Expression, processing, and glycosaminoglycan binding activity of the recombinant human 315-kDa hyaluronic acid receptor for endocytosis (HARE)
Expression, processing, and glycosaminoglycan binding activity of the recombinant human 315-kDa hyaluronic acid receptor for endocytosis (HARE)
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DOI：
10.1074/jbc.m607787200
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发表时间：
2007-02-02
影响因子：
4.8
通讯作者：
Weigel, Paul H.
中科院分区：
文献类型：
--
作者：
Harris, Edward N.;Kyosseva, Svetlana V.;Weigel, Paul H.
透明质酸(HA)内吞作用受体(HARE；又称稳定素-2和Feel-2)通过包被的小窝介导摄取，介导全身糖胺聚糖从循环系统和淋巴系统的清除。HARE主要以两种亚型(315 kDa和190 kDa)存在于肝、淋巴结和脾的肝窦内皮细胞中。在这里，我们鉴定了在FLP-in 293细胞系中稳定表达的大的315-Hare异构体的配体特异性和功能。与人脾静脉窦内皮细胞一样，在293细胞系中，FLP-1基因以3-4：1的比例表达315-kDa和截短的190-kDa异构体。由315-kDa Hare和315-kDa Hare特异结合I-125-HA。像190 kDa的野兔单独表达(Harris，E.N.，Weigel，J.A.和Weigel，P.H.(2004)J.Biol.化学。在315HARE细胞系中表达的190kDa和315kDa的HARE亚型可被抗HARE单抗30、154和159识别。所有315-Hare细胞系均能内吞和降解I-125-HA。与活细胞的竞争研究表明，190-HARE和315-HARE与HA的结合具有比A、C、D或E型硫酸软骨素更高的表观亲和力(Kd值类似于10-20 nM)，仅与CS-B和软骨素有轻微的竞争内吞作用。与315-Hare胞外结构域的直接结合分析表明，与CS-C、CS-D和CS-E的结合亲和力高，结合亲和力低。每一种HARE亚型的大部分都在细胞内，在内吞系统内，这表明瞬时的表面滞留是活跃的内吞循环受体的典型特征。
The hyaluronic acid ( HA) receptor for endocytosis ( HARE; also designated stabilin-2 and FEEL-2) mediates systemic clearance of glycosaminoglycans from the circulatory and lymphatic systems via coated pit-mediated uptake. HARE is primarily found as two isoforms (315- and 190-kDa) in sinusoidal endothelial cells of the liver, lymph node, and spleen. Here we characterize the ligand specificity and function of the large stably expressed 315- HARE isoform in Flp-In 293 cell lines. Like human spleen sinusoidal endothelial cells, Flp-In 293 cell lines transfected with a single cDNA encoding the full-length 315- HARE express both the 315-kDa and the proteolytically truncated 190-kDa isoforms in a ratio of similar to 3-4:1. The 190-kDa HARE isoform generated from the 315-kDa HARE and the 315-kDa HARE specifically bound I-125-HA. Like the 190-kDa HARE expressed alone (Harris, E. N., Weigel, J. A., and Weigel, P. H. (2004) J. Biol. Chem. 279, 36201-36209), the 190- and 315-kDa HARE isoforms expressed in 315-HARE cell lines were recognized by anti-HARE monoclonal antibodies 30, 154, and 159. All 315-HARE cell lines could endocytose and degrade I-125-HA. Competition studies with live cells indicate that 190- HARE and 315- HARE bind HA with higher apparent affinity (Kd similar to 10-20 nM) than chondroitin sulfate (CS) types A, C, D, or E. Only slight competition of HA endocytosis was observed with CS-B (dermatan sulfate) and chondroitin. Direct binding assays with the 315-HARE ectodomain revealed high affinity HA binding, and lower binding affinities for CS-C, CS-D, and CS-E. A majority of each HARE isoform was intracellular, within the endocytic system, suggesting transient surface residency typical of an active endocytic recycling receptor.