Studies on laminin domains involved in the epithelial organogenesis.

参与上皮器官发生的层粘连蛋白结构域的研究。

基本信息

批准号：
13670023
负责人：
KADOYA Yuichi
金额：
$ 2.24万
依托单位：
Kitasato University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13670023/
关键词：
antibody cell adhesion laminin organogenesis peptide ペプチド抗体活性ペプチド唾液腺毛包

项目摘要

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.

层粘连蛋白是在各种器官的基底膜（BMS）中发现的多功能细胞外糖蛋白。至少已经确定了15种粘胺同工型。其中，发现层粘连蛋白1和-10在胚胎小鼠下颌腺（SMG）的上皮BMS中共定位，这表明它们的上皮器官发生至关重要。为了鉴定层粘连蛋白-1和-10的功能结构域，我们筛选了50多个可支持HT1080细胞粘附的已知的细胞粘附12-mer合成层粘连蛋白-1和-10肽，该肽是在有力的SMG或毛囊的器官中，或使用上皮细胞纤维细胞细胞系的细胞粘附测定。结果，我们确定了四种功能性层粘着蛋白肽，AG73肽（RKRLQVQLSIRT）和A5G77F肽（LVLFLNHGH），抑制上皮分支形态发生：和A5G33肽（AskaiQVfllag）和A5G65肽（AskaiQVfllag）和A5G65肽（HQNMG65 Peptide（HQNMGNMGNMGNMGNM）序列（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。合成了AG73，A5G77F和A5G33肽，并结合到钥匙孔Limpet Hemocyanin（KLH）。以2周的间隔向兔子注射3至5次肽-KLH。在某些实验中，使用豚鼠代替兔子。然而，这些肽都没有增加足够数量的特定抗体。因此，我们合成了A5G77F的多聚体形式，并与之合成了免疫性的兔子。抗血清R3对A5G77F 100-1000倍的亲和力高于A5G77FT，这是A5G77F的对照争夺肽。使用器官培养和细胞粘附测定，我们现在正在测试R3的生物学功能。