The Heparin-Binding Activity of Secreted Modular Calcium-Binding Protein 1 (SMOC-1) Modulates Its Cell Adhesion Properties

Secreted modular calcium-binding proteins 1 and 2 (SMOC-1 and SMOC-1) are extracellular calcium- binding proteins belonging to the BM-40 family of proteins. In this work we have identified a highly basic region in the extracellular calcium-binding (EC) domain of the SMOC-1 similar to other known glycosaminoglycan-binding motifs. Size-exclusion chromatography shows that full length SMOC-1 as well as its C-terminal EC domain alone bind heparin and heparan sulfate, but not the related chondroitin sulfate or dermatan sulfate glycosaminoglycans. Intrinsic tryptophan fluorescence measurements were used to quantify the binding of heparin to full length SMOC-1 and the EC domain alone. The calculated equilibrium dissociation constants were in the lower micromolar range. The binding site consists of two antiparallel alpha helices and mutagenesis experiments have shown that heparin-binding residues in both helices must be replaced in order to abolish heparin binding. Furthermore, we show that the SMOC-1 EC domain, like the SMOC-2 EC domain, supports the adhesion of epithelial HaCaT cells. Heparin-binding impaired mutants failed to support S1EC-mediated cell adhesion and together with the observation that S1EC in complex with soluble heparin attenuated cell adhesion we conclude that a functional and accessible S1EC heparin-binding site mediates adhesion of epithelial cells to SMOC-1.分泌型模块化钙结合蛋白1与2（Secreted modular calcium-binding proteins 1 and 2，SMOC-1和SMOC-2）属于BM-40蛋白家族的细胞外钙结合蛋白。本研究在SMOC-1的细胞外钙结合（extracellular calcium-binding, EC）结构域中鉴定出一段高度碱性区域，该区域与其他已知的糖胺聚糖（glycosaminoglycan）结合基序相似。尺寸排阻色谱（Size-exclusion chromatography）结果显示，全长SMOC-1及其单独的C端EC结构域均可结合肝素（heparin）与硫酸乙酰肝素（heparan sulfate），但无法结合硫酸软骨素（chondroitin sulfate）或硫酸皮肤素（dermatan sulfate）这类相关糖胺聚糖。本研究采用内源色氨酸（tryptophan）荧光测定法，定量分析了全长SMOC-1及其单独的EC结构域与肝素的结合情况，计算得到的平衡解离常数（equilibrium dissociation constants）处于低微摩尔浓度范围。该结合位点由两条反向平行的α螺旋（alpha helices）组成，诱变实验（mutagenesis experiments）证实，需同时替换两条螺旋中的肝素结合残基，才能完全阻断肝素结合。此外，本研究发现，与SMOC-2的EC结构域类似，SMOC-1的EC结构域可支持上皮HaCaT细胞的黏附。肝素结合功能受损的突变体无法介导S1EC依赖的细胞黏附；结合可溶性肝素可减弱S1EC介导的细胞黏附这一实验结果，我们由此得出结论：具备功能且可及的S1EC肝素结合位点，可介导上皮细胞与SMOC-1的黏附。