CHARACTERIZATION OF LAMININ-5 ADHESION/MIGRATION DOMAINS

层粘连蛋白-5是介导上皮细胞粘附到下面的基底膜（BM）的细胞外基质（ECM）蛋白。层粘连蛋白-5通过其同时结合多种细胞表面和ECM配体的能力介导这种细胞-ECM附着。层粘连蛋白-5的强粘附特性负责为皮肤提供其完整性和承受创伤力的能力。层粘连蛋白-5功能的重要性最好通过观察具有这种蛋白质遗传缺陷的患者来说明。这些患者患有称为交界性大疱性表皮病（JEB）的病症，其特征在于在所有表皮和粘膜表面上形成广泛的水疱。层粘连蛋白-5的主要细胞和ECM结合结构域的鉴定和测序对于开发针对JEB患者的基因治疗具有主要的治疗潜力，除了稳定用于治疗烧伤患者的皮肤移植物，以及提高角膜和胰岛细胞移植物的存活率之外。然而，迄今为止尚未鉴定出特异性配体结合序列。在定位和测序这些关键的结合结构域设计的多种方法被提出。首先，将在固相结合测定中筛选一系列重叠的肽片段，以确定它们与细胞和ECM配体结合的能力。其次，将测定具有层粘连蛋白-5的α 3链的连续C-末端截短的肽与整联蛋白结合的能力。C-末端残基的逐渐消除将干扰配体结合，从而鉴定并因此鉴定关键序列。第三，将使用疑似配体结合结构域的定点诱变来确认这些结合序列的位置。

Laminin-5 is the extracellular matrix (ECM) protein that mediates epithelial adhesion to the underlying basement membrane (BM). Laminin-5 mediates this cell-ECM attachment through its ability to simultaneously bind to multiple cell-surface and ECM ligands. The strong adhesive properties of laminin-5 are responsible for providing the skin with its integrity and ability to withstand traumatic forces. The importance of laminin-5's function is best exemplified through observations of patients who possess a genetic deficiency of this protein. These patients suffer from a disorder known as Junctional Epidermolysis Bullosa (JEB), characterized by extensive blister formation on all epidermal and mucosal surfaces. Identification and sequencing of the major ell- and ECM-binding domains of laminin-5 has major therapeutic potentials for developing gene therapy for JEB patients, in addition to stabilizing skin grafts for the treatment of burn patients, and improving graft survival in corneal and pancreatic islet cell transplants. The specific ligand-binding sequences, however, have not been identified to date. Multiple approaches designed at locating and sequencing these critical binding domains are proposed. First, a series of overlapping peptide fragments will be screened in sold-phase binding assays for their ability to bind to cell and ECM ligands. Secondly, peptides with sequential C-terminal truncations of the alpha3 chain of laminin-5 will be assayed for their ability to bind to integrins. Progressive elimination of C-terminal residues will perturb ligand-binding and thus identify and thus identify key sequences. Thirdly, site-directed mutagenesis of suspected ligand- binding domains will be used to confirm the location of these binding sequences.