RECEPTOR OF HELICOBACTER PYLORI VACA TOXIN AND ITS SIGNAL FOR TOXIXITY

幽门螺杆菌，一种革兰氏阴性胃细菌，分泌VacA，一种细胞毒素，引起易感细胞的空泡变性。暴露于酸或碱刺激VacA与AZ-521细胞结合。先前暴露于酸和碱处理的VacA的AZ-521细胞的免疫沉淀含有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 -乙酰氨基葡萄糖胺。通过花生凝集素亲和柱和Superose 6层析纯化得到的p250分别含有YRQQRKLVEEIGWSYT和LIIQDEILEATQDDY的n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/PTPζ）的序列相同，p250与抗人RPTPβ单克隆抗体反应。与VacA或热灭活VacA孵育的增溶膜制剂的抗人RPTPβ抗体免疫沉淀研究表明，RPTPβ结合了天然VacA，但不结合变性VacA。这些数据表明，酸性和碱性处理诱导VacA的分子变化，这与其在靶细胞上的活化和与RPTPβ的结合增加有关。为了确定RPTPβ是否作为VacA受体，我们在HL-60细胞中研究了诱导VacA敏感性与RPTPβ表达的关系。通过RT-PCR和间接免疫荧光研究发现，磷肉豆酸酯（PMA， TPA）能诱导人白血病细胞系HL-60分化为具有巨噬细胞特征的细胞，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可以刺激HL-60细胞向单核细胞样细胞的分化，也可以诱导VacA敏感性和RPTPβ mRNA的表达，而1.2% DMSO和维甲酸可以刺激HL-60向粒细胞样细胞的成熟，但效果不明显。PMA加入RPTPβ反义寡核苷酸可特异性抑制VacA敏感性的诱导以及RPTPβ的表达。双重免疫染色研究也表明，在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后，这些细胞获得了VacA敏感性。所有数据都与pma处理的HL-60细胞获得VacA敏感性的结论一致，这是由于RPTPβ的诱导，RPTPβ是一种作为VacA受体的蛋白质。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