A polymorphism in thrombospondin-1 associated with familial premature coronary artery disease alters Ca2+ binding
A polymorphism in thrombospondin-1 associated with familial premature coronary artery disease alters Ca2+ binding
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DOI：
10.1074/jbc.m409632200
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发表时间：
2004-12-10
影响因子：
4.8
通讯作者：
Mosher, DF
中科院分区：
文献类型：
--
作者：
Hannah, BLA;Misenheimer, TM;Mosher, DF
导致血小板反应蛋白-1中丝氨酸（Ser-700）残基700替换天冬酰胺（Asn-700）的单核苷酸多态性与家族性早发冠状动脉疾病相关。多态性位于13个Ca 2+结合基序中的第一个，在一个共有序列中，Asn-700可能与Ca 2+配位。由相邻的表皮生长因子样模块和Ca 2+结合区（E3 Ca）组成的构建体的平衡透析表明，在低[Ca 2 +]下，E3 Ca Ser-700比E3 Ca Asn-700结合的Ca 2+显著更少。假设这种差异是由于Ser-700蛋白中的结合位点的损失进行了测试与截断E3 Ca含有四个（Tr 4），三个（Tr 3），两个（Tr 2），或一个（Tr 1）N-末端Ca 2+结合基序。Ser-700截短构建体比匹配的Asn-700构建体少结合1个Ca 2+，并表现出降低的结合亲和力。通过向Asn-700 Tr 2、Tr 3和Tr 4构建体中加入Ca 2+，协同淬灭了最N-末端基序中残基698处色氨酸（Trp-698）的固有荧光。在Ser-700构建体中，Trp-698的淬灭在Tr 2和Tr 3构建体中是不完全的，并且仅在Tr 4构建体中是完全的。Ca 2+诱导的Ser-700结构的淬灭需要更高的[Ca 2 +]，并且如停流实验中所示比Asn-700结构的淬灭更慢。Tb 3+对Asn-700和Ser-700构建体的荧光猝灭作用相当，但Tb 3+没有发现这种差异。因此，Ser-700多态性改变了第一个Ca 2+结合基序中快速填充的高亲和力Ca 2+结合位点。较慢的Ca 2+结合到相邻的图案部分补偿的变化。
A single nucleotide polymorphism that results in substitution at residue 700 of a serine (Ser-700) for an asparagine (Asn-700) in thrombospondin-1 is associated with familial premature coronary artery disease. The polymorphism is located in the first of 13 Ca2+-binding motifs, within a consensus sequence in which Asn-700 likely coordinates Ca2+. Equilibrium dialysis of constructs comprised of the adjoining epidermal growth factor-like module and the Ca2+-binding region (E3Ca) demonstrated that E3Ca Ser-700 binds significantly less Ca2+ than E3Ca Asn-700 at low [Ca2+]. The hypothesis that this difference is due to loss of a binding site in Ser-700 protein was tested with truncations of E3Ca containing four (Tr4), three (Tr3), two (Tr2), or one (Tr1) N-terminal Ca2+-binding motifs. The Ser-700 truncation constructs bound 1 fewer Ca2+ than matching Asn-700 constructs and exhibited decreased binding affinities. Intrinsic fluorescence of a tryptophan at residue 698 (Trp-698) in the most N-terminal motif was cooperatively quenched by the addition of Ca2+ to Asn-700 Tr2, Tr3, and Tr4 constructs. In Ser-700 constructs, quenching of Trp-698 was incomplete in the Tr2 and Tr3 constructs and complete only in the Tr4 construct. Ca2+-induced quenching of Ser-700 constructs required higher [Ca2+] and was slower as shown in stopped-flow experiments than quenching of Asn-700 constructs. Such differences were not found with Tb3+, which quenched the fluorescence of Asn-700 and Ser-700 constructs equivalently. Thus, the Ser-700 polymorphism alters a rapidly filled, high affinity Ca2+-binding site in the first Ca2+-binding motif. Slower Ca2+ binding to adjoining motifs partly compensates for the change.