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.
