RECEPTOR OF HELICOBACTER PYLORI VACA TOXIN AND ITS SIGNAL FOR TOXIXITY

幽门螺杆菌是一种革兰氏阴性胃细菌，分泌VacA，一种引起易感细胞空泡变性的细胞毒素。VacA暴露于酸或碱刺激其与AZ-521细胞的结合。AZ-521细胞经酸、碱处理后，用VacA多克隆抗体免疫沉淀出250 kDa的糖蛋白，含有半乳糖-_（1-3）-N-乙酰半乳糖胺和半乳糖-_（1-4）-N-乙酰葡萄糖胺。通过花生凝集素（PNA）亲和柱和Superose 6柱层析纯化的p250分别含有YRQQRKLVEEIGWSYT和LIIQDEILEATQDDY的N-末端和内部氨基酸序列。这些序列与受体蛋白酪氨酸磷酸酶（RPTPβ/PTP β）的序列完全相同，p250与抗人RPTPβ单克隆抗体反应。用抗人RPTPβ_抗体免疫沉淀法研究了与VacA或热灭活VacA孵育的可溶性膜制品，结果表明RPTPβ与天然VacA结合 关于我们 为了确定RPTPβ是否作为VacA的受体，我们研究了诱导分化试剂处理HL-60细胞后VacA敏感性和RPTPβ表达之间的关系。RT PCR和间接免疫荧光研究表明，佛波醇酯（PMA，TPA）能诱导HL-60细胞分化为巨噬细胞样细胞，增强HL-60细胞对VacA的敏感性，诱导RPTPβ mRNA和蛋白的表达。维生素D3和IFN-γ可诱导HL-60细胞向单核细胞样细胞分化，但对VacA敏感性和RPTPβ mRNA表达无明显影响，而1.2%DMSO和视黄酸则不能诱导HL-60细胞向粒细胞样细胞分化。RPTPβ反义寡核苷酸与PMA共同作用可特异性抑制VacA敏感性的诱导及RPTPβ的表达。免疫双染显示，PMA处理的HL-60细胞中新表达的RPTPβ与VacA共定位，将RPTPβ基因转染缺乏VacA敏感性的仓鼠肾细胞系BKN-21，使其获得VacA敏感性。所有数据都与PMA处理的HL-60细胞获得VacA敏感性是由于诱导RPTPβ（一种作为VacA受体的蛋白质）的结论一致。少

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