Identification in collagen type I of an integrin α2β1-binding site containing an essential GER sequence
Identification in collagen type I of an integrin α2β1-binding site containing an essential GER sequence
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DOI：
10.1074/jbc.273.50.33287
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发表时间：
1998-12-11
影响因子：
4.8
通讯作者：
Barnes, MJ
中科院分区：
文献类型：
--
作者：
Knight, CG;Morton, LF;Barnes, MJ
已知I型胶原衍生片段α（1）（I）CB 3与母体胶原一样有效地识别血小板胶原受体整联蛋白α（2）β（1），尽管它缺乏血小板聚集活性。我们已经合成了七个重叠的肽片段，这些肽自发地组装成三螺旋。基于它们结合纯化的α（2）β（1）和重组的α（2）A结构域的能力，以及它们支持α（2）β（1）介导的细胞粘附的能力，我们鉴定了两种肽，CB 3（I）-5和-6，其含有α（2）β（1）识别位点。合成含有肽5和6之间的重叠序列的肽CB 3（I）-5/6，使我们能够在15个残基序列GFP*GERGVEGPP*GPA（其中P* 代表羟脯氨酸）内定位结合位点，对应于I型胶原α（1）链的残基502-516。发现格尔三联体中的Glu和Arg残基对于识别是必需的，因为用Ala取代任一残基都会导致α（2）A结构域结合的丧失。相比之下，GVE中Glu的取代并没有减少结合，而是略微增强了结合。我们不能检测到含有推定的α（2）β（1）识别序列DGEA的肽CB 3（I）-2对α（2）β（1）的显著识别。肽CB 3（I）-1至-6与肽CB 3（I）-5/6一起表现出良好的血小板聚集活性，在某些情况下优于胶原蛋白。然而，肽CB 3（I）-7是无活性的，表明存在抑制元件，其可能解释了母体α（1）（I）CB 3片段缺乏聚集活性。
The collagen type I-derived fragment alpha(1)(I)CB3 is known to recognize the platelet collagen receptor integrin alpha(2)beta(1) as effectively as the parent collagen, although it lacks platelet-aggregatory activity. We have synthesized the fragment as seven overlapping peptides that spontaneously assemble into triple helices. On the basis of their capacity to bind purified alpha(2)beta(1) and the recombinant alpha(2) A-domain, and their ability to support alpha(2)beta(1)-mediated cell adhesion, we identified two peptides, CB3(I)-5 and -6, which contain an alpha(2)beta(1) recognition site. Synthesis of the peptide CB3(I)-5/6, containing the overlap sequence between peptides 5 and 6, allowed us to locate the binding site within the 15-residue sequence, GFP*GERGVEGPP*GPA (where P* represents hydroxyproline), corresponding to residues 502-516 of the collagen type I alpha(1) chain. The Glu and Arg residues in the GER triplet were found to be essential for recognition since substitution of either residue with Ala caused a loss of alpha(2) A-domain binding. By contrast, substitution of the Glu in GVE did not reduce binding, but rather enhanced it slightly. We were unable to detect significant recognition of alpha(2)beta(1) by the peptide CB3(I)-2 containing the putative alpha(2)beta(1) recognition sequence DGEA. Peptides CB3(I)-1 to -6, together with peptide CB3(I)-5/6, exhibited good platelet-aggregatory activity, in some cases better than collagen. However, peptide CB3(I)-7 was inactive, suggesting the presence of an inhibitory element that might account for the lack of aggregatory activity of the parent alpha(1)(I)CB3 fragment.