Multiple binding sites in collagen type I for the integrins alpha1beta1 and alpha2beta1.
Multiple binding sites in collagen type I for the integrins alpha1beta1 and alpha2beta1.
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发表时间：
2000
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通讯作者：
Y. Xu;S. Gurusiddappa;R. Rich;R. Owens;D. Keene;R. Mayne;A. Höök;M. Höök
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作者：
Y. Xu;S. Gurusiddappa;R. Rich;R. Owens;D. Keene;R. Mayne;A. Höök;M. Höök
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）。在这里，我们使用表面等离子体共振比较了α（2）β（1）（α（2）A）的重组A结构域与I型胶原的结合和α（1）A的结合，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，每个胶原分子约3个结合位点的K（D）约为10 μ m。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II有效地抑制了α（1）A和α（2）A与I型胶原的结合，而肽III则适度地抑制了α（1）A和α（2）A与I型胶原的结合。I型胶原蛋白的N-末端位点在所有三条链中都具有序列GLOGER。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., et 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 approximately 10 microm at approximately 3 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., et 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 alpha(1)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.