Yang B, Zhang L, Turley E A
Manitoba Institute of Cell Biology, University of Manitoba, Winnipeg, Canada.
J Biol Chem. 1993 Apr 25;268(12):8617-23.
We have identified two discrete hyaluronan- (HA) binding domains in the HA receptor RHAMM (Receptor for HA-Mediated Motility) that mediates the locomotion of H-ras transformed fibroblasts. A complete RHAMM cDNA (1.43 kilobases (kb)) was expressed as a fusion protein with pGEX-2T in Escherichia coli HB101 and was shown to bind specifically to both biotin-labeled HA in a transblot assay and to HA-Sepharose. The complete cDNA was truncated with restriction endonucleases from the 3' end resulting in 1.30-, 1.02-, 0.71-, and 0.41-kb cDNAs which were then expressed in HB101. Only the fusion peptide expressed from the complete cDNA and the 1.30-kb cDNA bound to HA indicating that the region located between 1.02-1.30 kb of RHAMM cDNA was critical for recognition of this glycosaminoglycan. Deletion of 114 bases in this region virtually eliminated HA binding activity thus defining the major glycosaminoglycan binding region to amino acids 400-434 located near the carboxyl terminus of RHAMM. Two domains containing clusters of basic amino acids were identified within this region. Synthetic peptides mimicking these two domains both inhibited HA binding to the complete 1.43-kb expressed glutathione s-transferase-RHAMM fusion protein, and also directly bound to HA-Sepharose. Random peptides and peptides mimicking other regions in RHAMM did not inhibit HA-RHAMM interactions and bound weakly to HA-Sepharose. Oligonucleotides encoding either of these two peptides were linked to the NH2-terminal 0.71 kb of RHAMM which encoded a peptide that did not contain HA binding activity. Fusion proteins containing either of these recombinant peptides acquired HA binding activity as assessed with a transblot assay. Thus, we have identified two domains within RHAMM that are responsible for its HA binding activity.
我们在介导H-ras转化成纤维细胞运动的HA受体RHAMM（透明质酸介导的运动受体）中鉴定出两个离散的透明质酸（HA）结合结构域。完整的RHAMM cDNA（1.43千碱基（kb））在大肠杆菌HB101中作为与pGEX-2T的融合蛋白表达，并在转印分析中显示与生物素标记的HA以及HA-琼脂糖特异性结合。用限制性内切酶从3'端截断完整的cDNA，得到1.30-、1.02-、0.71-和0.41-kb的cDNA，然后在HB101中表达。只有从完整cDNA和1.30-kb cDNA表达的融合肽与HA结合，表明RHAMM cDNA中位于1.0₂-1.30 kb之间的区域对于识别这种糖胺聚糖至关重要。该区域中114个碱基的缺失几乎消除了HA结合活性，从而将主要的糖胺聚糖结合区域定义为RHAMM羧基末端附近的氨基酸400-434。在该区域内鉴定出两个含有碱性氨基酸簇的结构域。模拟这两个结构域的合成肽既抑制HA与完整的1.43-kb表达的谷胱甘肽S-转移酶-RHAMM融合蛋白的结合，也直接与HA-琼脂糖结合。随机肽和模拟RHAMM其他区域的肽不抑制HA-RHAMM相互作用，并且与HA-琼脂糖弱结合。编码这两种肽中任何一种的寡核苷酸与RHAMM的NH₂末端0.71 kb连接，该0.71 kb编码一种不具有HA结合活性的肽。用转印分析评估，含有这两种重组肽中任何一种的融合蛋白都获得了HA结合活性。因此，我们在RHAMM中鉴定出两个负责其HA结合活性的结构域。
