Miles A J, Skubitz A P, Furcht L T, Fields G B
Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis 55455.
J Biol Chem. 1994 Dec 9;269(49):30939-45.
Several regions within the triple-helical domain of type IV collagen function as cellular recognition sites. We have demonstrated previously that melanoma cell activities promoted by the alpha 1(IV)1263-1277 sequence are enhanced by triple helicity (Fields, C. G., Mickelson, D. J., Drake, S.L., McCarthy, J.B., and Fields, G.B. (1993) J. Biol. Chem. 268, 14153-14160), whereas Eble et al. reached similar conclusions for alpha 1 beta 1 integrin-mediated fibrosarcoma cell adhesion to [alpha 1(IV)]2 alpha 2(IV)434-472 (Eble, J. A., Golbik, R., Mann, K., and Kühn, K. (1993) EMBO J. 12, 4795-4802). In the present study, we have examined the cell adhesion activities of a third region in type IV collagen. A single-stranded peptide (SSP) incorporating the alpha 1(IV)531-543 sequence promoted the adhesion of melanoma, ovarian carcinoma, and Jurkat cells in a dose-dependent manner, with 40% cell adhesion observed at [SSP] = 1.8, 11.5, and 42.2 microM, respectively. Nearly identical results were obtained for cell adhesion to an all-D-enantiomer of the SSP, suggesting that the cell surface receptor(s) for this site do not discriminate based on chirality. The alpha 1(IV)531-543 sequence maintained its cell adhesion promoting activity when incorporated into a homotrimeric triple-helical polypeptide, although relative levels of adhesion were either slightly enhanced or slightly diminished compared with the SSP. Triple-helical conformation was thus not critical for cellular recognition of the alpha 1(IV)531-543 sequence. Single-site substitution experiments of the SSP showed no overall correlation between the biological effects of substitutions and SSP conformation. The SSP, D-SSP, triple-helical polypeptide, and SSP substitution results suggest that cell recognition of the alpha 1(IV)531-543 sequence is generally independent of substrate conformation. The present and prior studies indicate that "conformationally dependent" and "conformationally independent" cellular recognition sites exist within the triple-helical domain of type IV collagen.
IV型胶原三螺旋结构域内的几个区域可作为细胞识别位点。我们之前已经证明，由α1(IV)1263 - 1277序列促进的黑色素瘤细胞活性会因三螺旋结构而增强（菲尔兹，C.G.，米克尔森，D.J.，德雷克，S.L.，麦卡锡，J.B.，以及菲尔兹，G.B.（1993年）《生物化学杂志》268卷，14153 - 14160页），而埃布尔等人对于α1β1整合素介导的纤维肉瘤细胞与[α1(IV)]2α2(IV)434 - 472的黏附也得出了类似结论（埃布尔，J.A.，戈尔比克，R.，曼，K.，以及库恩，K.（1993年）《欧洲分子生物学组织杂志》12卷，4795 - 第4802页）。在本研究中，我们检测了IV型胶原中第三个区域的细胞黏附活性。一种包含α1(IV)531 - 543序列的单链肽（SSP）以剂量依赖方式促进黑色素瘤细胞、卵巢癌细胞和Jurkat细胞的黏附，在[SSP]分别为1.8、11.5和42.2微摩尔时，细胞黏附率分别为40%。对于细胞与SSP的全D - 对映体的黏附，得到了几乎相同的结果，这表明该位点的细胞表面受体不会基于手性进行区分。当α1(IV)531 - 543序列被整合到同三聚体三螺旋多肽中时，其仍保持细胞黏附促进活性，尽管与SSP相比，黏附的相对水平要么略有增强，要么略有降低。因此，三螺旋构象对于α1(IV)531 - 543序列的细胞识别并非至关重要。SSP的单点取代实验表明，取代的生物学效应与SSP构象之间没有总体相关性。SSP、D - SSP、三螺旋多肽以及SSP取代结果表明，α1(IV)531 - 543序列的细胞识别通常与底物构象无关。本研究及之前的研究表明，IV型胶原三螺旋结构域内存在“构象依赖性”和“构象独立性”的细胞识别位点。
