Weigel Janet A, Raymond Robert C, McGary Carl, Singh Anil, Weigel Paul H
Department of Biochemistry and Molecular Biology and the Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190, USA.
J Biol Chem. 2003 Mar 14;278(11):9808-12. doi: 10.1074/jbc.m211462200.
Hyaluronan (HA) and chondroitin sulfate clearance from lymph and blood is mediated by the hyaluronan receptor for endocytosis (HARE). The purification and molecular cloning (Zhou, B., Weigel, J. A., Saxena, A., and Weigel, P. H. (2002) Mol. Biol. Cell 13, 2853-2868) of this cell surface receptor were finally achieved after we developed monoclonal antibodies (mAbs) against HARE. There are actually two independent isoreceptors for HA, which in rat are designated the 175-kDa HARE and 300-kDa HARE. Only one mAb (number 174) effectively and completely blocked the specific uptake of 125I-HA at 37 degrees C by rat liver sinusoidal endothelial cells. 125I-HA binding to both the 175-kDa and 300-kDa HARE proteins in a ligand blot assay was almost completely inhibited by <1 microg/ml mAb-174, whereas mouse IgG had little or no effect. MAb-174 also performed very well in Western analysis, indirect fluorescence microscopy, and a variety of immuno-procedures. Immunohistochemistry using mAb-174 localized HARE to the sinusoidal cells of rat liver, spleen, and lymph node. Western analysis using mAb-174 revealed that the sizes of both HARE glycoproteins were the same in these three tissues. 125I-HA was taken up and degraded by excised rat livers that were continuously perfused ex vivo with a recirculating medium. This HA clearance and metabolism by liver, which is a physiological function of HARE, was very effectively blocked by mAb-174 but not by mouse IgG. The results indicate that mAb-174 will be a useful tool to study the functions of HARE and the physiological significance of HA clearance.
透明质酸（HA）和硫酸软骨素从淋巴和血液中的清除是由用于内吞作用的透明质酸受体（HARE）介导的。在我们开发出针对HARE的单克隆抗体（mAb）后，最终实现了这种细胞表面受体的纯化和分子克隆（周，B.，韦格尔，J. A.，萨克塞纳，A.，和韦格尔，P. H.（2002年）《分子生物学与细胞》13卷，2853 - 2868页）。实际上存在两种独立的HA同型受体，在大鼠中它们被命名为175 kDa的HARE和300 kDa的HARE。只有一种单克隆抗体（编号174）能有效且完全地阻断大鼠肝窦内皮细胞在37℃时对125I - HA的特异性摄取。在配体印迹分析中，<1微克/毫升的单克隆抗体 - 174几乎完全抑制了125I - HA与175 kDa和300 kDa的HARE蛋白的结合，而小鼠IgG几乎没有影响。单克隆抗体 - 174在蛋白质免疫印迹分析、间接荧光显微镜检查以及各种免疫程序中也表现出色。使用单克隆抗体 - 174进行免疫组织化学分析将HARE定位到大鼠肝脏、脾脏和淋巴结的窦状细胞。使用单克隆抗体 - 174进行蛋白质免疫印迹分析表明，这三种组织中两种HARE糖蛋白的大小相同。125I - HA被离体连续灌注循环培养基的大鼠肝脏摄取并降解。肝脏对HA的这种清除和代谢是HARE 的一种生理功能，单克隆抗体 - 174能非常有效地阻断它，但小鼠IgG则不能。结果表明，单克隆抗体 - 174将是研究HARE功能以及HA清除的生理意义的有用工具。
