Syndecan binding sites in the laminin α1 chain G domain
Syndecan binding sites in the laminin α1 chain G domain
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DOI：
10.1021/bi030014s
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发表时间：
2003-11-04
期刊：
影响因子：
2.9
通讯作者：
Nomizu, M
中科院分区：
文献类型：
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作者：
Suzuki, N;Ichikawa, N;Nomizu, M
层粘连蛋白α 1链G结构域具有多种生物活性。在此之前，我们通过筛选113个合成肽-聚苯乙烯珠来确定层粘连蛋白α 1链G结构域的细胞结合序列。在这里，我们使用层粘连蛋白α 1G结构域的重组蛋白（rec- α 1g）和大量的合成肽来进一步鉴定和表征层粘连蛋白α 1链G结构域的肝素、细胞和syndecan-4结合位点。rec-alpha1G蛋白促进细胞附着和肝素结合（K-D = 19 nM）。肝素和EDTA分别抑制60%和30% rec-alpha1G蛋白的细胞附着。通过在溶液中与110个合成肽竞争肝素与rec-alpha1G蛋白的结合，确定了肝素的结合位点。只有AG73 （IC50 = 147 muM）和AG75 （IC50 = 206 muM）两种肽抑制肝素与rec-alpha1G的结合。当肽在固相肝素结合实验中进行比较时，AG73比AG75显示出更多的肝素结合。AG73也能抑制成纤维细胞对rec-alpha1G蛋白的附着，而AG75则没有。利用肽包被板和肽偶联的蔗糖球研究细胞对肽的附着。AG73在两种实验中均能促进细胞附着，而AG75仅在细胞头实验中表现出细胞附着活性。此外，在器官培养中，AG73抑制小鼠下颌腺分支形态的形成，而AG75没有。此外，rec-alpha1G蛋白结合syndecan-4， AG73和AG75都抑制这种结合。这些结果表明，AG73和AG75位点对层粘连蛋白α 1链G域的肝素和syndecan-4结合很重要。这些位点可能在涉及肝素和syndecan-4结合的多种生物活性中发挥关键作用。
The laminin alpha1 chain G domain has multiple biological activities. Previously, we identified cell binding sequences in the laminin alpha1 chain G domain by screening 113 synthetic peptide-polystyrene beads for cell attachment activity. Here, we have used a recombinant protein of the laminin alpha1 G domain (rec-alpha1G) and a large set of synthetic peptides to further identify and characterize heparin, cell, and syndecan-4 binding sites in the laminin alpha1 chain G domain. The rec-alpha1G protein promoted both cell attachment and heparin binding (K-D = 19 nM). Cell attachment to the rec-alpha1G protein was inhibited 60% by heparin and 30% by EDTA. The heparin binding sites were identified by competing heparin binding to the rec-alpha1G protein with 110 synthetic peptides in solution. Only two peptides, AG73 (IC50 = 147 muM) and AG75 (IC50 = 206 muM), inhibited heparin binding to rec-alpha1G. When the peptides were compared in a solid-phase heparin binding assay, AG73 showed more heparin binding than AG75. AG73 also inhibited fibroblast attachment to the rec-alpha1G protein, but AG75 did not. Cell attachment to the peptides was studied using peptide-coated plates and peptide-conjugated sepharose beads. AG73 promoted cell attachment in both assays, but AG75 only showed cell attachment activity in the bead assay. Additionally, AG73, but not AG75, inhibited branching morphogenesis of mouse submandibular glands in organ culture. Furthermore, the rec-alpha1G protein bound syndecan-4, and both AG73 and AG75 inhibited this binding. These results suggest that the AG73 and AG75 sites are important for heparin and syndecan-4 binding in the laminin alpha1 chain G domain. These sites may play a critical role in the diverse biological activities involving heparin and syndecan-4 binding.