Varner J A, Burger M M, Kaufman J F
Biozentrum, University of Basel, Switzerland.
J Biol Chem. 1988 Jun 15;263(17):8498-508.
Two extracellular matrix cell surface proteins which bind the proteoglycan-like aggregation factor from the marine sponge Microciona prolifera (MAF) and which may function as physiological receptors for MAF were identified and characterized for the first time. By probing nitrocellulose blots of nonreducing sodium dodecyl sulfate gels containing whole sponge cell protein with iodinated MAF, a 210- and a 68-kDa protein, which have native molecular masses of approximately 200-400 and 70 kDa, were identified. MAF binding to blots is species-specific. It is also sensitive to reduction and is completely abolished by pretreatment of live cells with proteases, as was cellular aggregation, indicating that the 210- and 68-kDa proteins may be located on the cell surface. The additional observations that the 68 kDa is an endoglycosidase F-sensitive glycoprotein and that antisera against whole sponge cells or membranes can immunoprecipitate the 210 kDa when prebound to intact cells are consistent with a cell surface location. Both proteins can be isolated from sponge cell membranes and from the sponge skeleton (insoluble extracellular matrix), but the 210-kDa MAF-binding protein can also be found in the soluble extracellular matrix (buffer washes of cells and skeleton) as well. A third MAF-binding protein of molecular mass 95 kDa was also found in the sponge extracellular matrix but rarely on cells. Both of the cell-associated 210- and 68-kDa proteins are nonintegral membrane proteins, based on Triton X-114 phase separation, flotation of liposomes containing sponge membrane lysates, and their extraction from membranes by buffer washes. Both proteins bind MAF affinity resins, indicating that they each exhibit a moderate affinity for MAF under native conditions. They can also be separated from each other and from the bulk of the protein in an octylpolyoxyethylene extract of membranes by fast protein liquid chromatography Mono Q anion exchange chromatography, as assessed by native dot blot and denaturing Western blot assays. Although neither protein bound to heparin, gelatin, hexosamine, or uronic acid-Sepharose resins, their affinity for an invertebrate proteoglycan, their roles in sponge cell adhesion, and their peripheral membrane protein natures suggest that they may represent early invertebrate analogs of cell-associated vertebrate extracellular matrix adhesion proteins, such as fibronectin or vitronectin, or else an entirely novel set of cell adhesion molecules.
首次鉴定并表征了两种细胞外基质细胞表面蛋白，它们能结合来自海洋海绵微小多细胞海绵（MAF）的蛋白聚糖样聚集因子，可能作为MAF的生理受体。通过用碘化MAF探测含有整个海绵细胞蛋白的非还原十二烷基硫酸钠凝胶的硝酸纤维素印迹，鉴定出了一种210 kDa和一种68 kDa的蛋白，它们的天然分子量分别约为200 - 400 kDa和70 kDa。MAF与印迹的结合具有物种特异性。它对还原也敏感，并且用蛋白酶预处理活细胞会完全消除这种结合，细胞聚集也同样被消除，这表明210 kDa和68 kDa的蛋白可能位于细胞表面。另外的观察结果表明，68 kDa是一种对内切糖苷酶F敏感的糖蛋白，并且针对整个海绵细胞或膜的抗血清在预结合到完整细胞时可以免疫沉淀210 kDa，这与细胞表面定位一致。这两种蛋白都可以从海绵细胞膜和海绵骨架（不溶性细胞外基质）中分离出来，但210 kDa的MAF结合蛋白也可以在可溶性细胞外基质（细胞和骨架的缓冲液洗涤液）中找到。在海绵细胞外基质中还发现了第三种分子量为95 kDa的MAF结合蛋白，但在细胞上很少见。基于Triton X - 114相分离、含有海绵膜裂解物的脂质体浮选以及通过缓冲液洗涤从膜中提取，细胞相关的210 kDa和68 kDa的蛋白都是非整合膜蛋白。这两种蛋白都能结合MAF亲和树脂，表明它们在天然条件下对MAF都表现出中等亲和力。通过天然斑点印迹和变性蛋白质印迹分析评估，它们还可以通过快速蛋白质液相色谱Mono Q阴离子交换色谱从膜的辛基聚氧乙烯提取物中的彼此以及大部分蛋白质中分离出来。尽管这两种蛋白都不与肝素、明胶、己糖胺或糖醛酸 - 琼脂糖树脂结合，但它们对无脊椎动物蛋白聚糖的亲和力、在海绵细胞粘附中的作用以及它们的外周膜蛋白性质表明，它们可能代表细胞相关的脊椎动物细胞外基质粘附蛋白（如纤连蛋白或玻连蛋白）的早期无脊椎动物类似物，或者是一组全新的细胞粘附分子。
