The secretory granules of mature mast cells from many species are known to contain serine proteases which can constitute more than 50% of the total cellular protein. For rats as well as humans, two different mast cell subclasses have been identified based on differences in content of two identified serine proteases. It is shown here that the secretory granules of murine mast cells contain a minimum of 6 distinct serine proteases, 5 of which we have recently described. It is postulated that, like the protease repertoire, the extent of mast cell subclass diversity has not yet been fully defined. The broad, long-term objectives of this investigator are to define the full extent of the repertoire of proteases in murine mast cell secretory granules and to use probes for each of the proteases to define subclasses of murine mast cells in normal tissues and during parasite infestation. This laboratory has employed a Kirsten murine sarcoma virus to produce immortalized murine mast cell lines KiSV-MC) which are an excellent source for mast cell proteases. KiSV-MC and in vivo-derived murine mast cells were examined by high resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis, amino-terminal amino add protein sequencing, cDNA sequencing, and RNA blot analyses. A total of 6 distinct serine proteases were found, and different mast cell subclasses possessed different sets of the proteases. Complete cDNAs for {4} of the 6 serine proteases have been sequenced. The initial goal is to clone and sequence cDNAs for the (2) remaining serine proteases. This will require the construction and screening of two new cDNA libraries, from mouse peritoneal connective tissue mast cells and from mouse mucosal mast cells. Redundant oligodeoxynucleotide probes, based on partial amino acid sequences, will be used to screen the cDNA libraries for each of the proteases. RNA blot analyses and in situ hybridization studies will be utilized to examine tissues from normal and parasite-infected mice for the presence of mRNA for each of the mast cell proteases. These studies will provide the necessary probes and techniques to allow a more complete description of mast cell subclasses than has been available previously for any species and will provide a model for future studies of human mast cells. (Finally, it may be possible to identify and clone other mast cell proteases not recognized by the amino-terminal protein sequencing. Should time permit, each cDNA library will be rescreened under a combination of low- and high-stringency conditions to identify unique protease cDNAs.)
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