Taraboletti G, Rao C N, Krutzsch H C, Liotta L A, Roberts D D
Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
J Biol Chem. 1990 Jul 25;265(21):12253-8.
A sulfatide-binding site on the globular end region of the long arm of laminin has been identified. Following proteolytic digestion with thermolysin, an intact fragment of the laminin A chain carboxyl-terminal domain exhibiting sulfatide-binding activity was isolated using gel filtration and heparin affinity chromatography. This fragment is composed of two peptides that are covalently linked by at least one disulfide bond and encompass the carboxyl-terminal 394 amino acids of the A chain. The clusters of charged residues in the primary structure of these fragments are sufficient for heparin-binding activity but not sulfatide binding since reduction and alkylation of the fragments abolished sulfatide binding under conditions in which heparin binding was retained. Thus, sulfatide binding requires an intact three-dimensional structure. The iodinated fragment bound to A2058 melanoma and T47D breast carcinoma cells and could be displaced by the unlabeled fragment. Based on incorporation of [35S] sulfate, both cell lines synthesize sulfated glycolipids that bind to laminin. In agreement with previous data that indicate a synergistic interaction of the sulfatide-binding domain with other laminin-binding sites on melanoma cells during attachment, the isolated sulfatide-binding fragment significantly inhibited interaction of labeled intact laminin with melanoma and breast carcinoma cells in direct binding assays.
已确定层粘连蛋白长臂球状末端区域存在一个硫苷结合位点。用嗜热菌蛋白酶进行蛋白水解消化后，通过凝胶过滤和肝素亲和色谱法分离出具有硫苷结合活性的层粘连蛋白A链羧基末端结构域的完整片段。该片段由两个通过至少一个二硫键共价连接的肽组成，包含A链的羧基末端394个氨基酸。这些片段一级结构中的带电残基簇足以产生肝素结合活性，但不能产生硫苷结合活性，因为在保留肝素结合的条件下，片段的还原和烷基化消除了硫苷结合。因此，硫苷结合需要完整的三维结构。碘化片段与A2058黑色素瘤细胞和T47D乳腺癌细胞结合，且可被未标记的片段取代。基于[35S]硫酸盐的掺入，两种细胞系均合成与层粘连蛋白结合的硫酸化糖脂。与先前表明硫苷结合结构域在附着过程中与黑色素瘤细胞上其他层粘连蛋白结合位点存在协同相互作用的数据一致，在直接结合试验中，分离出的硫苷结合片段显著抑制标记的完整层粘连蛋白与黑色素瘤细胞和乳腺癌细胞的相互作用。
