Mohri H, Katoh K, Iwamatsu A, Okubo T
First Department of Internal Medicine, Yokohama City University School of Medicine, Japan.
Exp Cell Res. 1996 Feb 1;222(2):326-32. doi: 10.1006/excr.1996.0042.
The hematopoietic cell recognition sites of human fibronectin (FN) are the Arg-Gly-Asp-Ser (RGDS) sequence recognized by widely distributed integrin receptor alpha 5 beta 1 and the type III connecting segment (III CS) containing two cell-binding sites, designated CS1 and CS5, that are recognized by the alpha 4 beta 1 receptor. The C-terminal heparin-binding domain of FN (Hep II) has recently been demonstrated to support adhesion of alpha 4 beta 1-dependent melanoma cells [A. P. Mould and M. J. Humphries (1991) EMBO J. 10, 4089-4095]. Previously we demonstrated that this region of FN mediated binding of FN to HL-60 cells (acute promyelocytic leukemia cell line) by direct interaction independently of RGD and CS1 [H. Fujita et al., (1995) Exp. Cell Res. 217, 484-488]. In this study we have characterized a novel site in the Hep II region for binding to HL-60 cells. alpha 4 beta 1 and alpha 5 beta 1 were expressed on HL-60 cells, while alpha 2 beta 1 and alpha 3 beta 1 were not present, as shown by flow cytometry using monoclonal antibodies specific for the different integrins. Anti-alpha 4 beta 1 (P4C2) and anti-beta 1 (JB1a) antibodies inhibited binding of a 29-kDa dispase-digestive fragment of FN to HL-60 cells. This fragment contains the C-terminal heparin-binding domain of FN but lacks CS1 and CS5. Only the peptide representing the sequence from Val1866 to Arg1880, designated E1, inhibited the binding of the 29-kDa fragment to HL-60 cells. The active region of this peptide was a sequence of Thr-Asp-Ile-Asp-Ala-Pro-Ser (TAI-DAPS), which is homologous to Leu-Asp-Val-Pro-Ser (LDVPS) derived from the active site of CS1. Furthermore, labeled E1 peptide directly bound to HL-60 cells. The anti-alpha 4 beta 1 antibody (P4C2) inhibited this interaction. These results indicate that the site of binding to hematopoietic cells is present in the Hep II region of FN and the definition of the chemical structure of FN clarifies a fundamental mechanism of cell invasion of the extracellular matrix.
人纤连蛋白（FN）的造血细胞识别位点包括被广泛分布的整合素受体α5β1识别的精氨酸 - 甘氨酸 - 天冬氨酸 - 丝氨酸（RGDS）序列，以及含有两个细胞结合位点（称为CS1和CS5）的III型连接段（III CS），这两个位点被α4β1受体识别。最近已证明FN的C端肝素结合结构域（Hep II）可支持α4β1依赖性黑色素瘤细胞的黏附[A. P. Mould和M. J. Humphries（1991年），《欧洲分子生物学组织杂志》10，4089 - 4095]。此前我们证明，FN的该区域通过直接相互作用介导FN与HL - 60细胞（急性早幼粒细胞白血病细胞系）的结合，且不依赖于RGD和CS1 [H. Fujita等人，（1995年），《细胞实验研究》217，484 - 488]。在本研究中，我们鉴定了Hep II区域中一个与HL - 60细胞结合的新位点。通过使用针对不同整合素的单克隆抗体进行流式细胞术检测表明，HL - 60细胞表达α4β1和α5β1，而不表达α2β1和α3β1。抗α4β1（P4C2）和抗β1（JB1a）抗体抑制了FN的29 kDa分散酶消化片段与HL - 60细胞的结合。该片段包含FN的C端肝素结合结构域，但缺乏CS1和CS5。只有代表从Val1866到Arg1880序列的肽（称为E1）抑制了29 kDa片段与HL - 60细胞的结合。该肽的活性区域是苏氨酸 - 天冬氨酸 - 异亮氨酸 - 天冬氨酸 - 丙氨酸 - 脯氨酸 - 丝氨酸（TAI - DAPS）序列，它与源自CS1活性位点的亮氨酸 - 天冬氨酸 - 缬氨酸 - 脯氨酸 - 丝氨酸（LDVPS）同源。此外，标记的E1肽直接与HL - 60细胞结合。抗α4β1抗体（P4C2）抑制了这种相互作用。这些结果表明，与造血细胞结合的位点存在于FN的Hep II区域，FN化学结构的确定阐明了细胞侵袭细胞外基质的基本机制。
