CHARACTERIZATION OF HUMAN NEUTROPHIL COLLAGENASE

中性粒细胞胶原酶(NC)参与了 类风湿关节炎患者结缔组织的变化 特发性肺纤维化、角膜溃疡和慢性牙周炎。 尽管这种酶是最早发现的金属蛋白酶之一 它的主要结构和调控机制还没有得到很好的描述 具有金属蛋白酶家族其他成员的特征。我们 已分离出编码中性粒细胞胶原酶的cdna，并将使用Site 中性粒细胞胶原酶基因的定向诱变 研究不同功能域在主服务器中的作用 结构。突变的c DNA将被导入真核细胞 并且表达的蛋白具有与酶的特性 活性、潜伏期和底物专一性。并行的研究： 脉冲追逐标记及后续培养的转基因细胞 免疫沉淀将被用来表征翻译后 表达的蛋白质与合成的蛋白质相比的修饰 正常的人骨髓细胞。此外，转基因细胞系将 用来评价宿主细胞表型对 该酶在细胞内靶向分泌颗粒。这个 建议的研究应提供有关这一关系的基本信息 中性粒细胞胶原酶的一级结构及其功能 酶除了有助于理解 中性粒细胞成熟的基本要素。 我们还将研究中性粒细胞胶原酶的组织 利用我们为该酶分离的基因组克隆。这个 这些克隆中的外显子/内含子边界，以及起始点和 转录单位的3‘端，将被识别。这个 这种基因的表达被认为是特异的和紧密的 受控制的，与特定的成熟和成熟阶段相关的 差异化。我们将通过以下方式开展对其监管要素的研究 对第一外显子5‘侧翼区进行测序。我们将使用我们的 人骨髓细胞原位杂交的cDNA克隆 确定中性粒细胞转录的成熟阶段 这一基因的开始。基因转录调控的研究进展 中性粒细胞胶原酶应该有助于我们对 中性粒细胞成熟并阐明与 以成熟停滞为特征的状态。

Neutrophil collagenase (NC) has been implicated in the destructive changes seen in connective tissue of patients with rheumatoid arthritis, idiopathic pulmonary fibrosis, corneal ulceration and chronic gingivitis. Although this enzyme was one of the first metalloproteinases to be described, its primary structure and regulation have not been as well characterized as have other members of the metalloproteinase family. We have isolated a cDNA encoding neutrophil collagenase and will use site directed mutagenesis of the cDNA for neutrophil collagenase to investigate the role of various functional domains within the primary structure. The mutated cDNA will be transfected into eucaryotic cells and the expressed protein characterized with respect to enzymatic activity, latency and substrate specificity. Parallel studies of the transfected cells by pulse-chase labeling and subsequent immunoprecipitation will be used to characterize post-translational modifications of the expressed protein as compared to that synthesized by normal human bone marrow cells. In addition, transfected cell lines will be used to evaluate the influence of the host cell phenotype on intracellular targeting of the enzyme to secretion granules. The proposed studies should provide basic information about the relationship of the primary structure of neutrophil collagenase and its function as an enzyme in addition to contributing to the understanding of the rudimentary elements of neutrophil maturation. We will also study the organization of the neutrophil collagenase gene using genomic clones we have isolated for this enzyme. The exon/intron borders within these clones, as well as the start site and the 3' end of the transcriptional unit, will be identified. The expression of this gene is believed to be specifically and tightly controlled and associated with a defined phase of maturation and differentiation. We will initiate studies of its regulatory elements by sequencing the 5' flanking region of the first exon. We will use our cDNA clone for in situ hybridization of cells from human bone marrow to determine the maturational stage of the neutrophil at which transcription of this gene begins. The study of the transcriptional control of neutrophil collagenase should contribute to our understanding of neutrophil maturation and shed light on mechanisms associated with conditions characterized by maturation arrest.