Structure/function of 105-KD basement membrane protein

描述：(逐字)皮肤基底膜区(BML)作为 界面屏障，并作为外层之间的附着连接 皮肤，表皮，和皮肤的内层，真皮。一些BMZ 8P230，BP180(XVI型胶原)等成分被发现是一种 被自身免疫反应作为目标的结果。用病人的血清 由于自身免疫性水泡病和针对BMZ成分的自身抗体， 编码BMZ成分的cDNA，如BP18O和III型胶原， 决心并在生物医学研究中变得非常有价值。这个 自身抗体描述的核苷酸序列允许合成大分子 大量的重组蛋白，这反过来又大大促进了 对起泡过程发病机制及正常连接的认识 BMZ组件的功能。我们现在有机会发现和 理解另一个以前未知的BMZ组件，称为p105，也 被确认为自身免疫反应的结果。这种新的自体免疫 水疱性皮肤病的特征是广泛的水泡和皮损。 临床表现为真皮下分离伴中性粒细胞浸润 组织学上，在BMZ的体内LGG沉积和循环中的LGG结合 盐裂皮肤基质真皮一侧的免疫病理学，活体LGG 下层透明膜沉积和循环中的LGG结合 识别10-5 kDa表皮的超微结构！Y和LGG自身抗体 蛋白质免疫化学。这款P105已经被进一步表征为它的 用免疫化学方法与HO5-kDa层粘连蛋白-5-y2链区分，其 等电点双向凝胶电泳法和免疫印迹法， 用Mono 0阴离子交换柱层析法测定其离子强度，并测定其离子强度。 蛋白质测序确定N-末端氨基酸序列。此外，一株单克隆 已产生针对该p105蛋白的抗体。克隆了两个cDNA 编码BMZ组件，首席研究员在以下方面获得了经验 分子克隆，现在准备提出以下工作来研究 该新型BMZ组分P105的结构和功能。在这项提案中，我们 目的分子克隆人p105基因，表达人p105基因。 P105重组蛋白的构建及其体外功能的研究 蛋白，分子克隆小鼠p105基因，并对其进行被动转移 针对新生小鼠的抗鼠p105抗体。对结构的理解和 这一新的BMZ组件P105的功能将揭示该综合体 皮肤BMZ的结构，不同成分之间的关系 皮肤BMZ及其在表皮-真皮粘连、人起泡皮肤中的作用 疾病、妊娠发育、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.