Dougherty G J, Cooper D L, Memory J F, Chiu R K
Terry Fox Laboratory for Hematology/Oncology, British Columbia Cancer Agency, Vancouver, Canada.
J Biol Chem. 1994 Mar 25;269(12):9074-8.
CD44 species of widely differing molecular mass have been identified on various normal and/or transformed cells. Recent studies have demonstrated that much of this heterogeneity is produced as a result of the alternative splicing of a series of 10 exons present within the CD44 gene generating a large number of CD44 isoforms containing additional peptide sequences of varying length inserted into a single site within the extracellular domain of the molecule. At present, the effect of such insertions on the ligand binding specificity of CD44 remains unclear. CD44H, the major CD44 isoform expressed by most resting cell types, has been shown to function as a receptor for the glycosaminoglycan hyaluronan. In contrast, CD44E, the major isoform expressed by the colon carcinoma cell line HT29, which contains a 132-amino acid insert, is unable to recognize and bind this ligand. In the present study we demonstrate that CD44R1, an isoform isolated from the myelomonocytic cell line KG1a, that differs from CD44E by just 3 amino acid substitutions, is fully capable of mediating the attachment of transfected COS7 cells to hyaluronan-coated plastic. In order to confirm that such binding was directly mediated by the introduced CD44 species, chimeric proteins containing the entire extracellular domain of CD44H or CD44R1 fused in-frame to human bone/liver/kidney alkaline phosphatase were prepared and tested for their ability to bind hyaluronan-coated plastic. Both fusion proteins bound equally well to hyaluronan and in each case their attachment could be readily inhibited by monoclonal antibodies directed against the hyaluronan-binding domain of CD44. These data indicate that the 132-amino acid insert present within the extracellular domain of CD44R1 does not interfere with the hyaluronan binding function of the molecule. Since CD44E contains an identically sized insert but is unable to bind hyaluronan, it is likely that mutation of one or more of the 3 amino acid residues that differ between CD44E and CD44R1 is responsible for the altered functional activity of this particular molecule.
在各种正常和/或转化细胞上已鉴定出分子量差异很大的CD44亚型。最近的研究表明，这种异质性大多是由于CD44基因中存在的一系列10个外显子的可变剪接所致，从而产生了大量的CD44异构体，这些异构体含有插入到分子胞外域内单个位点的不同长度的额外肽序列。目前，这种插入对CD44配体结合特异性的影响尚不清楚。CD44H是大多数静止细胞类型表达的主要CD44异构体，已被证明可作为糖胺聚糖透明质酸的受体。相比之下，结肠癌细胞系HT29表达的主要异构体CD44E含有一个132个氨基酸的插入序列，无法识别和结合这种配体。在本研究中，我们证明了从骨髓单核细胞系KG1a分离出的异构体CD44R1，它与CD44E仅相差3个氨基酸取代，完全能够介导转染的COS7细胞与包被透明质酸的塑料的附着。为了证实这种结合是由引入的CD44亚型直接介导的，制备了含有CD44H或CD44R1整个胞外域并与人骨/肝/肾碱性磷酸酶框内融合的嵌合蛋白，并测试它们结合包被透明质酸的塑料的能力。两种融合蛋白与透明质酸的结合同样良好，并且在每种情况下，它们的附着都可以很容易地被针对CD44透明质酸结合域的单克隆抗体抑制。这些数据表明，CD44R1胞外域中存在的132个氨基酸插入序列不会干扰该分子的透明质酸结合功能。由于CD44E含有相同大小的插入序列但无法结合透明质酸，因此很可能是CD44E和CD44R1之间不同的3个氨基酸残基中的一个或多个发生了突变，导致了这个特定分子功能活性的改变。
