Drake S L, Klein D J, Mickelson D J, Oegema T R, Furcht L T, McCarthy J B
Department of Laboratory Medicine, University of Minnesota, Minneapolis 55455.
J Cell Biol. 1992 Jun;117(6):1331-41. doi: 10.1083/jcb.117.6.1331.
Cell surface heparan sulfate proteoglycan (HSPG) from metastatic mouse melanoma cells initiates cell adhesion to the synthetic peptide FN-C/H II, a heparin-binding peptide from the 33-kD A chain-derived fragment of fibronectin. Mouse melanoma cell adhesion to FN-C/H II was sensitive to soluble heparin and pretreatment of mouse melanoma cells with heparitinase. In contrast, cell adhesion to the fibronectin synthetic peptide CS1 is mediated through an alpha 4 beta 1 integrin and was resistant to heparin or heparitinase treatment. Mouse melanoma cell HSPG was metabolically labeled with [35S]sulfate and extracted with detergent. After HPLC-DEAE purification, 35S-HSPG eluted from a dissociative CL-4B column with a Kav approximately 0.45, while 35S-heparan sulfate (HS) chains eluted with a Kav approximately 0.62. The HSPG contained a major 63-kD core protein after heparitinase digestion. Polyclonal antibodies generated against HSPG purified from mouse melanoma cells grown in vivo also identified a 63-kD core protein. This HSPG is an integral plasma membrane component by virtue of its binding to Octyl Sepharose affinity columns and that anti-HSPG antibody staining exhibited a cell surface localization. The HSPG is anchored to the cell surface through phosphatidylinositol (PI) linkages, as evidenced in part by the ability of PI-specific phospholipase C to eliminate binding of the detergent-extracted HSPG to Octyl Sepharose. Furthermore, the mouse melanoma HSPG core protein could be metabolically labeled with 3H-ethanolamine. The involvement of mouse melanoma cell surface HSPG in cell adhesion to fibronectin was also demonstrated by the ability of anti-HSPG antibodies and anti-HSPG IgG Fab monomers to inhibit mouse melanoma cell adhesion to FN-C/H II. 35S-HSPG and 35S-HS bind to FN-C/H II affinity columns and require 0.25 M NaCl for elution. However, heparitinase-treated 125I-labeled HSPG failed to bind FN-C/H II, suggesting that HS, and not HSPG core protein, binds FN-C/H II. These data support the hypothesis that a phosphatidylinositol-anchored HSPG on mouse melanoma cells (MPIHP-63) initiates recognition to FN-C/H II, and implicate PI-associated signal transduction pathways in mediating melanoma cell adhesion to this defined ligand.
来自转移性小鼠黑色素瘤细胞的细胞表面硫酸乙酰肝素蛋白聚糖（HSPG）启动细胞与合成肽FN-C/H II的黏附，FN-C/H II是一种来自纤连蛋白33-kD A链衍生片段的肝素结合肽。小鼠黑色素瘤细胞对FN-C/H II的黏附对可溶性肝素和用硫酸乙酰肝素酶预处理小鼠黑色素瘤细胞敏感。相比之下，细胞对纤连蛋白合成肽CS1的黏附是通过α4β1整合素介导的，并且对肝素或硫酸乙酰肝素酶处理具有抗性。小鼠黑色素瘤细胞HSPG用[35S]硫酸盐进行代谢标记，并用去污剂提取。经过HPLC-DEAE纯化后，35S-HSPG从解离性CL-4B柱上洗脱，其分配系数（Kav）约为0.45，而35S-硫酸乙酰肝素（HS）链以Kav约为0.62洗脱。硫酸乙酰肝素酶消化后，HSPG含有一种主要的63-kD核心蛋白。针对从体内生长的小鼠黑色素瘤细胞纯化的HSPG产生的多克隆抗体也鉴定出一种63-kD核心蛋白。这种HSPG是一种完整的质膜成分，因为它与辛基琼脂糖亲和柱结合，并且抗HSPG抗体染色显示出细胞表面定位。HSPG通过磷脂酰肌醇（PI）连接锚定在细胞表面，部分证据是PI特异性磷脂酶C能够消除去污剂提取的HSPG与辛基琼脂糖的结合。此外，小鼠黑色素瘤HSPG核心蛋白可以用3H-乙醇胺进行代谢标记。抗HSPG抗体和抗HSPG IgG Fab单体抑制小鼠黑色素瘤细胞对FN-C/H II的黏附能力也证明了小鼠黑色素瘤细胞表面HSPG参与细胞对纤连蛋白的黏附。35S-HSPG和35S-HS与FN-C/H II亲和柱结合，洗脱需要0.25 M NaCl。然而，硫酸乙酰肝素酶处理的125I标记的HSPG未能结合FN-C/H II，表明是HS而不是HSPG核心蛋白结合FN-C/H II。这些数据支持这样的假设，即小鼠黑色素瘤细胞上的磷脂酰肌醇锚定的HSPG（MPIHP-63）启动对FN-C/H II的识别，并暗示PI相关信号转导途径在介导黑色素瘤细胞对这种特定配体的黏附中起作用。
