Briggs J B, Larsen R A, Harris R B, Sekar K V, Macher B A
Glycomed, Inc., Alameda, CA 94501, USA.
Glycobiology. 1996 Dec;6(8):831-6. doi: 10.1093/glycob/6.8.831.
Previously, it was established that the peptide YYWIGIRK-NH2 inhibits both myeloid cell adhesion to selectins in vitro and neutrophil influx into inflammatory sites in vivo (Briggs et al., 1995). Initial structure/activity studies revealed that at least one Y residue at the N-terminus of the peptide was essential for these bioactivities but that the C-terminal K residue was unnecessary for inhibitory activity. We have now synthesized a new series of peptides which contain single residue substitutions at each position of the reference peptide, YYWIGIR-NH2, and have tested these peptides for inhibitory activity in a selectin cell binding assay. In addition, peptides containing single D-amino acids at selected positions, or an all D-configured reference peptide sequence, or the retro-inverso version (rigiwyy-NH2) of the reference peptide sequence have also been analyzed for inhibitory activity in the same assays. Finally, the ability of the reference peptide and a specifically designed control sequence (YY(AIB)IGIR-NH2) to discriminate between potential synthetic saccharide ligands, including sialyl-Lewis x, Lewis x, and sialyl-N-acetyl-lactosamine, was investigated using isothermal titration calorimetry. The results of these studies demonstrate that whereas many single amino acid substitutions are tolerated in the peptide without complete loss of inhibitory activity, substitution at some positions (e.g., the W residue) results in relatively inactive compounds, clearly pointing to the importance of these residues in making critical contacts with the appropriate saccharide ligand. Titration calorimetry revealed that the reference peptide does not discriminate between Lewis x or sialyl-Lewis x in vitro, but binds these saccharides with nearly 40-fold higher affinity (KD 25 microM) than the nonfucosylated trisaccharide, sialyl-N-acetyl-lactosamine. We can infer from these studies that the presence of a sialyl group per se, is not a requisite for complex formation between the reference peptide and its saccharide ligand. Substitution of single D-amino acid residues at various positions in the reference peptide sequence reduces or eliminates all inhibitory properties. However, the all D-configured peptide or the retro-inverso peptide sequence have greater activity than the all L-configured reference peptide in the in vitro biological assays, and each was an effective inhibitor of neutrophil infiltration in a thioglycolate-induced mouse peritonitis model. These results, combined with the results of titration, allow us to conclude that binding between the reference peptide and its saccharide ligand, which affords its inhibitory properties, is mediated by the presence of a contiguous, nonpolar surface, or face, presented at the N-terminus of the reference peptide, likely encompassing the sequence YYWI. Furthermore, the W plays a critical role in binding, probably through formation of an essential hydrogen bond with a suitably juxtaposed group carried on the saccharide ligand.
此前已证实，肽YYWIGIRK-NH2在体外可抑制髓样细胞与选择素的黏附，在体内可抑制中性粒细胞流入炎症部位（Briggs等人，1995年）。最初的结构/活性研究表明，该肽N端至少有一个Y残基对这些生物活性至关重要，但C端的K残基对抑制活性并非必需。我们现已合成了一系列新的肽，它们在参考肽YYWIGIR-NH2的每个位置都有单个残基取代，并在选择素细胞结合试验中测试了这些肽的抑制活性。此外，还分析了在选定位置含有单个D-氨基酸的肽、全D构型的参考肽序列或参考肽序列的反向异构体（rigiwyy-NH2）在相同试验中的抑制活性。最后，使用等温滴定量热法研究了参考肽和专门设计的对照序列（YY(AIB)IGIR-NH2）区分潜在合成糖配体（包括唾液酸化路易斯x、路易斯x和唾液酸化N-乙酰乳糖胺）的能力。这些研究结果表明，虽然肽中许多单个氨基酸取代可被耐受而不完全丧失抑制活性，但某些位置（如W残基）的取代会产生相对无活性的化合物，这清楚地表明这些残基在与适当糖配体进行关键接触中的重要性。滴定量热法显示，参考肽在体外不能区分路易斯x或唾液酸化路易斯x，但与这些糖的结合亲和力（KD 25 microM）比非岩藻糖基化三糖唾液酸化N-乙酰乳糖胺高近40倍。从这些研究中我们可以推断，唾液酸基团本身的存在并非参考肽与其糖配体形成复合物的必要条件。在参考肽序列的不同位置取代单个D-氨基酸残基会降低或消除所有抑制特性。然而，在体外生物学试验中，全D构型的肽或反向异构体肽序列比全L构型的参考肽具有更高的活性，并且在硫代乙醇酸盐诱导的小鼠腹膜炎模型中，它们都是中性粒细胞浸润的有效抑制剂。这些结果与滴定结果相结合，使我们得出结论，参考肽与其糖配体之间的结合赋予其抑制特性，这种结合是由参考肽N端呈现的连续非极性表面或面介导的，可能包括序列YYWI。此外，W在结合中起关键作用，可能是通过与糖配体上适当并列的基团形成必需的氢键。
