Rao Y, Wu X F, Yip P, Gariepy J, Siu C H
Banting and Best Department of Medical Research, University of Toronto, Ontario, Canada.
J Biol Chem. 1993 Sep 25;268(27):20630-8.
We have previously used synthetic peptides to identify a homophilic binding site between Lys-243 and Glu-252 (KYSFNYDGSE) in the third immunoglobulin-like domain of the chick neural cell adhesion molecule (NCAM). In this report, we show that the deletion of this decapeptide sequence from chick NCAM or the scrambling of the first 5 amino acid residues led to the abolition of the homophilic binding activity of NCAM, thus confirming the role of this sequence in NCAM-NCAM binding. To investigate the involvement of individual residues of this decapeptide in NCAM binding, competition experiments were carried out using peptide analogues with various amino acid substitutions. Substitution of both Lys-243 and Asp-249 with Ala or of the 3 aromatic residues with Ala led to a total loss of activity, highlighting the importance of these residues in NCAM binding. Site-directed mutagenesis was then employed to substitute individual amino acids within the decapeptide sequence with Ala. The homophilic binding activity of mutant NCAMs transiently expressed in COS-1 cells was determined using the NCAM-Covasphere binding assay. Substitution of the charged residues with alanine decreased NCAM binding activity, implicating electrostatic interactions in NCAM binding activity. Substitution of the aromatic residues Tyr-244 and Phe-246 with Ala abolished NCAM binding activity, suggesting that hydrophobic and/or aromatic interactions may play an important role in NCAM homophilic binding. Substitution of amino acids in the predicted beta-strand portion of the decapeptide with Pro, which would tend to disrupt beta-strand conformation, led to a substantial loss of activity. Thus, NCAM-NCAM binding may also depend on the beta-backbone structure of this site. These results are consistent with the involvement of multiple amino acids within the decapeptide sequence in NCAM homophilic interaction.
我们之前曾使用合成肽来鉴定鸡神经细胞黏附分子（NCAM）第三个免疫球蛋白样结构域中赖氨酸 - 243和谷氨酸 - 252（KYSFNYDGSE）之间的嗜同性结合位点。在本报告中，我们表明从鸡NCAM中删除此十肽序列或前5个氨基酸残基的打乱会导致NCAM嗜同性结合活性的丧失，从而证实了该序列在NCAM - NCAM结合中的作用。为了研究该十肽中各个残基在NCAM结合中的参与情况，使用具有各种氨基酸取代的肽类似物进行了竞争实验。用丙氨酸取代赖氨酸 - 243和天冬氨酸 - 249两者或用丙氨酸取代3个芳香族残基会导致活性完全丧失，突出了这些残基在NCAM结合中的重要性。然后采用定点诱变将十肽序列中的单个氨基酸替换为丙氨酸。使用NCAM - Covasphere结合测定法测定在COS - l细胞中瞬时表达的突变型NCAM的嗜同性结合活性。用丙氨酸取代带电荷的残基会降低NCAM结合活性，这表明静电相互作用参与了NCAM结合活性。用丙氨酸取代芳香族残基酪氨酸 - 244和苯丙氨酸 - 246会消除NCAM结合活性，这表明疏水和/或芳香族相互作用可能在NCAM嗜同性结合中起重要作用。用脯氨酸取代十肽预测的β - 链部分中的氨基酸，这往往会破坏β - 链构象，导致活性大幅丧失。因此，NCAM - NCAM结合也可能取决于该位点的β - 主链结构。这些结果与十肽序列中的多个氨基酸参与NCAM嗜同性相互作用一致。
