|Budget Amount *help
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2003: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2002: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2001: ¥2,000,000 (Direct Cost: ¥2,000,000)
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.