Jang Jun-Hyeog, Hwang Jung-Hee, Chung Chong-Pyoung, Choung Pill-Hoon
Intellectual Biointerface Engineering Center, Seoul National University College of Dentistry, Seoul 110-749, Korea.
J Biol Chem. 2004 Jun 11;279(24):25562-6. doi: 10.1074/jbc.M403170200. Epub 2004 Apr 6.
The interaction between tenascin-C (TN-C), a multi-subunit extracellular matrix protein, and heparin was examined using a surface plasmon resonance-based technique on a Biacore system. The aims of the present study were to examine the affinity of fibronectin type III repeats of TN-C fragments (TNIII) for heparin, to investigate the role of the TNIII4 domains in the binding of TN-C to heparin, and to delineate a sequence of amino acids within the TNIII4 domain, which mediates cooperative heparin binding. At a physiological salt concentration, and pH 7.4, TNIII3-5 binds to heparin with high affinity (K(D) = 30 nm). However, a major heparin-binding site in TNIII5 produces a modest affinity binding at a K(D) near 4 microm, and a second site in TNIII4 enhances the binding by several orders of magnitude, although it was far too weak to produce an observable binding of TNIII4 by itself. Moreover, mutagenesis of the KEDK sequence in the TNIII4 domain resulted in the significant reduction of heparin-binding affinity. In addition, residues in the KEDK sequences are conserved in TN-C throughout mammalian evolution. Thus the structure-based sequence alignment, mutagenesis, and sequence conservation data together reveal a KEDK sequence in TNIII4 suggestive of a minor heparin-binding site. Finally, we demonstrate that TNIII4 contains binding sites for heparin sulfate proteoglycan and enhances the heparin sulfate proteoglycan-dependent human gingival fibroblast adhesion to TNIII5, thus providing the biological significance of heparin-binding site of TNIII4. These results suggest that the heparin-binding sites may traverse TNIII4-5 and thus require KEDK in TNIII4 for optimal heparin-binding.
利用Biacore系统上基于表面等离子体共振的技术，研究了多亚基细胞外基质蛋白腱生蛋白-C（TN-C）与肝素之间的相互作用。本研究的目的是检测TN-C片段（TNIII）中III型纤连蛋白重复序列对肝素的亲和力，研究TNIII4结构域在TN-C与肝素结合中的作用，并确定TNIII4结构域内介导协同肝素结合的氨基酸序列。在生理盐浓度和pH 7.4条件下，TNIII3-5与肝素高亲和力结合（K(D)=30 nm）。然而，TNIII5中的一个主要肝素结合位点在K(D)接近4μm时产生适度亲和力结合，TNIII4中的第二个位点将结合增强了几个数量级，尽管其本身过于微弱以至于无法产生可观察到的TNIII4结合。此外，TNIII4结构域中KEDK序列的诱变导致肝素结合亲和力显著降低。此外，KEDK序列中的残基在整个哺乳动物进化过程中在TN-C中保守。因此，基于结构的序列比对、诱变和序列保守性数据共同揭示了TNIII4中一个暗示次要肝素结合位点的KEDK序列。最后，我们证明TNIII4含有硫酸乙酰肝素蛋白聚糖的结合位点，并增强了硫酸乙酰肝素蛋白聚糖依赖性人牙龈成纤维细胞对TNIII5的黏附，从而提供了TNIII4肝素结合位点的生物学意义。这些结果表明，肝素结合位点可能贯穿TNIII4-5，因此需要TNIII4中的KEDK来实现最佳肝素结合。
