Identification of Neurite Outgrowth Active Sites on the Laminin 4 Chain G Domain
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- 作者：Naoki Ichikawa ; Shingo Kasai ; Nobuharu Suzuki ; Norio Nishi ; Shinya Oishi ; Nobutaka Fujii ; Yuichi Kadoya ; Kozo Hatori ; Yoshikuni Mizuno ; Motoyoshi Nomizu ; and Eri Arikawa-Hirasawa
- 刊名：Biochemistry
- 出版年：2005
- 出版时间：April 19, 2005
- 年：2005
- 卷：44
- 期：15
- 页码：5755 - 5762
- 全文大小：670K
- 年卷期：v.44,no.15(April 19, 2005)
- ISSN：1520-4995
文摘
The laminin 
4 chain is widely distributed in various mesodermal tissues, including theperineurium of peripheral nerves, dorsal root ganglion (DRG), skeletal muscle, and capillaries, andplays important roles in synaptic specialization at the neuromuscular junction and in microvascularformation. The C-terminal globular domain (G domain) of the laminin 4 chain was previously found tobe critical for heparin binding and cell attachment activity. Here, we focused on neurite outgrowth activityof the laminin 4 chain G domain. We found that the recombinant 4 chain G domain protein (rec-4G)promoted neurite outgrowth of rat pheochromocytoma PC12 cells. When 114 overlapping synthetic peptidesthat covered the entire G domain were tested for neurite outgrowth activity, nine peptides were active,but the 105 remaining peptides did not exhibit activity. Three of the nine active peptides, A4G6(LAIKNDNLVYVY), A4G20 (DVISLYNFKHIY), and A4G107 (VIRDSNVVQLDV), strongly promotedneurite outgrowth of PC12 cells. A4G107 was found to form amyloid-like fibrils in Congo red, X-ray,and electron microscopy analyses. We also synthesized cyclic peptides to evaluate their conformationalrequirements. Cyclic peptide A4G82X (cyc-A4G82X;TLFLAHGRLVFX, where X is norleucine)significantly enhanced neurite outgrowth activity, but the rest of the cyclic peptides eliminated the activity.The A4G82 sequence is located on the loop region, suggesting that the activity of A4G82 is required fora loop conformation. These peptides also exhibited neurite outgrowth activity with dorsal root ganglion(DRG) explants and with DRG cells from E14.5 mouse embryos, indicating that they are active in bothneuronal cell lines and native neuronal cells. Taken together, the data suggest that the peptides from thelaminin 4 chain G domain promote neurite outgrowth activity via a specific conformation.
4 chain is widely distributed in various mesodermal tissues, including theperineurium of peripheral nerves, dorsal root ganglion (DRG), skeletal muscle, and capillaries, andplays important roles in synaptic specialization at the neuromuscular junction and in microvascularformation. The C-terminal globular domain (G domain) of the laminin 4 chain was previously found tobe critical for heparin binding and cell attachment activity. Here, we focused on neurite outgrowth activityof the laminin 4 chain G domain. We found that the recombinant 4 chain G domain protein (rec-4G)promoted neurite outgrowth of rat pheochromocytoma PC12 cells. When 114 overlapping synthetic peptidesthat covered the entire G domain were tested for neurite outgrowth activity, nine peptides were active,but the 105 remaining peptides did not exhibit activity. Three of the nine active peptides, A4G6(LAIKNDNLVYVY), A4G20 (DVISLYNFKHIY), and A4G107 (VIRDSNVVQLDV), strongly promotedneurite outgrowth of PC12 cells. A4G107 was found to form amyloid-like fibrils in Congo red, X-ray,and electron microscopy analyses. We also synthesized cyclic peptides to evaluate their conformationalrequirements. Cyclic peptide A4G82X (cyc-A4G82X;TLFLAHGRLVFX, where X is norleucine)significantly enhanced neurite outgrowth activity, but the rest of the cyclic peptides eliminated the activity.The A4G82 sequence is located on the loop region, suggesting that the activity of A4G82 is required fora loop conformation. These peptides also exhibited neurite outgrowth activity with dorsal root ganglion(DRG) explants and with DRG cells from E14.5 mouse embryos, indicating that they are active in bothneuronal cell lines and native neuronal cells. Taken together, the data suggest that the peptides from thelaminin 4 chain G domain promote neurite outgrowth activity via a specific conformation.