Mast Cell and Basophil Biology
Granule stored serine proteases of mast cells, basophils, NK cells, T cells and neutrophils.
Serine proteases are major constituents of the cytoplasmic granules in mast cells, neutrophils, T cells and NK cells. These proteases can account for up to 50% of the total cellular protein and they are stored in their active form in the cell (19, 36). The genes encoding the granule-stored proteases are arranged in four different loci, the mast cell chymase locus, the mast cell tryptase locus, the met-ase locus and the T cell tryptase locus. In almost all placental mammals investigated, the mast cell chymase locus holds at least one mast cell expressed enzyme; the α-chymase, one neutrophil protease; cathepsin G and two T cell and NK cell expressed genes: the granzymes B and H. The mast cell tryptase locus contains the mast cell tryptases and also membrane bound tryptases also expressed by other cell types (102). The met-ase locus contains granzyme M and several neutrophil proteases, i.e. N-elastase, proteinase 3 and the antibacterial but proteolytically inactive azurocidin. This locus also harbors the complement factor D (adipsin) and another serine protease of yet unknown function, Prss L1, that is expressed in islet cells of the pancreas. The T cell tryptase locus encodes granzymes A and K.
The T cell granzymes have been shown to be responsible for caspase dependent and caspase independent activation of apoptosis in target cells. The neutrophil proteases are involved in paving the way for these cells during their migration to inflamed tissue and several of them also have anti-bacterial properties. The mast cell chymase and the tryptases have been shown to cleave a number of substrates, in vitro. However their in vivo functions are less well understood.
In several mammalian species the chymase locus has expanded by numerous gene duplication events. In mice, instead of the 4 genes found in the human locus, there are 15 functional chymase locus genes and in rats there are 28 (84, 86). In rodents, two new subfamilies are also among these duplicated genes, the αchymases and the mouse mast cell protease (mMCP)-8 family. mMCP-8 was cloned by our group a number of years ago. This gene has been found to be expressed only by basophils and was the first cloned basophil specific marker in any species (40, 50, 55, 90, 97). The mMCP-8 gene is presently used as a marker for basophil development and also to construct basophil depleted animals.
One of our main areas of research is related to the emergence of these enzymes during vertebrate evolution (36, 53, 83). A more detailed view of their appearance can hopefully help us find the central evolutionary conserved function of these enzymes and also see if divergent, convergent or both processes have participated in their emergence and diversification. Both the chymase and the tryptases are presently evaluated as potential targets for the treatment of allergies and other mast cell dependent inflammatory disorders, which increase the interest for these enzymes from a medical point of view.
In addition to their tissue location and evolution, the most important characteristic of these proteases is their cleavage specificity and their in vivo targets. To obtain a detailed picture of their extended specificity we use a screening system involving a library of more than 50 million random nonamers presented on the surface of T7 phages. This phage display system makes it possible to obtain a detailed picture of the cleavage specificity involving up to nine amino acids surrounding the cleavage site. To be able to verify the result and to obtain kinetic data on the cleavage reaction we have recently developed a system of recombinant substrates. With these novel substrates we have been able to verify the result from the phage display and also get detailed information on the influence of individual amino acid substitutions on the cleavage rate.
Using these recombinant substrates we have recently determined the cleavage specificity of a number of different mast cell enzymes and also performed a detailed study of human thrombin. The thrombin study turned out to be very interesting as it showed that many natural substrates often are relatively poor substrates for this enzyme and are most likely highly dependent on exosite interactions to be actively cleaved in vivo (108).
The projects related to the various hematopoietic serine proteases are listed here below.
A. Evolution of gene loci
We have performed a detailed analysis on the structure of the chymase locus in mammals, including a panel of placental mammals, one marsupial, the American opossum and on one monotreme, the Australian platypus (84, 86). The results show that massive gene duplications have expanded this locus in several mammalian lineages. The most extensive expansion has occurred in rodents. Interestingly, independent duplications have resulted in a relatively similar expansion in rats and mice during the past 15-20 million years, indicating that environmental factors have been a strong force in this expansion. Dramatic changes and the appearance of new enzymes, the duodenases, have also occurred in ruminats (Fig 1)(86). Interestingly, the dueodenases have changed their tissue specificity and function; from originally being immune proteases they are now expressed in the intestinal region where they participate in food digestion. We then performed an analysis of the mast cell tryptase locus that showed that this locus has been relatively well preserved during the past 140-200 million years of mammalian evolution (102). Distantly related enzymes, both to the chymase and the tryptase locus genes, are also found in fish, however their tissue location and cleavage specificity is not yet known (83).
The ongoing project is presently focusing on the cleavage specificity of a few proteases from the chymase locus of the platypus, several chymases from a diverse set of placental mammals, the human neutrophil proteases, N-elastase, cathepsin G and proteinase 3, the human T cell granzymes A and K, the opossum granzyme B and several fish proteases distantly related to the chymase locus genes of mammals. We are also focusing on completing an evolutionary analysis of the T cell granzyme and the granzyme M loci.
B. Cleavage specificity
A major effort during the last 10 years has been the characterization of the extended specificity of a selected panel of hematopoietic serine proteases (63, 71, 91, 92, 95, 96, 101, 103). One of the first enzymes studied was the rat α-chymase (rMCP-5). This protease was found to have changed primary specificity, from a chymase to an elastase (71). The subsequent question was if rodents have a counterpart of the human mast cell chymase. Mice and rats have several additional closely related genes, the βchymases (Fig 1B). Analysis of one of the major mouse βchymases, mMCP-4, did indeed show that this protease has a cleavage specificity that is almost identical to the human chymase (91). The chymase specificity was later shown to have been a key feature of mast cells during more than 140 million years of mammalian evolution. By analysis of the mast cell chymase from the American opossum we were able to show that this enzyme is also a chymase with very similar specificity as the human chymase (92).
C. General mast cell and basophil biology
The granule-associated serine proteases are stored in an active form tightly bound to negatively charged polysaccharides. In mast cells and basophils, these carbohydrates are either present as heavily sulfated heparin or chondroitin sulfate. These carbohydrates are synthesized on a protein core, the serglycin core protein. The mouse serglycin cDNA and gene were cloned by us a number of years ago (17, 18). Interestingly, knocking out this core protein results in problems for the cell to store theses granule associated proteases (89). Knocking out one of the enzymes involved in the synthesis of heparin, NDST-2, also results in mast cells lacking heparin and cells that have difficulty in storing several of the proteases in their granula (47). The granule-associated proteases can also be used as markers to study mast cell differentiation and to characterize mast cell lines. Using these criteria we have identified a mouse cell line with both mast cell and basophil characteristics indicating an early bipotential precursor (52). In addition, these criteria have been used to study the kinetics of mast cell expansion during various parasite infections (43, 50) and the phenotype of a number of human mast cell, basophil and monocytic cell lines (21, 24, 25, 27, 28, 34, 35). A number of years ago we developed antisera against most of the different proteases stored in various mouse mast cell populations. These antisera have been used by a number of labs all over the world to study mast cell function and differentiation (47, 51, 57, 62, 89).
We have a long-term interest in basophil biology, which has resulted in one publication on establishing culture conditions and purification scheme for immature human basophils. This project was initiated to obtain sufficient amounts of transcriptionally active basophil precursors to study the transcriptome of human basophils (85). Mature blood basophils are mostly terminally differentiated and lack almost entirely functional mRNA (85). An additional manuscript is presently being completed on the analysis of the transcriptome of these in vitro differentiated human basophil precursors (manuscript in preparation).