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。

• 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

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 (BM) 中发现的多功能细胞外糖蛋白。至少已鉴定出 15 种层粘连蛋白亚型。其中，层粘连蛋白-1和-10被发现共定位于胚胎小鼠下颌下腺（SMG）的上皮BM中，表明它们对于上皮器官发生至关重要。为了鉴定层粘连蛋白-1和-10的功能域，我们在原始SMG或毛囊的器官培养物中筛选了超过50种已知支持HT1080细胞粘附的细胞粘附12聚体合成层粘连蛋白-1和-10肽，或使用上皮肝细胞系的细胞粘附测定。结果，我们鉴定了四种功能性层粘连蛋白肽：AG73肽（RKRLQVQLSIRT）和A5G77f肽（LVLFLNHGH），它们抑制上皮分支形态发生：以及A5G33肽（ASKAIQVFLLAG）和A5G65肽（HQNMGSVNVVSVG），它们支持上皮细胞扩散。根据这些发现，我们尝试建立针对功能性层粘连蛋白的抗体肽。合成 AG73、A5G77f 和 A​​5G33 肽并与匙孔血蓝蛋白 (KLH) 缀合。每隔两周给兔子注射肽-KLH 3 至 5 次。在一些实验中，使用豚鼠代替兔子。然而，这些肽都没有产生足够量的特异性抗体。因此，我们合成了 A5G77f 的多聚体形式，并用它免疫兔子。抗血清R3对A5G77f的亲和力比对A5G77fT（A5G77f的对照乱序肽）的亲和力高100-1000倍。我们现在正在使用器官培养和细胞粘附测定来测试 R3 的生物学功能。