Fields C G, Mickelson D J, Drake S L, McCarthy J B, Fields G B
Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis 55455.
J Biol Chem. 1993 Jul 5;268(19):14153-60.
A great variety of cells, such as melanoma cells, fibroblasts, platelets, keratinocytes, and epithelial cells, adhere to and migrate on specific regions within the triple-helical domains of types I, III, and IV collagen. The relative importance of collagen primary, secondary, and tertiary structures on these cellular activities has not been ascertained, as no general synthetic methodology exists to allow for the study of peptides incorporating biologically active sequences in triple-helical conformation. We have thus developed a novel, generally applicable solid-phase branching methodology for the synthesis of aligned, triple-helical collagen-model polypeptides (i.e. "mini-collagens"). Three nascent peptide chains are carboxyl-terminally linked through one N alpha-amino and two N epsilon-amino groups of Lys, while repeating Gly-Pro-Hyp triplets induce triple helicity. A homotrimeric triple-helical polypeptide (THP) of 124 amino acids, incorporating residues 1263-1277 of alpha 1 (IV) collagen, was synthesized. Highly metastatic mouse melanoma cells showed a profound preference for adhesion to this THP as compared with a single-stranded peptide (SSP) incorporating the same type IV collagen sequence or a branched peptide containing eight repeats of Gly-Pro-Hyp (designated GPP*). Specifically, 50% cell adhesion occurred at a THP concentration of 1.12 microM, while comparable levels of adhesion required [SSP] = 170 microM or [GPP*] > 100 microM. Melanoma cells also spread on the THP to a greater extent than on the SSP or GPP*. These results are the first direct demonstrations of the significance of triple helicity for cell adhesion to and spreading on a specific collagen sequence and support earlier conclusions of conformational dependency for cell adhesion to and migration on types I and IV collagen. In addition, the melanoma cell THP activities support the concept that tumor cell adhesion and spreading on type IV collagen involves multiple, distinct domains in triple-helical conformation. The triple-helical peptide synthetic protocol developed here will allow eventually for the study of both structure and biological activity of specific, glycosylated collagen sequences in homotrimeric and heterotrimeric forms.
多种细胞，如黑色素瘤细胞、成纤维细胞、血小板、角质形成细胞和上皮细胞，可黏附于I型、III型和IV型胶原三螺旋结构域内的特定区域并在其上迁移。由于不存在可用于研究具有三螺旋构象且包含生物活性序列的肽段的通用合成方法，因此尚未确定胶原一级、二级和三级结构对这些细胞活动的相对重要性。因此，我们开发了一种新颖的、普遍适用的固相分支方法，用于合成排列整齐的三螺旋胶原模型多肽（即“微型胶原”）。三条新生肽链通过赖氨酸的一个Nα-氨基和两个Nε-氨基在羧基末端相连，同时重复的甘氨酸-脯氨酸-羟脯氨酸三联体诱导三螺旋结构的形成。合成了一种包含α1(IV)胶原1263 - 1277位残基的124个氨基酸的同三聚体三螺旋多肽（THP）。与包含相同IV型胶原序列的单链肽（SSP）或含有八个甘氨酸-脯氨酸-羟脯氨酸重复序列（命名为GPP*）的分支肽相比，高转移性小鼠黑色素瘤细胞对这种THP表现出明显的黏附偏好。具体而言，在THP浓度为1.12微摩尔时，50%的细胞发生黏附，而达到可比黏附水平所需的[SSP] = 170微摩尔或[GPP*] > 100微摩尔。黑色素瘤细胞在THP上的铺展程度也比在SSP或GPP*上更大。这些结果首次直接证明了三螺旋结构对于细胞黏附于特定胶原序列并在其上铺展的重要性，并支持了早期关于细胞黏附于I型和IV型胶原并在其上迁移的构象依赖性的结论。此外，黑色素瘤细胞对THP的反应支持了这样一种概念，即肿瘤细胞在IV型胶原上的黏附和铺展涉及三螺旋构象中的多个不同结构域。这里开发的三螺旋肽合成方案最终将允许研究同三聚体和异三聚体形式的特定糖基化胶原序列的结构和生物活性。
