PURIFICATION & STUDY OF HUMAN KERATINOCYTE COLLAGENASE

脊椎动物胶原酶是一种中性金属内切蛋白酶， 天然间质胶原蛋白转化为特定的四分之三和一， 四分之一长度的片段，然后易于进一步 蛋白水解 在皮肤中，胶原酶的过度产生是 认为有助于遗传性疾病的发病机制 起泡病症，营养不良性大疱性表皮病（RDEB）。 真皮成纤维细胞一直被认为是主要的 胶原酶的来源，该酶已被 广泛研究。 我们最近记录了 培养的人角质形成细胞产生胶原酶。 角化细胞胶原酶在功能上似乎 结构上类似于成纤维细胞酶，尽管 生产的调节可能是不同的。 这个目标 建议是纯化，进一步表征和评估 正常和RDEB中胶原酶产生的调节 角质形成细胞 胶原酶将从条件化的 来自佛波醇刺激的角质形成细胞的培养基， 其特征在于关于等电点，钙 要求、热稳定性和部分氨基酸序列 数据 人类皮肤胶原酶的抗体将在 兔和ELISA开发。 底物特异性 纯化的酶将用IV型和VII型进行评价 胶原 将研究胶原酶产生的调节 使用已知的成纤维细胞胶原酶刺激剂和抑制剂， 蛋白激酶C途径类似物和生长因子。 的 细胞-细胞和细胞-基质相互作用的影响，细胞的 细胞迁移和细胞损伤对胶原酶合成的影响 评估角质形成细胞。 生产和调制 将研究RDEB角质形成细胞对胶原酶的影响， 创伤组织胶原酶免疫细胞化学定位 评价的人皮肤和皮肤等效模型。

Vertebrate collagenase, a neutral metalloendoproteinase, cleaves native interstitial collagen into specific three-quarter and one- quarter length fragments which are then susceptible to further proteolysis. In the skin, excessive production of collagenase is thought to contribute to the pathogenesis of the inherited blistering disorder, dystrophic epidermolysis bullosa (RDEB). The dermal fibroblast has been considered to be the primary source of collagenase in human skin and the enzyme has been extensively studied. We have recently documented the production of collagenase by human keratinocytes in culture. The keratinocyte collagenase appears functionally and structurally similar to the fibroblast enzyme, although modulation of production may be dissimilar. The goal of this proposal is to purify, further characterize, and evaluate the modulation of collagenase production in normal and RDEB keratinocytes. Collagenase will be purified from conditioned medium from phorbol-stimulated keratinocytes and characterized in regards to the isoelectric point, calcium requirements, thermal stability and partial amino acid sequence data. Antibodies to human skin collagenase will be produced in rabbits and an ELISA developed. Substrate specificity of the purified enzyme will be evaluated with type IV and type VII collagen. Modulation of collagenase production will be studied with known stimulators and inhibitors of fibroblast collagenase, protein kinase C pathway analogues and growth factors. The effect of cell-cell and cell-matrix interactions, cellular migration and cellular injury on synthesis of collagenase by keratinocytes will be assessed. The production and modulation of collagenase by RDEB keratinocytes will be studied and immunocytochemical localization of collagenase in wounded human skin and skin equivalent models evaluated.