Kovalchuk Svetlana N, Golotin Vasily A, Balabanova Larissa A, Buinovskaya Nina S, Likhatskaya Galina N, Rasskazov Valery A
G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Science, 159, Stoletya Vladivostoku Str., Vladivostok, 690022, Russia.
G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Science, 159, Stoletya Vladivostoku Str., Vladivostok, 690022, Russia; Far Eastern Federal University, 8, Sukhanova Str., Vladivostok, 690950, Russia.
Fish Shellfish Immunol. 2015 Nov;47(1):565-71. doi: 10.1016/j.fsi.2015.09.045. Epub 2015 Oct 9.
The GalNAc/Gal-specific lectin from the sea mussel Crenomytilus grayanus (CGL) was shown to represent a novel family of lectins and to be characterized by three amino acid tandem repeats with high (up to 73%) sequence similarities to each other. We have used homology modeling approach to predict CGL sugar-binding sites. In silico analysis of CGL-GalNAc complexes showed that CGL contained three binding sites, each of which included conserved HPY(K)G motif. In silico substitutions of histidine, proline and glycine residues by alanine in the HPY(K)G motifs of the Sites 1-3 was shown to lead to loss of hydrogen bonds between His and GalNAc and to the increasing the calculated CGL-GalNAc binding energies. We have obtained recombinant CGL and used site-specific mutagenesis to experimentally examine the role of HPK(Y)G motifs in hemagglutinating and carbohydrate binding activities of CGL. Substitutions of histidine, proline and glycine residues by alanine in the HPYG motif of Site 1 and Site 2 was found to led to complete loss of CGL hemagglutinating and mucin-binding activities. The same mutations in HPKG motif of the Site 3 resulted in decreasing the mucin-binding activity in 6-folds in comparison with the wild type lectin. The mutagenesis and in silico analysis indicates the importance of the all three HPY(K)G motifs in the carbohydrate-binding and hemagglutinating activities of CGL.
已证明海贻贝Crenomytilus grayanus的GalNAc/Gal特异性凝集素（CGL）代表一种新型凝集素家族，其特征在于具有三个氨基酸串联重复序列，彼此之间具有高度（高达73%）的序列相似性。我们使用同源建模方法来预测CGL的糖结合位点。对CGL-GalNAc复合物的计算机模拟分析表明，CGL包含三个结合位点，每个位点都包含保守的HPY(K)G基序。在计算机模拟中，将第1-3位点的HPY(K)G基序中的组氨酸、脯氨酸和甘氨酸残基替换为丙氨酸，结果显示导致His与GalNAc之间的氢键丧失，并增加了计算出的CGL-GalNAc结合能。我们获得了重组CGL，并使用位点特异性诱变实验性地研究HPK(Y)G基序在CGL的血凝和碳水化合物结合活性中的作用。发现在第1位点和第2位点的HPYG基序中将组氨酸、脯氨酸和甘氨酸残基替换为丙氨酸会导致CGL的血凝和粘蛋白结合活性完全丧失。与野生型凝集素相比，第3位点的HPKG基序中的相同突变导致粘蛋白结合活性降低6倍。诱变和计算机模拟分析表明所有三个HPY(K)G基序在CGL的碳水化合物结合和血凝活性中都很重要。
