Sato Miki, Nishi Nozomu, Shoji Hiroki, Seki Masako, Hashidate Tomomi, Hirabayashi Jun, Kasai Ki Ken-ichi, Hata Yuiro, Suzuki Shigehiko, Hirashima Mitsuomi, Nakamura Takanori
Department of Endocrinology, Kagawa Medical University, 761-0793, Japan.
Glycobiology. 2002 Mar;12(3):191-7. doi: 10.1093/glycob/12.3.191.
Human galectin-9 is a beta-galactoside-binding protein consisting of two carbohydrate recognition domains (CRDs) and a linker peptide. We have shown that galectin-9 represents a novel class of eosinophil chemoattractants (ECAs) produced by activated T cells. A previous study demonstrated that the carbohydrate binding activity of galectin-9 is indispensable for eosinophil chemoattraction and that the N- and C-terminal CRDs exhibit comparable ECA activity, which is substantially lower than that of full-length galectin-9. In this study, we investigated the roles of the two CRDs in ECA activity in conjunction with the sugar-binding properties of the CRDs. In addition, to address the significance of the linker peptide structure, we compare the three isoforms of galectin-9, which only differ in the linker peptide region, in terms of ECA activity. Recombinant proteins consisting of two N-terminal CRDs (galectin-9NN), two C-terminal CRDs (galectin-9CC), and three isoforms of galectin-9 (galectin-9S, -9M, and -9L) were generated. All the recombinant proteins had hemagglutination activity comparable to that of the predominant wild-type galectin-9 (galectin-9M). Galectin-9NN and galectin-9CC induced eosinophil chemotaxis in a manner indistinguishable from the case of galectin-9M. Although the isoform of galectin-9 with the longest linker peptide, galectin-9L, exhibited limited solubility, the three isoforms showed comparable ECA activity over the concentration range tested. The interactions between N- and C-terminal CRDs and glycoprotein glycans and glycolipid glycans were examined using frontal affinity chromatography. Both CRDs exhibited high affinity for branched complex type sugar chain, especially for tri- and tetraantennary N-linked glycans with N-acetyllactosamine units, and the oligosaccharides inhibited the ECA activity at low concentrations. These results suggest that the N- and C-terminal CRDs of galectin-9 interact with the same or a closely related ligand on the eosinophil membrane when acting as an ECA and that ECA activity does not depend on a specific structure of the linker peptide.
人半乳糖凝集素-9是一种β-半乳糖苷结合蛋白，由两个碳水化合物识别结构域（CRD）和一个连接肽组成。我们已经表明，半乳糖凝集素-9代表了一类由活化T细胞产生的新型嗜酸性粒细胞趋化因子（ECA）。先前的一项研究表明，半乳糖凝集素-9的碳水化合物结合活性对于嗜酸性粒细胞趋化作用是不可或缺的，并且N端和C端CRD表现出相当的ECA活性，但其活性明显低于全长半乳糖凝集素-9。在本研究中，我们结合CRD的糖结合特性，研究了两个CRD在ECA活性中的作用。此外，为了探讨连接肽结构的重要性，我们比较了仅在连接肽区域不同的半乳糖凝集素-9的三种同工型在ECA活性方面的差异。我们制备了由两个N端CRD（半乳糖凝集素-9NN）、两个C端CRD（半乳糖凝集素-9CC）和半乳糖凝集素-9的三种同工型（半乳糖凝集素-9S、-9M和-9L）组成的重组蛋白。所有重组蛋白的血凝活性与主要的野生型半乳糖凝集素-9（半乳糖凝集素-9M）相当。半乳糖凝集素-9NN和半乳糖凝集素-9CC诱导嗜酸性粒细胞趋化作用的方式与半乳糖凝集素-9M的情况难以区分。虽然连接肽最长的半乳糖凝集素-9同工型半乳糖凝集素-9L的溶解度有限，但在测试的浓度范围内，这三种同工型表现出相当的ECA活性。使用前沿亲和色谱法研究了N端和C端CRD与糖蛋白聚糖和糖脂聚糖之间的相互作用。两种CRD对分支复合型糖链均表现出高亲和力，尤其是对带有N-乙酰乳糖胺单元的三天线和四天线N-连接聚糖，并且这些寡糖在低浓度下抑制ECA活性。这些结果表明，半乳糖凝集素-9的N端和C端CRD在作为ECA起作用时与嗜酸性粒细胞膜上相同或密切相关的配体相互作用，并且ECA活性不依赖于连接肽的特定结构。
