Multiple binding sites in collagen type I for the integrins α1β1 and α2β1
Multiple binding sites in collagen type I for the integrins α1β1 and α2β1
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DOI：
10.1074/jbc.m007668200
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发表时间：
2000-12-15
影响因子：
4.8
通讯作者：
Höök, M
中科院分区：
文献类型：
--
作者：
Xu, Y;Gurusiddappa, S;Höök, M
整合素α（1）β（1）和α（2）β（1）是真核细胞表面上的两种主要胶原受体。与胶原蛋白的结合主要是由于α链N末端附近的A结构域。先前，我们报道了α（1）β（1）（α（1）A）的重组A-结构域在I型胶原中具有至少两种亲和力类别的结合位点（Rich，R. L.，等人（1999）J.Biol.Chem.274，24906-24913），在此，我们比较了α（2）β（1）的重组A-结构域的结合使用表面等离子体共振将α（2）A与I型胶原的结合位点与α（1）A的结合位点进行比较，并显示α（2）A在I型胶原中仅表现出一类可检测的结合位点，在每个胶原蛋白分子的3个结合位点处具有类似于10 μ M的K-D。我们进一步证明，α（1）A和α（2）A相互竞争结合I型胶原蛋白在酶联免疫吸附试验（ELISA），这表明，在胶原蛋白的结合位点的两个A-结构域重叠或彼此相邻。通过使用旋转阴影法，可观察到α（1）A-和α（2）A-前胶原的复合物。形态测定分析表明，两个A-结构域的I型前胶原分子沿着有三个主要结合区域（靠近N末端、中部和靠近C末端）。α（1）A和α（2）A各自结合区的位置彼此重叠或相邻，与ELISA结果一致。对I型胶原蛋白序列的分析表明，格尔或GER样基序存在于每个结合区域，值得注意的是，中心区域含有GFO格尔序列，其先前被鉴定为对α（1）A和α（2）A的高亲和力位点（Knight，C. G.，等人（2000）J.Biol.Chem.275，35-40）。合成含有GLOGERGRO（肽I，靠近N末端）、GFOGERGVQ（肽II，中间）和GASGERGRO（肽III，靠近C末端）的肽。肽I和肽II有效地抑制了α A、A和α（2）A与I型胶原的结合，而肽III则适度地抑制了这种结合。I型胶原的N-末端位点在所有三条链中都具有GLOGER序列。因此，似乎肽I代表了新发现的α（1）A和α（2）A的天然高亲和力位点。
Integrins alpha (1)beta (1) and alpha (2)beta (1) are two major collagen receptors on the surface of eukaryotic cells. Binding to collagen is primarily due to an A-domain near the N terminus of the alpha chains. Previously, we reported that recombinant A-domain of alpha (1)beta (1) (alpha (1)A) had at least two affinity classes of binding sites in type I collagen (Rich, R. L., ct al. (1999) J. Biol. Chem. 274, 24906-24913), Here, we compared the binding of the recombinant A-domain of alpha (2)beta (1) (alpha (2)A) to type I collagen with that of alpha (1)A using surface plasmon resonance and showed that alpha (2)A exhibited only one detectable class of binding sites in type I collagen, with a K-D of similar to 10 muM at similar to3 binding sites per collagen molecule. We further demonstrated that alpha (1)A and alpha (2)A competed with each other for binding to type I collagen in enzyme-linked immunosorbent assay (ELISA), suggesting that the binding sites in collagen for the two A-domains overlap or are adjacent to each other. By using rotary shadowing, the complexes of alpha (1)A- and alpha (2)A-procollagen were visualized. Morphometric analyses indicated three major binding regions (near the N terminus, in the central part, and near the C terminus) along the type I procollagen molecule for both A-domains. The positions of the respective binding regions for alpha (1)A and alpha (2)A were overlapping with or adjacent to each other, consistent with the ELISA results. Analysis of the sequences of type I collagen revealed that GER or GER-like motifs are present at each of the binding regions, and notably, the central region contains the GFOGER sequence, which was previously identified as a high affinity site for both alpha (1)A and alpha (2)A (Knight, C. G., ct al. (2000) J. Biol. Chem. 275, 35-40). Peptides containing GLOGERGRO (peptide I, near the N terminus), GFOGERGVQ (peptide II, central), and GASGERGPO (peptide III, near the C terminus) were synthesized. Peptides I and II effectively inhibited the binding of cli,A and alpha (2)A to type I collagen, while peptide III did so moderately, The N-terminal site in type I collagen has the sequence GLOGER in all three chains. Thus, it seems that peptide I represents a newly discovered native high affinity site for alpha (1)A and alpha (2)A.