Ishikawa D, Kikkawa H, Ogino K, Hirabayashi Y, Oku N, Taki T
Cellular Technology Institute, Otsuka Pharmaceutical Co. Ltd., Tokushima, Japan.
FEBS Lett. 1998 Dec 11;441(1):20-4. doi: 10.1016/s0014-5793(98)01511-7.
A novel peptide technology to produce mimicking peptides of carbohydrate moiety (which we propose to name glyco-replica peptides) is a useful tool to elucidate the functions of glycoconjugate. Carbohydrate moiety of ganglioside GD1alpha functions as a molecule involved in the adhesion between murine highly metastatic lymphoma RAW117-H10 cells and hepatic sinusoidal endothelial (HSE) cells. To prepare peptides which mimic the carbohydrate structure of GD1alpha, phage clones expressing peptides which bound to a monoclonal antibody against GD1alpha (KA17) were isolated from a phage-displayed random peptide library. Four phage clones having affinity to the monoclonal antibody KA17 were isolated, and these clones showed inhibitory effect on the binding of KA17 to GD1alpha. The amino acid sequences of the displayed pentadecamers were determined, and one of the phages displaying sequence WHWRHRIPLQLAAGR bound to HSE cells directly and showed the highest inhibitory effect on the adhesion between RAW117-H10 cells and HSE cells. The synthesized peptides having the same sequences to the displayed 15mers in the four isolated phage clones also showed the inhibitory effect on the adhesion of RAW117-H10 cells to HSE cells, and, again, the WHWRHRIPLQLAAGR peptide showed the highest inhibitory effect. Furthermore, intravenous injection of the peptide brought almost complete inhibition of the metastasis of RAW117-H10 cells to lung and spleen, and about 50% inhibition of the liver metastasis. These results indicate that GD1alpha plays an important role for metastasis of RAW117-H10 cells, and the peptides obtained by the present procedure are able to mimic the functional role of the glycoconjugate.
一种用于生产碳水化合物部分模拟肽（我们提议将其命名为糖复制品肽）的新型肽技术，是阐明糖缀合物功能的有用工具。神经节苷脂GD1α的碳水化合物部分作为一种分子，参与小鼠高转移性淋巴瘤RAW117-H10细胞与肝窦内皮（HSE）细胞之间的黏附。为了制备模拟GD1α碳水化合物结构的肽，从噬菌体展示的随机肽库中分离出表达与抗GD1α单克隆抗体（KA17）结合的肽的噬菌体克隆。分离出四个对单克隆抗体KA17具有亲和力的噬菌体克隆，这些克隆对KA17与GD1α的结合显示出抑制作用。测定了所展示的十五肽的氨基酸序列，其中一个展示序列为WHWRHRIPLQLAAGR的噬菌体直接与HSE细胞结合，并对RAW117-H10细胞与HSE细胞之间的黏附显示出最高的抑制作用。在四个分离的噬菌体克隆中，与所展示的15聚体具有相同序列的合成肽也对RAW117-H10细胞与HSE细胞的黏附显示出抑制作用，并且，同样，WHWRHRIPLQLAAGR肽显示出最高的抑制作用。此外，静脉注射该肽几乎完全抑制了RAW117-H10细胞向肺和脾的转移，以及对肝转移的约50%的抑制。这些结果表明GD1α在RAW117-H10细胞的转移中起重要作用，并且通过本方法获得的肽能够模拟糖缀合物的功能作用。
