Syndecan Binding Sites in the Laminin 1 Chain G Domain
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- 作者：Nobuharu Suzuki ; Naoki Ichikawa ; Shingo Kasai ; Masanori Yamada ; Norio Nishi ; Hiroshi Morioka ; Hironobu Yamashita ; Yasuo Kitagawa ; Atsushi Utani ; Matthew P. Hoffman ; and Motoyoshi Nomizu
- 刊名：Biochemistry
- 出版年：2003
- 出版时间：November 4, 2003
- 年：2003
- 卷：42
- 期：43
- 页码：12625 - 12633
- 全文大小：229K
- 年卷期：v.42,no.43(November 4, 2003)
- ISSN：1520-4995
文摘
The laminin 
1 chain G domain has multiple biological activities. Previously, we identifiedcell binding sequences in the laminin 1 chain G domain by screening 113 synthetic peptide-polystyrenebeads for cell attachment activity. Here, we have used a recombinant protein of the laminin 1 G domain(rec-1G) and a large set of synthetic peptides to further identify and characterize heparin, cell, andsyndecan-4 binding sites in the laminin 1 chain G domain. The rec-1G protein promoted both cellattachment and heparin binding (KD = 19 nM). Cell attachment to the rec-1G protein was inhibited60% by heparin and 30% by EDTA. The heparin binding sites were identified by competing heparinbinding to the rec-1G protein with 110 synthetic peptides in solution. Only two peptides, AG73 (IC50 =147 
M) and AG75 (IC50 = 206 M), inhibited heparin binding to rec-1G. When the peptides werecompared in a solid-phase heparin binding assay, AG73 showed more heparin binding than AG75. AG73also inhibited fibroblast attachment to the rec-1G protein, but AG75 did not. Cell attachment to thepeptides was studied using peptide-coated plates and peptide-conjugated sepharose beads. AG73 promotedcell 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 glandsin organ culture. Furthermore, the rec-1G protein bound syndecan-4, and both AG73 and AG75 inhibitedthis binding. These results suggest that the AG73 and AG75 sites are important for heparin and syndecan-4binding in the laminin 1 chain G domain. These sites may play a critical role in the diverse biologicalactivities involving heparin and syndecan-4 binding.
1 chain G domain has multiple biological activities. Previously, we identifiedcell binding sequences in the laminin 1 chain G domain by screening 113 synthetic peptide-polystyrenebeads for cell attachment activity. Here, we have used a recombinant protein of the laminin 1 G domain(rec-1G) and a large set of synthetic peptides to further identify and characterize heparin, cell, andsyndecan-4 binding sites in the laminin 1 chain G domain. The rec-1G protein promoted both cellattachment and heparin binding (KD = 19 nM). Cell attachment to the rec-1G protein was inhibited60% by heparin and 30% by EDTA. The heparin binding sites were identified by competing heparinbinding to the rec-1G protein with 110 synthetic peptides in solution. Only two peptides, AG73 (IC50 =147 M) and AG75 (IC50 = 206 M), inhibited heparin binding to rec-1G. When the peptides werecompared in a solid-phase heparin binding assay, AG73 showed more heparin binding than AG75. AG73also inhibited fibroblast attachment to the rec-1G protein, but AG75 did not. Cell attachment to thepeptides was studied using peptide-coated plates and peptide-conjugated sepharose beads. AG73 promotedcell 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 glandsin organ culture. Furthermore, the rec-1G protein bound syndecan-4, and both AG73 and AG75 inhibitedthis binding. These results suggest that the AG73 and AG75 sites are important for heparin and syndecan-4binding in the laminin 1 chain G domain. These sites may play a critical role in the diverse biologicalactivities involving heparin and syndecan-4 binding.