Weigel Janet A, Weigel Paul H
Department of Biochemistry & Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190.
J Biol Chem. 2003 Oct 31;278(44):42802-11. doi: 10.1074/jbc.M307201200. Epub 2003 Aug 20.
Hyaluronan (HA) and chondroitin sulfate (CS) clearance from lymph and blood in mammals is mediated by the HA receptor for endocytosis (HARE), which is present as two isoforms in rat and human (175/300 kDa and 190/315 kDa, respectively) in the sinusoidal endothelial cells of liver, spleen, and lymph nodes (Zhou, B., McGary, C. T., Weigel, J. A., Saxena, A., and Weigel, P. H. (2003) Glycobiology 13, 339-349). The small rat and human HARE proteins are not encoded directly by mRNA but are derived from larger precursors. Here we characterize the specificity and function of the 175-kDa HARE, expressed in the absence of the 300-kDa species, in stably transfected SK-Hep-1 cells. The HARE cDNA was fused with a leader sequence to allow correct orientation of the membrane protein. The recombinant rHARE contained approximately 25 kDa of N-linked oligosaccharides and, like the native protein, was able to bind HA in a ligand blot assay, even after de-N-glycosylation. SK-HARE cell lines demonstrated specific 125I-HA endocytosis, receptor recycling, and delivery of HA to lysosomes for degradation. The Kd for the binding of HA (number-average molecular mass approximately 133 kDa) to the 175-kDa HARE at 4 degrees C was 4.1 nm with 160,000 to 220,000 HA-binding sites per cell. The 175-kDa rHARE binds HA, dermatan sulfate, and chondroitin sulfates A, C, D, and E, but not chondroitin, heparin, heparan sulfate, or keratan sulfate. Surprisingly, recognition of glycosaminoglycans (GAGs) other than HA by native or recombinant HARE was temperature-dependent. Although competition was observed at 37 degrees C, none of the other GAGs competed for 125I-HA binding to SK-HARE cells at 4 degrees C. Anti-HARE monoclonal antibody-174 showed a similar temperature-dependence in its ability to block HA endocytosis. These data suggest that temperature-induced conformational changes may alter the GAG specificity of HARE. The results confirm that the 175-kDa rHARE does not require the larger HARE isoform to mediate endocytosis of multiple GAGs.
哺乳动物体内透明质酸（HA）和硫酸软骨素（CS）从淋巴和血液中的清除是由内吞作用的HA受体（HARE）介导的，该受体在大鼠和人类中以两种异构体形式存在（分别为175/300 kDa和190/315 kDa），位于肝脏、脾脏和淋巴结的窦状内皮细胞中（周，B.，麦加里，C.T.，韦格尔，J.A.，萨克森纳，A.，和韦格尔，P.H.（2003年）《糖生物学》13，339 - 349）。大鼠和人类的小HARE蛋白不是由mRNA直接编码的，而是来自更大的前体。在此，我们在稳定转染的SK - Hep - 1细胞中，对在不存在300 kDa异构体的情况下表达的175 kDa HARE的特异性和功能进行了表征。HARE cDNA与一个前导序列融合，以使膜蛋白具有正确的方向。重组rHARE含有约25 kDa的N - 连接寡糖，并且与天然蛋白一样，即使在去N - 糖基化后，在配体印迹分析中也能够结合HA。SK - HARE细胞系表现出特异性的12⁵I - HA内吞作用、受体循环以及将HA递送至溶酶体进行降解。在4℃时，HA（数均分子量约133 kDa）与175 kDa HARE结合的Kd为4.1 nM，每个细胞有160,000至220,000个HA结合位点。175 kDa的rHARE结合HA、硫酸皮肤素以及硫酸软骨素A、C、D和E，但不结合硫酸软骨素、肝素、硫酸乙酰肝素或硫酸角质素。令人惊讶的是，天然或重组的HARE对HA以外的糖胺聚糖（GAGs）的识别是温度依赖性的。尽管在37℃时观察到竞争，但在4℃时，其他GAGs均不竞争12⁵I - HA与SK - HARE细胞的结合。抗HARE单克隆抗体 - 174在阻断HA内吞作用的能力上表现出类似的温度依赖性。这些数据表明，温度诱导构象变化可能会改变HARE的GAG特异性。结果证实，175 kDa的rHARE不需要更大的HARE异构体来介导多种GAGs的内吞作用。
