期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 279, 期 50, 页码 51915-51922出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M409632200
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资金
- NHLBI NIH HHS [HL54462] Funding Source: Medline
- NIGMS NIH HHS [T32 GM08688] Funding Source: Medline
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.
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