CHARACTERIZATION OF LAMININ-5 ADHESION/MIGRATION DOMAINS

层粘连蛋白-5是细胞外基质(ECM)蛋白，介导上皮细胞与基底膜(BM)的黏附。层粘连蛋白-5通过同时结合多个细胞表面和细胞外基质配体的能力来介导这种细胞-细胞外基质的附着。层粘连蛋白-5具有很强的粘附性，为皮肤提供了完整性和承受创伤性力量的能力。层粘连蛋白-5‘S功能的重要性通过对具有该蛋白遗传缺陷的患者的观察得到了最好的例证。这些患者患有一种称为交界性大疱性表皮松解症(JEIB)的疾病，其特征是所有表皮和粘膜表面都有广泛的水泡形成。层粘连蛋白-5的主要细胞结合区和细胞外基质结合区的鉴定和测序，除了稳定烧伤患者的皮肤移植，提高角膜和胰岛细胞移植的移植物存活率外，还具有开发JEB患者基因治疗的主要治疗潜力。然而，到目前为止，还没有鉴定出特定的配体结合序列。提出了多种用于定位和测序这些关键结合结构域的方法。首先，一系列重叠的多肽片段将在单相结合分析中筛选出它们与细胞和细胞外基质配体结合的能力。其次，具有层粘连蛋白-5α3链C端连续截短的多肽将被检测其与整合素的结合能力。C末端残基的逐渐消除将扰乱配体结合，从而识别并识别关键序列。第三，对可疑的配体结合域进行定点突变，以确定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.