Localization of the laminin α4 chain in the skin and identification of a heparin-dependent cell adhesion site within the laminin α4 chain C-terminal LG4 module
Localization of the laminin α4 chain in the skin and identification of a heparin-dependent cell adhesion site within the laminin α4 chain C-terminal LG4 module
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DOI：
10.1111/j.0022-202x.2004.22325.x
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发表时间：
2004-03-01
影响因子：
6.5
通讯作者：
Utani, A
中科院分区：
文献类型：
--
作者：
Matsuura, H;Momota, Y;Utani, A
层粘连蛋白α 4链是层粘连蛋白-8/9的组分，在内皮细胞、外周神经和肌纤维的基底膜中表达。层粘连蛋白α 4链在皮肤中的定位和功能尚未阐明。通过用特异性抗体进行免疫染色，我们在这里证明了α 4链位于血管的基底膜区，并且还与真皮中的成纤维细胞样细胞相关。Western blot显示培养的成纤维细胞分泌含有α 4链的层粘连蛋白三聚体。我们还关注了人层粘连蛋白α 4 LG 4模块的细胞粘附活性，因为层粘连蛋白α 3的相应LG 4模块先前被鉴定为具有细胞粘附活性。重组人α 4 LG 4对肝素依赖性成纤维细胞粘附具有活性。用覆盖整个α 4 LG 4模块的19种合成肽进行的筛选测定鉴定出三种肽（HA 4G 82：TLFLAHGRLVYM; HA 4G 83：LVYMFNVGHKKL;和HA 4G 90：TEATWKIKGPIYL）作为肝素和硫酸乙酰肝素依赖性细胞粘附的活性位点。丝氨酸取代的肽表明，两个基本的残基，组氨酸和精氨酸，在HA 4G 82细胞粘附活性是必不可少的。细胞表面硫酸乙酰肝素蛋白聚糖（HSPG）、多配体蛋白聚糖-2、-4和磷脂酰肌醇蛋白聚糖在293 T细胞中稳定表达以评估它们是否作为细胞粘附受体起作用。过表达多配体蛋白聚糖-2或-4的293 T细胞与重组α 4 LG 4和HA 4 G82结合，但亲本或过表达磷脂酰肌醇蛋白聚糖-1的293 T细胞不结合。因此，多配体蛋白聚糖-2和多配体蛋白聚糖-4可以介导细胞与层粘连蛋白a4 LG 4模块的粘附。我们的研究表明，层粘连蛋白α 4 LG 4模块可能通过与syndecan-2和/或-4相互作用在皮肤中的细胞粘附和/或血管壁形成中发挥重要作用。
The laminin alpha4 chain, a component of laminin-8/9, is expressed in basement membranes of endothelial cells, the peripheral nerves, and muscle fibers. The localization and functions of laminin alpha4 chain in the skin have not been elucidated. By immunostaining with specific antibodies, we demonstrate here that the alpha4 chain is located in the basement membrane zones of blood vessels and is also associated with fibroblast-like cells in the dermis. Western blot showed that cultured fibroblasts secreted a laminin trimer containing the alpha4 chain. We have also focused on the cell adhesion activities of the human laminin alpha4 LG4 module since the corresponding LG4 module of laminin alpha3 was previously identified as active for cell adhesion. Recombinant human alpha4 LG4 was active for heparin-dependent fibroblast adhesion. Screening assays with 19 synthetic peptides covering the entire alpha4 LG4 module identified three peptides (HA4G82: TLFLAHGRLVYM; HA4G83: LVYMFNVGHKKL; and HA4G90: TEATWKIKGPIYL) as active sites for heparin- and heparan sulfate-dependent cell adhesion. Serine-substituted peptides demonstrated that two basic residues, His and Arg, within HA4G82 were essential for cell adhesion activity. The cell surface heparan sulfate proteoglycans (HSPGs), syndecan-2, -4, and glypican were stably expressed in 293T cells to estimate whether they function as cell adhesion receptors. 293T cells overexpressing syndecan-2 or -4 bound to recombinant alpha4 LG4 and to HA4G82, but parental or glypican-1-overexpressing 293T cells did not. Therefore, syndecan-2 and -4 could mediate cell adhesion to the laminin a4 LG4 module. Our study suggests that the laminin alpha4 LG4 module may play an important role in cell adhesion and/or vessel wall formation in the skin by interacting with syndecan-2 and/or -4.