Yannariello-Brown J, Zhou B, Weigel P H
Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City 73190, USA.
Glycobiology. 1997 Feb;7(1):15-21. doi: 10.1093/glycob/7.1.15.
The rat liver sinusoidal endothelial cell (LEC) hyaluronan (HA) receptor was previously identified using a photoaffinity HA derivative (J. Biol. Chem., 267, 20451-20456, 1992). Two polypeptides with M(r) = 175,000 and 166,000, were consistently crosslinked, suggesting that the LEC HA receptor is an oligomer. Whether one or both subunits participate in HA binding, was not determined. Here we investigate the HA-subunit interactions and the potential oligomeric nature of the LEC HA receptor. When Sephacryl-400 gel filtration chromatography was used to enrich the HA receptor, the 175 kDa polypeptide was the major band seen by SDS-PAGE analysis. Little staining was seen at 166 kDa, suggesting that the 175 kDa protein could be separated from the 166 kDa protein and still retain HA-binding activity. A ligand blot assay was used to determine if each individual subunit could bind HA. LEC proteins were separated by nonreducing SDS-PAGE, and then immobilized onto nitrocellulose. 125I-HA bound to a 175 kDa polypeptide but not to the 166 kDa protein. A high molecular weight band of approximately 300,000 also bound 125I-HA. 125I-HA binding to the 175 and 300 kDa proteins showed the same specificity of competition with a panel of carbohydrates as the bona fide LEC HA receptor. The 175 kDa HA-binding subunit may be nonglobular (asymmetric), since its apparent size by SDS-PAGE is dependent on the polyacrylamide gel pore size; M(r) increases as porosity decreases. LECs were crosslinked to an 125I-labeled photoaffinity HA derivative and the HA saccharides were then released with hyaluronidase. After SDS-PAGE without reduction, radiolabeled bands were seen at 175 and 166 kDa (3:1 ratio), and a high MW (approximately 300,000) species was also detected. These data support an oligomeric model of the LEC HA receptor, and show that the 175 kDa protein possesses HA-binding activity independent from the 166 kDa polypeptide.
大鼠肝窦内皮细胞（LEC）透明质酸（HA）受体先前是使用光亲和性HA衍生物鉴定出来的（《生物化学杂志》，267卷，20451 - 20456页，1992年）。两种分子量分别为175,000和166,000的多肽始终被交联在一起，这表明LEC HA受体是一种寡聚体。但未确定是一个还是两个亚基参与HA结合。在此我们研究HA - 亚基相互作用以及LEC HA受体潜在的寡聚性质。当使用Sephacryl - 400凝胶过滤色谱法富集HA受体时，通过SDS - PAGE分析可见175 kDa多肽是主要条带。在166 kDa处几乎没有染色，这表明175 kDa蛋白可以与166 kDa蛋白分离，并且仍保留HA结合活性。使用配体印迹分析来确定每个单独的亚基是否能结合HA。LEC蛋白通过非还原SDS - PAGE分离，然后固定在硝酸纤维素膜上。125I - HA与175 kDa多肽结合，但不与166 kDa蛋白结合。一条大约300,000的高分子量条带也结合125I - HA。125I - HA与175和300 kDa蛋白的结合显示出与一组碳水化合物竞争的相同特异性，如同真正的LEC HA受体一样。175 kDa的HA结合亚基可能是非球形的（不对称），因为其通过SDS - PAGE显示的表观大小取决于聚丙烯酰胺凝胶孔径；分子量随着孔隙率降低而增加。将LEC与125I标记的光亲和性HA衍生物交联，然后用透明质酸酶释放HA糖链。在不进行还原的SDS - PAGE后，在175和166 kDa处可见放射性标记条带（比例为3:1），并且还检测到一个高分子量（约300,000）的条带。这些数据支持LEC HA受体的寡聚体模型，并表明175 kDa蛋白具有独立于166 kDa多肽的HA结合活性。
