Heremans A, De Cock B, Cassiman J J, Van den Berghe H, David G
Center for Human Genetics, University of Leuven, Belgium.
J Biol Chem. 1990 May 25;265(15):8716-24.
The extracellular matrix of cultured human lung fibroblasts contains one major heparan sulfate proteoglycan. This proteoglycan contains a 400-kDa core protein and is structurally and immunochemically identical or closely related to the heparan sulfate proteoglycans that occur in basement membranes. Because heparitinase does not release the core protein from the matrix of cultured cells, we investigated the binding interactions of this heparan sulfate proteoglycan with other components of the fibroblast extracellular matrix. Both the intact proteoglycan and the heparitinase-resistant core protein were found to bind to fibronectin. The binding of 125I-labeled core protein to immobilized fibronectin was inhibited by soluble fibronectin and by soluble cold core protein but not by albumin or gelatin. A Scatchard plot indicates a Kd of about 2 x 10(-9) M. Binding of the core protein was also inhibited by high concentrations of heparin, heparan sulfate, or chrondroitin sulfate and was sensitive to high salt concentrations. Thermolysin fragmentation of the 125I-labeled proteoglycan yielded glycosamino-glycan-free core protein fragments of approximately 110 and 62 kDa which bound to both fibronectin and heparin columns. The core protein-binding capacity of fibronectin was very sensitive to proteolysis. Analysis of thermolytic and alpha-chymotryptic fragments of fibronectin showed binding of the intact proteoglycan and of its isolated core protein to a protease-sensitive fragment of 56 kDa which carried the gelatin-binding domain of fibronectin and to a protease-sensitive heparin-binding fragment of 140 kDa. Based on the NH2-terminal amino acid sequence analyses of the 56- and 140-kDa fragments, the core protein-binding domain in fibronectin was tentatively mapped in the area of overlap of the two fragments, carboxyl-terminally from the gelatin-binding domain, possibly in the second type III repeat of fibronectin. These data document a specific and high affinity interaction between fibronectin and the core protein of the matrix heparan sulfate proteoglycan which may anchor the proteoglycan in the matrix.
培养的人肺成纤维细胞的细胞外基质含有一种主要的硫酸乙酰肝素蛋白聚糖。这种蛋白聚糖含有一个400 kDa的核心蛋白，在结构和免疫化学上与基底膜中存在的硫酸乙酰肝素蛋白聚糖相同或密切相关。由于肝素酶不能从培养细胞的基质中释放核心蛋白，我们研究了这种硫酸乙酰肝素蛋白聚糖与成纤维细胞外基质其他成分的结合相互作用。完整的蛋白聚糖和抗肝素酶的核心蛋白均被发现与纤连蛋白结合。可溶性纤连蛋白和可溶性冷核心蛋白可抑制125I标记的核心蛋白与固定化纤连蛋白的结合，但白蛋白或明胶则无此作用。Scatchard图表明解离常数约为2×10(-9)M。高浓度的肝素、硫酸乙酰肝素或硫酸软骨素也可抑制核心蛋白的结合，且其对高盐浓度敏感。125I标记的蛋白聚糖经嗜热菌蛋白酶裂解产生了约110和62 kDa的无糖胺聚糖的核心蛋白片段，这些片段可与纤连蛋白柱和肝素柱结合。纤连蛋白的核心蛋白结合能力对蛋白水解非常敏感。对纤连蛋白的嗜热菌蛋白酶和α-胰凝乳蛋白酶片段的分析表明，完整的蛋白聚糖及其分离的核心蛋白与一个56 kDa的蛋白酶敏感片段结合，该片段携带纤连蛋白的明胶结合结构域，还与一个140 kDa的蛋白酶敏感肝素结合片段结合。根据对56 kDa和140 kDa片段的氨基末端氨基酸序列分析，纤连蛋白中的核心蛋白结合结构域初步定位在两个片段重叠的区域，位于明胶结合结构域的羧基末端，可能在纤连蛋白的第二个III型重复序列中。这些数据证明了纤连蛋白与基质硫酸乙酰肝素蛋白聚糖的核心蛋白之间存在特异性的高亲和力相互作用，这种相互作用可能将蛋白聚糖锚定在基质中。
