Studies on laminin domains involved in the epithelial organogenesis.

层粘连蛋白是存在于各种器官基底膜（BMs）中的多功能细胞外糖蛋白。至少有15种层粘连蛋白同种异构体已被鉴定。其中，laminin-1和-10被发现在胚胎小鼠颌下腺（SMG）上皮脑转移瘤中共定位，提示它们在上皮器官发生中起重要作用。为了鉴定层粘连蛋白-1和-10的功能域，我们筛选了50多种细胞粘连蛋白-1和-10合成肽，已知它们支持HT1080细胞粘附，在初级SMG或毛囊的器官培养中，或在上皮性肝细胞系的细胞粘附试验中。因此，我们鉴定出4种功能性层合蛋白肽，AG73肽（RKRLQVQLSIRT）和A5G77f肽（LVLFLNHGH）抑制上皮分支形态发生，A5G33肽（ASKAIQVFLLAG）和A5G65肽（HQNMGSVNVSVG）支持上皮细胞扩散。基于这些发现，尝试建立针对功能性层粘连蛋白肽的抗体。合成AG73、A5G77f和A5G33多肽，并与锁孔帽贝血青素（KLH）偶联。每2周给家兔注射3 ~ 5次肽- klh。在一些实验中，用豚鼠代替兔子。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A5G77f的多聚体形式，并用它免疫家兔。抗血清R3对A5G77f的亲和力是A5G77fT （A5G77f的对照肽）的100-1000倍。通过器官培养和细胞粘附试验，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.