that acidic and alkaline treatments induce a molecular change in VacA that is associated with its activation and increased binding to RPTPβ on target cells.
To define whether RPTPβ serves as the VacA receptor, we investigated the relationship between induction of VacA sensitivity and RPTPβ expression in HL-60 cells treated with differentiation-promoting reagents. Phorbolmyristate (PMA, TPA), which induces differentiation of the human leukemic cell line HL-60 into cells with macrophage-like characteristics, enhanced the susceptibility of HL-60 cells to VacA and induced expression of RPTPβ mRNA and protein as determined by RT-PCR and indirect immunofluorescence studies. Vitamin D3 and IFN-γ, which are known to stimulate differentiation of HL-60 cells into monocyte-like cells, also induced VacA sensitivity and expression of RPTPβ mRNA, whereas 1.2% DMSO and retinoic acid, which stimulated the maturation of HL-60 into granulocyte-like cells were ineffective. Addition of RPTPβ anti-sense oligonucleotide with PMA specifically inhibited induction of VacA sensitivity as well as expression of RPTPβ. Double immunostaining studies also indicated that newly expressed RPTPβ colocalized with VacA in PMA-treated HL-60 cells.
Transfection of RPTPβ gene into hamster kidney cell line, BKN-21, which lacks VacA sensitivity, resulted in acquisition of VacA sensitivity by these cells. All data are consistent with the conclusion that acquisition of VacA sensitivity by PMA-treated HL-60 cells results from induction of RPTPβ, a protein that functions as the VacA receptor. Less
|
