Thompson P, Lu J, Kaplan G G
Laboratory of Hepatitis Viruses, Division of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892, USA.
J Virol. 1998 May;72(5):3751-61. doi: 10.1128/JVI.72.5.3751-3761.1998.
The hepatitis A virus cellular receptor 1 (HAVcr-1) cDNA codes for a class I integral membrane glycoprotein, termed havcr-1, of unknown natural function which serves as an African green monkey kidney (AGMK) cell receptor for HAV. The extracellular domain of havcr-1 has an N-terminal Cys-rich region that displays homology with sequences of members of the immunoglobulin superfamily, followed by a Thr/Ser/Pro (TSP)-rich region characteristic of mucin-like O-glycosylated proteins. The havcr-1 glycoprotein contains four putative N-glycosylation sites, two in the Cys-rich region and two in the TSP-rich region. To characterize havcr-1 and define region(s) involved in HAV receptor function, we expressed the TSP-rich region in Escherichia coli fused to glutathione S-transferase and generated antibodies (Ab) in rabbits (anti-GST2 Ab). Western blot analysis with anti-GST2 Ab detected 62- and 65-kDa bands in AGMK cells and 59-, 62-, and 65-kDa bands in dog cells transfected with the HAVcr-1 cDNA (cr5 cells) but not in dog cells transfected with the vector alone (DR2 cells). Treatment of AGMK and cr5 cell extracts with peptide-N-glycosidase F resulted in the collapse of the havcr-1-specific bands into a single band of 56 kDa, which indicated that different N-glycosylated forms of havcr-1 were expressed in these cells. Treatment of AGMK and cr5 cells with tunicamycin reduced binding of protective monoclonal Ab (MAb) 190/4, which suggested that N-glycans are required for binding of MAb 190/4 to havcr-1. To test this hypothesis, havcr-1 mutants lacking the N-glycosylation motif at the first site (mut1), second site (mut2), and both (mut3) sites were constructed and transfected into dog cells. Binding of MAb 190/4 and HAV to mut1 and mut3 cells was highly reduced, while binding to mut2 cells was not affected and binding to dog cells expressing an havcr-1 construct containing a deletion of the Cys-rich region (d1- cells) was undetectable. HAV-infected cr5 and mut2 cells but not mut1, mut3, d1-, and DR2 cells developed the characteristic cytoplasmic granular fluorescence of HAV-infected cells. These results indicate that the Cys-rich region of havcr-1 and its first N-glycosylation site are required for binding of protective MAb 190/4 and HAV receptor function.
甲型肝炎病毒细胞受体1（HAVcr-1）cDNA编码一种I类整合膜糖蛋白，称为havcr-1，其天然功能未知，它作为甲型肝炎病毒在非洲绿猴肾（AGMK）细胞上的受体。havcr-1的细胞外结构域有一个N端富含半胱氨酸的区域，该区域与免疫球蛋白超家族成员的序列具有同源性，随后是富含苏氨酸/丝氨酸/脯氨酸（TSP）的区域，这是粘蛋白样O-糖基化蛋白的特征。havcr-1糖蛋白含有四个推定的N-糖基化位点，两个在富含半胱氨酸的区域，两个在富含TSP的区域。为了表征havcr-1并确定参与甲型肝炎病毒受体功能的区域，我们在大肠杆菌中表达了与谷胱甘肽S-转移酶融合的富含TSP的区域，并在兔中产生了抗体（抗GST2 Ab）。用抗GST2 Ab进行的蛋白质印迹分析在AGMK细胞中检测到62 kDa和65 kDa的条带，在用HAVcr-1 cDNA转染的犬细胞（cr5细胞）中检测到59 kDa、62 kDa和65 kDa的条带，但在仅用载体转染的犬细胞（DR2细胞）中未检测到。用肽-N-糖苷酶F处理AGMK和cr5细胞提取物导致havcr-1特异性条带塌陷为一条56 kDa的单一条带，这表明在这些细胞中表达了不同N-糖基化形式的havcr-1。用衣霉素处理AGMK和cr5细胞可降低保护性单克隆抗体（MAb）190/4的结合，这表明N-聚糖是MAb 190/4与havcr-1结合所必需的。为了验证这一假设，构建了在第一个位点（mut1）、第二个位点（mut2）和两个位点（mut3）均缺乏N-糖基化基序的havcr-1突变体，并将其转染到犬细胞中。MAb 190/4和甲型肝炎病毒与mut1和mut3细胞的结合显著降低，而与mut2细胞的结合不受影响，与表达缺失富含半胱氨酸区域的havcr-1构建体的犬细胞（d1-细胞）的结合未检测到。感染甲型肝炎病毒的cr5和mut2细胞出现了甲型肝炎病毒感染细胞特有的细胞质颗粒荧光，而mut1、mut3、d1-和DR2细胞则未出现。这些结果表明，havcr-1的富含半胱氨酸区域及其第一个N-糖基化位点是保护性MAb 190/4结合和甲型肝炎病毒受体功能所必需的。
