Expression and function of laminin isoforma during organogenesis

层粘连蛋白α 1和α 2链羧基末端球状结构域（Gdomain）的活性序列已经通过筛选重叠的合成肽在许多生物实验中被鉴定出来（Nomizu et al., j.b oll . chem .）。27: 20583-20590,1995；《科学通报》（英文版），1996年第3期。我们在胚胎第13天小鼠的下颌骨腺外植体中测试了这些肽的活性，以确定参与器官发育的功能部位。层粘连蛋白α 1链肽RKRLQVQLSIRT（残基2719-2730和指定AG-73）显著抑制上皮分支形态发生。相比之下，包括AASIKVAVSADR和NRWHSIYITRFG在内的其他细胞粘附层粘连蛋白α 1链肽未能抑制分支。MG-73是层粘连蛋白α - 2链上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天的颌下腺中该层粘连蛋白链水平较低所致。层粘连蛋白α 2链特异性单局部抗体与发育的腺泡细胞基底膜有强烈反应，但仅对胚胎第13天的下颌骨上皮基底膜有微弱染色。免疫荧光检测结果显示，AG-73和对照scramble肽处理的外植体上皮细胞中E-cadherin和alpha6整合素的表达均未发生变化。nidogen/entactin免疫染色显示AG-73处理3 d的外植体基底膜不连续。对照肽处理3 d后，外植体基底膜恢复正常。这些结果表明含有层粘连蛋白α 1链AG-73序列的区域对早期胚胎阶段颌下腺的发育至关重要。ag -73处理的外植体基底膜不连续可能表明该区域在基底膜组装中起重要作用。

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.