Studies on laminin domains involved in the epithelial organogenesis.

层粘连蛋白是一种多功能的胞外糖蛋白，存在于各种器官的基底膜上。目前已鉴定出至少15种层粘连蛋白亚型。其中，层粘连蛋白-1和层粘连蛋白-10共定位于胚胎小鼠颌下腺(SMG)的上皮性BMS，提示它们在上皮性器官发生中起关键作用。为了确定层粘连蛋白-1和-10的功能结构域，我们筛选了50多个支持HT1080细胞黏附的12聚体合成层粘连蛋白-1和-10多肽，在残存的SMG或毛囊的器官培养中，或者用上皮肝细胞系进行细胞黏附实验。因此，我们鉴定了四个功能性层粘连蛋白多肽：抑制上皮细胞分支形态发生的AG73肽(RKRLQVQLSIRT)和A5G77f肽(LVLFLNHGH)，以及支持上皮细胞扩散的A5G33肽(ASKAIQVFLLAG)和A5G65肽(HQNMGSVNVSVG)。合成了AG73、A5G77f和A5G33三种多肽，并将其与锁孔帽状血蓝蛋白(KLH)偶联。每隔2周给兔注射KLH3~5次。在一些实验中，用豚鼠代替了兔子。然而，这些多肽都不能产生足够数量的特异性抗体。因此，我们合成了一种多聚体A5G77f，并用其免疫兔。抗血清R3对A5G77f的亲和力是A5G77fT的100-1000倍，A5G77fT是A5G77f的对照扰乱肽。利用器官培养和细胞黏附试验，我们现在正在测试R3的生物学功能。

Laminins are multifunctional extracellular glycoproteins found in the basement membranes (BMs) of various organs. At least 15 laminin isoforms have been identified. Among them, laminin-1 and -10 were found to co-localize in the epithelial BMs of embryonic mouse submandibular gland (SMG), suggesting their crucial rotes for epithelial organogenesis. In order to identify functional domains of laminin-1 and -10, we screened more than 50 cell-adhesive 12-mer synthetic laminin-1 and -10 peptides known to support HT1080 cell adhesion, in the organ cultures of rudimental SMG or hair follicles, or a cell adhesion assay using epithelial liver cell line. As a result, we identified four functional laminin peptides, AG73 peptide (RKRLQVQLSIRT) and A5G77f peptide (LVLFLNHGH), which inhibited epithelial branching morphogenesis : and A5G33 peptide (ASKAIQVFLLAG) and A5G65 peptide (HQNMGSVNVSVG), which supported epithelial cell spreading.Based on these findings, attempts were made to establish antibodies against the functional laminin peptides. AG73, A5G77f, and A5G33 peptides were synthesized and conjugated to keyhole limpet hemocyanin (KLH). Rabbits were injected with the peptide-KLH 3 to 5 times at 2-week intervals. In some experiments, guinea pigs were used instead of rabbits. None of these peptides, however, raised sufficient amount of specific antibodies. We therefore synthesized a multimeric form of A5G77f and immunized rabbits with it. An antiserum R3 showed affinity for A5G77f 100-1000 times as high as that for A5G77fT, a control scramble peptide for A5G77f. Using an organ culture and cell adhesion assays, we are now testing biological functions of R3.