Wallon U M, Overall C M
Faculty of Dentistry and Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of British Columbia, 2199 Wesbrook Mall, Vancouver, British Columbia V6T 1Z3, Canada.
J Biol Chem. 1997 Mar 14;272(11):7473-81. doi: 10.1074/jbc.272.11.7473.
The binding properties of the COOH-terminal hemopexin-like domain (C domain) of human gelatinase A (matrix metalloproteinase-2, 72-kDa gelatinase) were investigated to determine whether the C domain has binding affinity for extracellular matrix and basement membrane components. Recombinant C domain (rC domain) (Gly417-Cys631) was expressed in Escherichia coli, and the purified protein, identified using two antipeptide antibodies, was determined by electrospray mass spectrometry to have a mass of 25,925 Da, within 0.1 Da of that predicted. As assessed by microwell substrate binding assays and by column affinity chromatography, the matrix proteins laminin, denatured type I collagen, elastin, SPARC (secreted protein that is acidic and rich in cysteine), tenascin, and MatrigelTM were not bound by the rC domain. Unlike the hemopexin-like domains of collagenase and stromelysin, the rC domain also did not bind native type I collagen. Nor were native or denatured types II, IV, V, and X collagen, or the NC1 domain of type VII collagen bound. However, binding to heparin and fibronectin (Kd, 1.1 x 10(-6) M) could be disrupted by 0.58-0.76 and 0.3 M NaCl, respectively. Using nonoverlapping chymotrypsin-generated fragments of fibronectin, binding sites for the rC domain were found on both the 40-kDa heparin binding and the 120-kDa cell binding fibronectin domains (Kd values, approximately 4-6 x 10(-7) M). The Ca2+ ion, but not the potential structural Zn2+ ion, were found to be essential for maintaining the binding properties of the protein. The apo-form of the rC domain did not bind heparin, and both ethylenediaminetetraacetic acid and the specific Ca2+ ion chelator 1, 2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid, but not the Zn2+ ion chelator 1,10-phenanthroline, eluted the holo form of the rC domain from both heparin-Sepharose and fibronectin. Inductive coupled plasma mass spectrometry also did not detect a Zn2+ ion in the rC domain. In contrast, reduction with 65 mM dithiothreitol did not interfere with heparin binding, further emphasizing the crucial structural role played by the Ca2+ ion. Together, these data demonstrate for the first time that the hemopexin-like domain of gelatinase A has a binding site for fibronectin and heparin, and that Ca2+ ions are important in maintaining the structure and function of the domain.
研究了人明胶酶A（基质金属蛋白酶-2，72 kDa明胶酶）COOH末端类血红素结合蛋白结构域（C结构域）的结合特性，以确定C结构域是否对细胞外基质和基底膜成分具有结合亲和力。重组C结构域（rC结构域）（Gly417-Cys631）在大肠杆菌中表达，使用两种抗肽抗体鉴定的纯化蛋白，通过电喷雾质谱法测定其质量为25,925 Da，与预测值相差在0.1 Da以内。通过微孔板底物结合试验和柱亲和色谱评估，基质蛋白层粘连蛋白、变性I型胶原、弹性蛋白、SPARC（酸性且富含半胱氨酸的分泌蛋白）、腱生蛋白和基质胶均不与rC结构域结合。与胶原酶和基质溶解素的类血红素结合蛋白结构域不同，rC结构域也不结合天然I型胶原。天然或变性的II、IV、V和X型胶原，或VII型胶原的NC1结构域也不与之结合。然而，分别用0.58 - 0.76和0.3 M NaCl可破坏与肝素和纤连蛋白的结合（解离常数，1.1×10⁻⁶ M）。使用纤连蛋白经胰凝乳蛋白酶产生的不重叠片段，在40 kDa肝素结合和120 kDa细胞结合纤连蛋白结构域上均发现了rC结构域的结合位点（解离常数约为4 - 6×10⁻⁷ M）。发现Ca²⁺离子而非潜在的结构Zn²⁺离子对于维持该蛋白的结合特性至关重要。rC结构域的脱辅基形式不结合肝素，乙二胺四乙酸和特异性Ca²⁺离子螯合剂1,2 - 双（2 - 氨基苯氧基）乙烷 - N,N,N',N' - 四乙酸，但不是Zn²⁺离子螯合剂1,10 - 菲咯啉，可将rC结构域的全蛋白形式从肝素 - 琼脂糖和纤连蛋白上洗脱下来。电感耦合等离子体质谱法也未在rC结构域中检测到Zn²⁺离子。相反，用65 mM二硫苏糖醇还原并不干扰肝素结合，进一步强调了Ca²⁺离子所起的关键结构作用。总之，这些数据首次证明明胶酶A的类血红素结合蛋白结构域具有纤连蛋白和肝素的结合位点，并且Ca²⁺离子对于维持该结构域的结构和功能很重要。
