Expression and function of laminin isoforma during organogenesis

已经通过在许多生物测定中筛选重叠的合成肽来鉴定来自层粘连蛋白α 1和α 2链羧基末端球状结构域（G结构域）的活性序列（Nomizu et al.，J.Biol.Chem.270：20583- 20590，1995; Nomizu等，FEBS Lett.396：37- 42，1996）。我们已经测试了这些肽在胚胎第13天小鼠的下颌下腺外植体中的活性，以确定参与器官发育的功能位点。层粘连蛋白α 1链肽RKRLQVQLSIRT（残基2719-2730-，命名为AG-73）显著抑制上皮分支形态发生。相比之下，其他细胞粘附层粘连蛋白α 1链肽，包括AASIKVAVSADR和NRWHSIYITRFG未能抑制分支。MG-73是来自层粘连蛋白α 2链的AG-73的同源物，不抑制分支。α 2链肽没有影响，这可能是由于胚胎第13天下颌下腺中层粘连蛋白链的低水平。层粘连蛋白α 2链特异性单域抗体强烈反应的基底膜发达的腺泡，但只有弱染色的胚胎第13天下颌下上皮基底膜。如通过免疫荧光检测的，E-钙粘蛋白和α 6整联蛋白的表达在AG-73和对照杂乱肽处理的外植体的上皮细胞中均未改变。相反，巢蛋白/巢蛋白的免疫染色显示，用AG-73处理3天的外植体具有不连续的基底膜。用对照肽处理3天的外植体显示正常的基底膜。这些结果表明，含有层粘连蛋白α 1链的AG-73序列的区域对于胚胎早期下颌下腺的发育至关重要。AG-73处理的外植体中的不连续基底膜可能表明该区域在基底膜组装中的重要作用。

Active sequences from the laminin alpha1 and alpha2 chain carboxyl-terminal globular domains (Gdomain)have been identified by screening overlapping synthetic peptides in a number of biological assays (Nomizu et al., J.Biol.Chem.270 : 20583-20590,1995 ; Nomizu et al., FEBS Lett.396 : 37-42,1996). We have tested the activity of these peptides in submandibular gland explants of embryonic day-13 mice to determine the functional sites involved in organ development. The laminin alpha1 chain peptide, RKRLQVQLSIRT (residues 2719-2730-and designated AG-73), significantly inhibited epithelial branching morphogenesis. In contrast, other cell adhesive laminin alpha1 chain peptides including the AASIKVAVSADR and NRWHSIYITRFG failed to inhibit the branching. MG-73, a homologue of AG-73 from the laminin alpha2 chain, did not inhibit the branching. The alpha2 chain peptide had no effect, which may be due to the low levels of this laminin chain in embryonic day 13 submandibular gland. Laminin alpha2 chain-specific monoclocal antibodies strongly reacted with the basement membranes of developed acini but only weakly stained embryonic day 13 submandibular epithelial basement membrane. The expression of E-cadherin and alpha6 integrin, as detected by immunofluorescence, were unchanged in both AG-73 and control scramble peptide treated epithelial cells of the explants. In contrast, immunostaining of nidogen/entactin showed that explants treated with AG-73 for 3 days had a discontinuous basement membrane. Explants treated for 3 days with control peptide showed a normal basement membrane. These results suggest that the region containing the AG-73 sequence of the laminin alpha1 chain is crucial for development of submandibular gland at early embryonic stages. The discontinuous basement membrane in AG-73-treated explants may indicate an important role for this region in basement membrane assembly.