Two of the major rat mast cell proteases, rat mast cell protease 1 (RMCP-1) and RMCP-2, have for many years served as important phenotypic markers for studies of various aspects of mast cell (MC) biology. However, except for these proteases only fragmentary information has been available on the structure and complexity of proteases expressed by different subpopulations of rat MCs. To address these questions, cDNA libraries were constructed from freshly isolated rat peritoneal MCs and from the rat mucosal MC line RBL-1. cDNA clones for 10 different serine proteases (RMCP-1-10), and the MC carboxypeptidase A were isolated and characterized. Six of these proteases have not been isolated previously. Based on their protease content, three separate subpopulations of MCs were identified. Connective tissue MCs (CTMCs) from the ear and peritoneum express the chymases RMCP-1 and -5, the tryptases RMCP-6, and -7 and the carboxypeptidase A. However, based on a large difference in the level of expression of RMCP-7, CTMCs of these two organs may be regarded as two separate subpopulations. RMCP-2 and the three closely related proteases of the RMCP-8 subfamily were identified as the major mucosal MC proteases in rat. In contrast to what has been reported for human MCs, no expression of cathepsin G or cathepsin G–like proteases was detected in any of the rat MC populations. To determine mRNA frequencies for the various proteases expressed by normal tissue MCs, an unamplified peritoneal MC cDNA library was screened with a panel of monospecific cDNA probes. These results showed that peritoneal MCs are highly specialized effector cells with mRNA frequencies for the major proteases in the range of several percent of the total mRNA pool.

Using a recently developed PCR-based strategy, a cDNA encoding a novel mouse mast cell (MC) serine protease (MMCP-8) was isolated and characterized. The MMCP-8 mRNA contains an open reading frame of 247 amino acids (aa), divided into a signal sequence of 18 aa followed by a 2-aa activation peptide (Gly-Glu) and a mature protease of 227 aa. The mature protease has an M(r) of 25072, excluding post-translational modifications, a net positive charge of +12 and six potential N-glycosylation sites. MMCP-8 showed a high degree of homology with mouse granzyme B in the critical regions for determining substrate cleavage specificity, indicating that MMCP-8, similar to granzyme B, preferentially cleaves after Asp residues. A comparative analysis of the aa sequence of MMCP-8 with other hematopoietic serine proteases shows that it is more closely related to cathepsin G and T cell granzymes than to the MC chymases. We therefore conclude that MMCP-8 belongs to a novel subfamily of mouse MC proteases distinct from both the classical chymases and tryptases. Southern blot analysis of BALB/c genomic DNA indicated that only one MMCP-8 gene (or MMCP-8 like gene) is present in the mouse genome. Northern blot analysis of rodent hematopoietic cell lines revealed high levels of MMCP-8 mRNA in a mouse connective tissue MC-like tumor line. However, MMCP-8 mRNA could not be detected in mouse liver, intestine, lung or ears, indicating very low expression in normal tissues. Analysis of the expression of different MMCP in the tissues of Schistosoma mansoni-infected BALB/c mice showed a strong increase in MMCP-8 levels in the lungs but not in the intestines of infected animals, suggesting the presence of a novel subpopulation of MC in the lungs that expressed MMCP-8, either alone or in combination with MMCP-5 and carboxypeptidase A. The dramatic increase in MMCP-1 and MMCP-2 levels but not of MMCP-8 in the intestines of parasitized animals also shows that MMCP-8 is not expressed in mucosal MC in the mouse. This latter is in clear contrast to what has been observed in the rat where the MMCP-8 homologues, RMCP-8, -9 and -10, can be considered as true mucosal MC proteases.

The knowledge about the differentiation of basophilic leukocytes is fragmentary. This report discusses a detailed phenotypic characterization of molecular markers for hematopoietic differentiation in a basophilic leukemia cell line, KU812. The expression of markers for lymphoid, erythroid, neutrophil, eosinophil, monocytic, megakaryocytic, mast cell and basophil differentiation was analyzed at the mRNA level by Northern blots in the KU812 cells, and for reference, in a panel of human cell lines representative of the different hematopoietic differentiation lineages. KU812 was found to express a number of mast cell and basophil-related proteins, i.e. mast cell tryptase, mast cell carboxypeptidase A, high-affinity immunoglobulin (IgE) receptor alpha and gamma chains and the core protein for heparin and chondroitin sulphate synthesis. We found no expression of a number of monocyte/-macrophage or neutrophil leukocyte markers except for lysozyme. From earlier studies, it has been shown that lysozyme is not expressed in murine mucosal mast cell lines. This finding, together with the expression of the mast cell carboxypeptidase in KU812 might distinguish the phenotype of this cell line from that typical of mucosal mast cell lines in rodents. We found a low level of expression of the eosinophil and basophil marker, major basic protein, which might indicate a relationship between basophils and eosinophils. No expression is, however, detected with the eosinophil-specific markers eosinophil cationic protein, eosinophil-derived neurotoxin or eosinophil peroxidase. We also report an extensive screening for inducers of basophilic differentiation of the KU812 cells. The most efficient protocol of induction included serum starvation which led to a dramatic increase in a number of markers specific for mast cells and basophils such as tryptase, carboxypeptidase A and the heparin core protein. Finally, diisopropylfluorophosphate analysis of total protein extracts from KU812 show four labeled protein bands with sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that this cell line expresses at least three previously undescribed serine proteases of which one or more could be a potential basophil-specific marker(s).

Expression of a mast cell tryptase mRNA was detected in two human monocytic cell lines, the U-937 and the Mono Mac 6, and in normal human peripheral blood (PB) monocytes. In the U-937 cell line but not in normal PB monocytes, the tryptase expression was upregulated 3-50 fold following phorbol ester (PMA)-induced differentiation, but no such induction was seen after retinoic acid, interferon-gamma or vitamin D3 exposure. The tryptases expressed in PMA-induced and non-induced U-937 and in Mono Mac 6 were characterized by PCR amplification and nucleotide sequence analysis. The U-937 cell line was found to express a tryptase identical to one of the previously cloned mast-cell beta tryptases (Tryptase I), and the tryptase expressed in Mono Mac 6 was found to be nearly identical to the previously cloned alpha tryptase. By northern blot analysis with oligonucleotide probes specific for the alpha and beta tryptases both cell lines were found to express only one type of tryptase. Densitometric quantifications of tryptase mRNA levels, in the two cell lines, showed approximately 80 times higher mRNA levels in Mono Mac 6 compared to non-induced U-937. Immunohistochemical staining for tryptase showed a marked heterogeneity in the Mono Mac 6 cell line. Only one out of 10 cells were positive for the protein but the levels in these cells were very high, equivalent, or even higher than the levels seen in the human mast cell line HMC-1. This shows that the expression of a single tryptase, in this case the alpha tryptase, is sufficient for the production of a stable protein and probably also a stable proteolytically active tetramer. The family of human mast-cell tryptases has been considered to represent a class of proteases specifically expressed in mast cells and basophilic leucocytes. The expression of tryptases in two monocytic cell lines and in normal PB monocytes indicate that in humans, the lineage specificity of these serine proteases is less restricted than earlier expected. The cloning of a full length cDNA for the murine counterpart to the human mast cell tryptases, the MMCP-6, is presented. No expression of the MMCP-6 was detected in a panel of mouse monocyte or macrophage cell lines indicating a species difference in the lineage specificity of the 'mast cell tryptases'.

No abstract

In this work, to study the emergence of the H chain V region repertoire during mammalian evolution, we present an analysis of 25 independent H chain V regions from a monotreme, the Australian duck-billed platypus, Ornithorhynchus anatinus. All the sequences analyzed were found to form a single branch within the clan III of mammalian V region sequences in a distance tree. However, compared with a classical V gene family this branch was more diversified in sequence. Sequence analysis indicates that the apparent lack of diversity in germline V segments is well compensated for by relatively long and highly diversified D and N nucleotides. In addition, extensive sequence variation was observed in the framework region 3. Furthermore, at least five and possibly seven different J segments seem to be actively used in recombination. Interestingly, internal cysteine bridges in the complementarity-determining region (CDR)3 loop, or between the CDR2 and CDR3 loops, are found in ∼36% of the platypus VH sequences. Such cysteine bridges have also been observed in cow, camel, and shark. Internal cysteine bridges may play a role in stabilizing long and diversified CDR3 and thereby have a role in increasing the affinity of the Ab-Ag interaction.

In birds, reptiles and amphibians the IgY isotype exhibits the functional characteristics of both of IgG and IgE. Hence, the gene for IgY most likely duplicated some time during early mammalian evolution and formed the ancestor of present day IgG and IgE. To address the question of when IgY duplicated and formed two functionally distinct isotypes, and to study when IgG and IgA lost their second constant domains, we have examined the Ig expression in a non-placental mammal, the marsupial Monodelphis domestica (grey short-tailed opossum). Screening of an opossum spleen cDNA library revealed the presence of all three isotypes in marsupials. cDNA clones encoding the entire constant regions of opossum IgE (epsilon chain), IgG (gamma chain) and IgA (alpha chain) were isolated, and their nucleotide sequences were determined. A comparative analysis of the amino acid sequences for IgY, IgA, IgE and IgG from various animal species showed that opossum IgE, IgG and IgA on the phylogenetic tree form branches clearly separated from their eutherian counterparts. However, they still conform to the general structure found in eutherian IgE, IgG and IgA. Our findings indicate that all the major evolutionary changes in the Ig isotype repertoire, and in basic Ig structure that have occurred since the evolutionary separation of mammals from the early reptile lineages, occurred prior to the evolutionary separation of marsupials and placental mammals.

To trace the emergence of the modern post‐switch immunoglobulin (Ig) isotypes in vertebrate evolution we have studied Ig expression in mammals distantly related to eutherians. We here present an analysis of the Ig expression in an egg‐laying mammal, a monotreme, the duck‐billed platypus (Ornithorhynchus anatinus). Fragments of platypus IgG and IgE cDNA were obtained by a PCR‐based screening using degenerate primers. The fragments obtained were used as probes to isolate full‐length cDNA clones of three platypus post‐switch isotypes, IgG1, IgG2, and IgE. Comparative amino acid sequence analysis against IgY, IgE and IgG from various animal species revealed that platypus IgE and IgG form branches that are clearly separated from those of their eutherian (placental) counterparts. However, the platypus IgE and IgG still conform to the general structure displayed by the respective Ig isotypes of eutherian and marsupial mammals. According to our findings, all of the major evolutionary changes in the expression array and basic Ig structure that have occurred since the evolutionary separation of mammals from the early reptile lineages, occurred prior to the separation of monotremes from marsupial and placental mammals. Hence, our results indicate that the modern post‐switch isotypes appeared very early in the mammalian lineage, possibly already 310–330 million years ago.