Záková Lenka, Barth Tomislav, Jirácek Jirí, Barthová Jana, Zórad Stefan
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of Czech Republic, Flemingovo 2, 16610 Prague, Czech Republic.
Biochemistry. 2004 Mar 2;43(8):2323-31. doi: 10.1021/bi036001w.
The role of three highly conserved insulin residues PheB24, PheB25, and TyrB26 was studied to better understand the subtleties of the structure-function relationship between insulin and its receptor. Ten shortened insulin analogues with modifications in the beta-strand of the B-chain were synthesized by trypsin-catalyzed coupling of des-octapeptide (B23-B30)-insulin with synthetic peptides. Insulin analogues with a single amino acid substitution in the position B26 and/or single N-methylation of the peptide bond at various positions were all shortened in the C-terminus of the B-chain by four amino acids. The effect of modifications was followed by two types of in vitro assays, i.e., by the binding to the receptor of rat adipose plasma membranes and by the stimulation of the glucose transport into the isolated rat adipocytes. From our results, we can deduce several conclusions: (i) the replacement of tyrosine in the position B26 by phenylalanine has no significant effect on the binding affinity and the stimulation of the glucose transport of shortened analogues, whereas the replacement of TyrB26 by histidine affects the potency highly positively; [HisB26]-des-tetrapeptide (B27-B30)-insulin-B26-amide and [NMeHisB26]-des-tetrapeptide (B27-B30)-insulin-B26-amide show binding affinity 529 and 5250%, respectively, of that of human insulin; (ii) N-methylation of the B24-B25 peptide bond exhibits a disruptive effect on the potency of analogues in both in vitro studies regardless the presence of amino acid in the position B26; (iii) N-methylation of the B23-B24 peptide bond markedly reduces the binding affinity and the glucose transport of respective analogue [NMePheB24]-des-tetrapeptide (B27-B30)-insulin-B26-amide.
研究了胰岛素三个高度保守的残基苯丙氨酸B24、苯丙氨酸B25和酪氨酸B26的作用，以更好地理解胰岛素与其受体之间结构-功能关系的微妙之处。通过胰蛋白酶催化去八肽（B23-B30）-胰岛素与合成肽的偶联，合成了10种在B链β-链中有修饰的缩短胰岛素类似物。在B26位有单个氨基酸取代和/或在不同位置肽键有单个N-甲基化的胰岛素类似物，其B链C端均缩短了四个氨基酸。通过两种体外试验跟踪修饰的效果，即与大鼠脂肪细胞质膜受体的结合以及对分离的大鼠脂肪细胞中葡萄糖转运的刺激。从我们的结果中，可以得出几个结论：（i）B26位的酪氨酸被苯丙氨酸取代对缩短类似物的结合亲和力和葡萄糖转运刺激没有显著影响，而组氨酸取代酪氨酸B26对效力有高度正向影响；[HisB26]-去四肽（B27-B30）-胰岛素-B26-酰胺和[NMeHisB26]-去四肽（B27-B30）-胰岛素-B26-酰胺的结合亲和力分别为人胰岛素的529%和5250%；（ii）B24-B25肽键的N-甲基化在两种体外研究中均对类似物的效力有破坏作用，无论B26位是否存在氨基酸；（iii）B23-B24肽键的N-甲基化显著降低了相应类似物[NMePheB24]-去四肽（B27-B30）-胰岛素-B26-酰胺的结合亲和力和葡萄糖转运。
