Roles of cell adhosive peptides from laminins in organogenesis

1.用免疫组织化学方法研究了层粘连蛋白-5和层粘连蛋白-α5链在小鼠颌下腺发育过程中的分布。层粘连蛋白-5在发育中的导管基底膜中有表达，而在上皮性终芽中未见表达。相反，在胚胎早期，层粘连蛋白-α5链在整个上皮基底膜中均有表达，提示层粘连蛋白-α5链在上皮形态发生中的作用。为了阐明层粘连蛋白α5链在器官发生中的作用，我们在颌下腺器官培养中检测了15个来自层粘连蛋白-α5链序列的羧基末端球状结构域的细胞粘附性合成肽。在250μg/ml的浓度范围内，我们发现了一个氨基酸残基为3307-3318的氨基酸序列，命名为A5G-77，对器官发育有很强的抑制作用。羧基末端缺失多肽A5G-77F抑制…的作用而氨基末端缺失多肽A5G-77c(FLNHGHFVA)不能抑制器官发育。A5G-77处理的外植体的形态与AG-73处理的不同，表明单个层粘连蛋白α链在器官发育中的作用是不同的。我们建立了胚胎第13天小鼠的无血清器官培养系统。该系统允许我们测试22个层粘连蛋白-1多肽在触觉生长和分化中的作用。我们发现RKRLQVQLSIRT序列定位于层粘连蛋白-α1-链羧基末端G区的LG-4亚区，命名为AG-73，它干扰了体外的触须生长。其他细胞粘附层粘连蛋白-1多肽和对照扰乱多肽LQQRRSVLRTKI未能显示出对触须生长的任何显著影响。在AG-73处理的触须表皮中，观察到基底膜形成受损，细胞质泡泡形成异常，以及不寻常的肌动蛋白束基底部形成，这表明AG-73信号参与了细胞骨架组织。较少

1. The distributions of laminin-5 and laminin-α5 chain, were studied immunohistochemically in the developing submandibular glands of mice. The expression of laminin-5 was found in the basement membrane of developing duct but not epithelial end bud. In contrast, laminin-α5 chain was expressed in the entire epithelial basement membrane at the early embryonic stage, suggesting the role of laminin-α5 chain on the epithelial morphogenesis.2. To elucidate roles of the laminin α5 chain on organogenesis, we tested 15 cell-adhesive synthetic peptides from a carboxy-terminal globular domain of laminin-α5-chain sequence in submandibular gland organ culture. We found a peptide sequence LVLFLNHGHFVA (residues 3307-3318 and designated A5G-77) strongly inhibits the organ development at 250μg/ml. Various amino- and carboxy-terminal truncated peptides of A5G-77 were synthesized and their activities were also tested in the same system. A carboxy-treminal deletion peptide A5G-77f (LVLFLNHGH) worked inhib … More itory, whereas an amino-treminal deletion peptide A5G-77c (FLNHGHFVA) failed to inhibit the organ development. The morphology of A5G-77-treated explants differed from those of AG-73-treated, indicating distinct role of individual laminin α chains for organ development.3. We established a serum-free organ culture system of isolated vibrissal rudiments of embryonic day-13 mice. This system allowed us to test 22 laminin-1 peptides for their roles on the vibrissal growth and differentiation. We found that RKRLQVQLSIRT sequence, which is mapped at LG-4 subdomain of laminin-α1-chain carboxyl-terminal G-domain and designated AG-73, perturbed vibrissal growth in vitro. Other cell-adhesive laminin-1 peptides and a control scrambled-peptide, LQQRRSVLRTKI, failed to show any significant effects on vibrissal growth. An impaired basement membrane formation, an abnormal cytoplasmic bleb formation, and an unusual basal formation of actin bundles were noted in AG-73-treated vibrissal epidermis, indicating that AG-73 signaling is involved in cytoskeletal organization. Less