CHARACTERIZATION OF HUMAN NEUTROPHIL COLLAGENASE

神经元胶原酶（NC）参与了 类风湿性关节炎患者结缔组织的变化， 特发性肺纤维化、角膜溃疡和慢性牙龈炎。 虽然这种酶是最早的金属蛋白酶之一， 描述，其主要结构和监管还没有 其特征在于具有金属蛋白酶家族的其他成员。 我们 已经分离出编码中性粒细胞胶原酶的cDNA，并将使用位点 定向诱变中性粒细胞胶原酶的cDNA， 研究主要功能域中各种功能域的作用 结构 将突变的cDNA转染到真核细胞中 并且表达的蛋白质的特征在于酶促 活性、潜伏期和底物特异性。 平行研究 通过脉冲追踪标记和随后的 免疫沉淀将用于表征翻译后 表达的蛋白质的修饰，与通过合成的蛋白质相比， 正常人骨髓细胞 此外，转染的细胞系将 用于评价宿主细胞表型对 酶的细胞内靶向分泌颗粒。 的 建议的研究应提供有关关系的基本信息， 中性粒细胞胶原酶的一级结构及其作为 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 基因使用基因组克隆，我们已经为这种酶分离。 的 这些克隆内的外显子/内含子边界，以及起始位点和 将鉴定转录单位的3 ′端。 的 该基因的表达被认为是特异性的， 控制并与成熟的确定阶段相关联， 分化 我们会展开研究其规管元素， 对第一外显子的5'侧翼区进行测序。 我们将用我们 人骨髓细胞与人成纤维细胞原位杂交的cDNA克隆 确定中性粒细胞的成熟阶段， 这个基因开始。 转录调控的研究 中性粒细胞胶原酶应该有助于我们理解 中性粒细胞成熟，并阐明与 以成熟停滞为特征的病症。

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.