Basically charged, serine proteases and carboxypeptidases that are enzymatically active at neural pH are major constituents of the secretory granules of human mast cells. Although only two serine proteases have so far been identified in human mast cells, we recently reported that the secretory granules of mouse mast cells contain various combinations of at least six serine proteases. These mouse mast cell proteases are derived from distinct genes, but they have regions within their peptide sequences that are 100% identical. This latter finding raised concerns that antibodies produced using a 26-32 kDa specific human mast cell serine protease will cross react against a homologous but distinct serine protease because an epitope recognized by that antibody resides in the conserved region of the protease. DNAs and anti-peptide antibodies that recognize different human mast cell proteins and their corresponding transcripts are critical reagents for addressing a number of questions regarding the relationship of mRNA content to translation and post-translational modification of specific proteins in these immune cells. Thus, it is proposed in this research project to make anti-peptide antibodies and gene- specific oligonucleotide probes that recognize human carboxypeptidase A and distinct serine proteases that are present in the secretory granules of human mast cells. These reagents will then be used to phenotype human mast cells in vivo in patients with physical allergies, systemic mastocytosis, and bullous pemphigoid, and to study various biochemical aspects of these enzymes in cultured human mast cells.
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