HUMAN SKIN MAST CELL GRANULE ENZYMES: SKIN DISEASES

人类皮肤肥大细胞颗粒酶：皮肤病

• 批准号: 3156147
• 负责人: BRUCE U WINTROUB
• 金额: $ 23.1万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1982
• 资助国家: 美国
• 起止时间: 1982-07-01 至 1993-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3156147
• 关键词: affinity chromatography; angiotensins; carboxypeptidase; clone cells; complementary DNA; enzyme complex; enzyme linked immunosorbent assay; enzyme mechanism; fibrinogen; genetic library; granule; high performance liquid chromatography; human subject; laminin; mast cell; monoclonal antibody; nucleic acid hybridization; pathologic process; pemphigus; peptidases; peptides; physical chemical interaction; protein biosynthesis; protein sequence; proteoglycan; proteolysis; radioimmunoassay; skin disorder; skin transplantation; substance P; urticaria; vesicular skin disorder

The goals of these studies are to isolate and characterize carboxypeptidase, a newly identified human mast cell secretory granule protease, and to localize and determine the pathobiologic role of mast cell proteolytic enzymes in mast cell mediated skin diseases. Proposed studies will address: 1) purification and physicochemical characterization of human skin mast cell carboxypeptidase; 2) characterization and in vitro reconstitution of the human skin mast cell secretory granule with respect to protease and proteoglycan components; 3) investigation of the biochemical action of purified carboxypeptidase, and enzyme- proteoglycan complexes on purified proteins, peptides, and potential cellular substrates in skin; and 4) examination of the pathobiologic role of human skin mast cell secretory granule proteases in mast cell-associated cutaneous diseases. These studies will add to understanding of the human skin mast cell, and the role of mast cell secretory granule proteases in mediating tissue damage in cutaneous disorders. To accomplish these goals, human skin mast cell carboxypeptidase, identified by an HPLC assay system, will be purified from whole skin extracts by affinity chromatography. A monospecific, polyclonal anticarboxypeptidase antibody will be developed. To understand regulation of carboxypeptidase expression, mast cells from various tissues will be assessed for carboxypeptidase activity by HPLC, and protein by ELISA or RIA, and a human mast cell cDNA library will be made to identify recombinant clones corresponding to carboxypeptidase. To understand their biochemical and biologic activity, enzyme-proteoglycan complexes from dispersed human mast cells or reconstituted from purified components, will be incubated with protein, peptide, and potential cellular substrates such as fibrinogen, laminin, substance P, angiotensin I, and cultured human umbilical vein endothelial cells, respectively. The proteolytic activity of these complexes will be examined in vitro using skin explants. The in situ release, localization and fate of carboxypeptidase will be examined in vitro using skin explants, and in vivo after intradermal injection of human C5a, and in a skin chamber model after challenge with allergens. Finally, the deposition of mast cell granule carboxypeptidase will be examined in vivo in diseases marked by increased vascular permeability, i.e. cold urticaria, and by tissue destruction, i.e. bullous pemphigoid.

这些研究的目的是分离和表征 羧肽酶，一种新发现的人肥大细胞分泌型羧肽酶， 颗粒蛋白酶，并定位和确定病理生物学 肥大细胞蛋白水解酶在肥大细胞介导的皮肤中的作用 疾病 拟议的研究将解决：1）纯化和 人皮肤肥大细胞理化特性 羧肽酶; 2）表征和体外重构 人皮肤肥大细胞分泌颗粒的相对于 蛋白酶和蛋白多糖组分; 3）调查 纯化的羧肽酶的生化作用，和酶- 蛋白聚糖复合物上的纯化蛋白质，肽，和 皮肤中潜在的细胞基质;以及4）检查 人皮肤肥大细胞分泌颗粒病理生物学作用 蛋白酶在肥大细胞相关皮肤疾病中的作用。 这些 研究将增加对人类皮肤肥大细胞的了解， 肥大细胞分泌颗粒蛋白酶介导 皮肤疾病中的组织损伤。 为了实现这些目标， 高效液相色谱法鉴定人皮肤肥大细胞羧肽酶 分析系统，将从整个皮肤提取物中纯化，通过亲和层析， 层析 一种单特异性，多克隆 抗羧肽酶抗体将被开发。 了解 羧肽酶表达的调节，肥大细胞从 通过以下方法评估各种组织的羧肽酶活性 HPLC法测定蛋白质，ELISA或RIA法测定蛋白质，以及人肥大细胞cDNA 将制备文库以鉴定相应的重组克隆 到羧肽酶。 为了了解它们的生物化学和 生物活性，酶-蛋白聚糖复合物从分散的 人肥大细胞或由纯化组分重构的人肥大细胞将 与蛋白质、肽和潜在的细胞一起孵育， 底物如纤维蛋白原、层粘连蛋白、P物质、血管紧张素I， 和培养的人脐静脉内皮细胞。 这些复合物的蛋白水解活性将在 使用皮肤外植体进行体外培养。 原位释放、定位和 将使用皮肤在体外检查羧肽酶的命运 外植体，以及在皮内注射人C5 a后的体内， 以及在用过敏原激发后的皮肤室模型中。 最后，肥大细胞颗粒羧肽酶的沉积将 在以血管生成增加为标志的疾病中进行体内检查 渗透性，即冷荨麻疹，和组织破坏，即 大疱性类天疱疮