Structure/function of 105-KD basement membrane protein

描述：（逐字）皮肤基底膜区（BML）作为一个 界面屏障，并作为外层之间的粘附连接， 皮肤，即表皮，和皮肤的内层，即真皮。一些BMZ 8 P230、BP 180（XVII型胶原）等成分被发现是 是自身免疫反应的结果使用患者的血清 对于自身免疫性起泡疾病和BMZ成分的自身抗体， 编码BMZ组分，如BP 18 O和VII型胶原的cDNA， 并在生物医学研究中变得非常有价值。的 自身抗体描绘的核苷酸序列允许合成大的 大量的重组蛋白，这反过来又大大促进了 了解水疱过程的发病机制和正常连接 BMZ组件的功能。我们现在有机会发现和 了解另一个以前未知的BMZ组分，称为p105，也 是自身免疫反应的结果这种新型的自身免疫性疾病 水疱性皮肤病的特征是广泛的水疱和糜烂 临床上，表皮下分离伴嗜中性浸润 在组织学上，BMZ处的体内IgG沉积和循环IgG与 盐裂皮肤基质的真皮侧免疫病理学上，体内IgG 沉积和循环lgG结合到下透明板 超微结构！γ和IgG自身抗体识别105-kDa的表皮 蛋白免疫化学该p105已被进一步表征为其 通过免疫化学方法与HO 5-kDa层粘连蛋白-5-γ 2链的区别， 通过二维凝胶电泳和免疫印迹法测定等电点， 通过Mono 0阴离子交换柱色谱法测定其离子强度， 通过蛋白质测序确定N-末端氨基酸序列。此外，单克隆 已经产生了针对该p105蛋白的抗体。克隆了两个cDNA 编码BMZ组件，主要研究者获得了以下经验： 分子克隆，现在准备提出以下工作，研究 BMZ新组分p105结构和功能。在本提案中，我们 目的克隆人p105基因cDNA序列，表达人p105蛋白， p105重组蛋白，研究人p105的体外功能 蛋白，分子克隆小鼠p105 cDNA，并被动转移 抗小鼠p105抗体对新生小鼠的作用。了解结构和 这个新的BMZ组件p105的功能将揭示复杂的 皮肤BMZ的结构，不同成分之间的关系， 皮肤BMZ及其在表皮-真皮粘附、人皮肤起泡中的作用 疾病、妊娠期发育、表皮癌转移和皮肤 伤口愈合

DESCRIPTION: (Verbatim) Skin basement membrane zone (BML) serves as an interface barrier and as an adherent connection between the outer layer of skin, the epidermis, and inner 'layer of skin, the dermis. Some of the BMZ components, such as 8P230, BP180 (type XVII collagen) were discovered as a result of being targeted by an autoimmune reaction. Using sera from patients with autoimmune blistering diseases and autoantibodies to BMZ components, the cDNAs encoding BMZ components, such as BP18O and type VII collagen, were determined and became very valuable in biomedical research. The autoantibody-delineated nucleotide sequences allow synthesis of large quantities of recombinant proteins, which in turn significantly facilitates the understanding of pathogenesis of blistering process and the normal connecting function of BMZ components. We now have the opportunity to discover and understand another previously unknown BMZ component, termed p105, also identified as a result of an autoimmune reaction. This novel autoimmune blistering skin disease is characterized by extensive blisters and erosions clinically, subepidermal separation with neutrophilic infiltration histologically, in vivo lgG deposition at BMZ and circulating lgG binding to the dermal side of salt-split skin substrate immunopathologically, in vivo lgG deposition and circulating lgG binding to the lower lamina lucida ultrastructural!y, and lgG autoantibodies recognizing a 1O5-kDa epidermal protein immunochemically. This p105 has been further characterized for its distinction from the HO5-kDa laminin-5 -y2 chain by immunochemical methods, its isoelectric point by two-dimensional gel electrophoresis and immunoblotting, its ionic strength by Mono 0 anion-exchange column chromatography, and its N-terminal amino acid sequence by protein sequencing. Furthermore, a monoclonal antibody has been generated against this p105 protein. Having cloned two cDNAs encoding BMZ components, the principal investigator has gained experience in molecular cloning and is now ready to propose the following works to study the structure and function of this novel BMZ component p105. In this proposal, we aim to molecularly clone the human p105 cDNA sequence, to express the human p105 recombinant protein, to study the in vitro functions of human p105 protein, to molecular clone the mouse p105 cDNA, and to passive transfer of anti-mouse p105 antibodies to new born mice. Understanding of the structure and function of this new BMZ component p105 will shed light on the complex structure of the skin BMZ, the relationship between different components of skin BMZ, and their roles in epidermal-dermal adhesion, human blistering skin diseases, gestational development, epidermal cancer metastasis, and cutaneous wound healing.