Maria Aveskogh

Evidence for an early appearance of modern post‐switch isotypes in mammalian evolution; cloning of IgE, IgG and IgA from the marsupial Monodelphis domestica

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 (ϵ chain), IgG (γ chain) and IgA (α 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.

Characterization of mouse mast cell protease-8, the first member of a novel subfamily of mouse mast cell serine proteases, distinct from both the classical chymases and tryptases

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 Mr 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.

Evidence for an early appearance of modern post‐switch immunoglobulin isotypes in mammalian evolution (II); cloning of IgE, IgG1 and IgG2 from a monotreme, the duck‐billed platypus, Ornithorhynchus anatinus

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.

Cloning of IgE from the echidna (Tachyglossus aculeatus) and a comparative analysis of ε chains from all three extant mammalian lineages

In continuation of our evolutionary studies of immunoglobulin (Ig) expression, we present here the cloning of IgE from a monotreme, the short-beaked echidna (Tachyglossus aculeatus). Including echidna IgE, 15 epsilon chain sequences have been isolated and each of the three mammalian lineages (placentals, marsupials and monotremes) is now represented by at least two sequences. Phylogenetic analyses based on all available epsilon chains and a selection of other mammalian Ig isotypes (IgM, IgA and IgG) were generated using three different algorithms. The resulting trees strongly support the Theria hypothesis, which states that the monotreme lineage was the first of the three extant mammalian lineages to appear in evolution. Furthermore, to increase our understanding of IgE we have done a detailed comparative analysis, with focus on primary structure, potential N-glycosylation, charge distribution and conservation of residues in the putative receptor-binding site. The overall structure of IgE, i.e. four constant domains and the positions of putative disulfide-bridge formations, are conserved, as is an N-glycosylation site in the third constant domain. An increased homology was observed in the putative receptor-binding site, which suggests an important function for the IgE/Fc epsilon RI interaction. IgE has been found exclusively in mammals, but it is present in all extant mammalian lineages. This, together with the overall conservation of structure, indicates that IgE appeared as a separate isotype early in mammalian evolution and that structural maintenance may have a selective advantage.

Heavy Chain V Region Diversity in the Duck-Billed Platypus (Ornithorhynchus anatinus): Long and Highly Variable Complementarity-Determining Region 3 Compensates for Limited Germline Diversity

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.

Expression of a Mast Cell Tryptase in the Human Monocytic Cell Lines U‐937 and Mono Mac 6

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‐γ or vitamin D3 exposure.

Characterization of cDNA clones encoding mouse proteinase 3 (myeloblastine) and cathepsin G

Abstract Serine proteases are the most abundant granule constituents of several major hematopoietic cell lineages. Due to their high abundance and their strict tissue specificity they have become important phenotypic cell markers used for studies of various aspects of hematopietic cell development. Using a polymerase chain reaction (PCR)-based strategy for the isolation of trypsin-related serine proteases, we were able to isolate cDNAs for two of the major neutrophil and monocyte serine proteases in the mouse, cathepsin G and mouse protease 3 (myeloblastin). The internal PCR fragments were used as probes to screen a mouse mast cell cDNA library and a cDNA library originating from a mouse monocytic cell line (WEHI-274.1). Full-length cDNAs for mouse cathepsin G and proteinase 3 were isolated and their complete sequences were determined. Northern blot analysis revealed expression of cathepsin G in immature cells of the monocyte macrophage lineage but also in the connective tissue mast cell line MTC. Proteinase 3 was expressed in several cell lines of myelo-monocytic origin and in one B-cell line, but not in any of the other cell lines tested. The isolation of cDNAs for mouse cathepsin G and mouse proteinase 3, together with the previous characterization of the gene for mouse N-elastase, and the entire or partial amino acid sequences for porcine azurocidine, equine N-elastase and proteinase 3, rat, dog, and rabbit cathepsin Gs in evolutionary relatively distantly related mammalian species, indicates that these four members of the serine protease family have been maintained for more than 100 million years of mammalian evolution. This latter finding indicates a strong evolutionary pressure to maintain specific immune functions associated with these neutrophil and monocyte proteases. All amino acid positions of major importance for the cleavage site selection have also been fully conserved between mouse and human proteinase 3 and a few minor changes have occurred between mouse and human cathepsin G.

Cloning and structural analysis of IgM (mu chain) and the heavy chain V region repertoire in the marsupial Monodelphis domestica.

To address the question of the Ig isotype repertoire of non placental mammals, we have examined the Ig expression in the marsupial Monodelphis domestica (grey short tailed opossum). Screening of an opossum spleen cDNA library has previously led to the isolation of full length clones for opossum IgG (gamma chain), IgE (epsilon chain) and IgA (alpha chain). We now present the isolation of several cDNA clones encoding the entire constant regions of the opossum IgM (mu chain). A comparative analysis of the amino acid sequences for IgM from various animal species showed that opossum IgM, within the various animals studied, is the most divergent member of its Ig class. However, it still conforms to the general structure of IgM in other vertebrates. Four Ig classes have now been identified in opossum and only one isotype is apparently present within each Ig class, IgM, IgG, IgA and IgE. Opossum has previously been shown to have a limited VH region diversity, with only two V gene families. Both of these belong to the group III of mammalian VH sequences. This limitation in variability is to some extent compensated for by a large variation in D, P and N regions, both in size and in sequence. However, evidence for the expression of only two functional J segments has so far been detected, which indicates a rather limited diversity also of the J segments in the opossum.

Cloning and structural analysis of a gene encoding a mouse mastocytoma proteoglycan core protein; analysis of its evolutionary relation to three cross hybridizing regions in the mouse genome

Serglycin (SGC) is a Ser-Gly-repeat-containing protein, used as a proteoglycan core protein in the parietal yolk sac and in mast cells, where glycosaminoglycan side chains are attached to the serine residues of the repeat region. In this article, the structure of the gene SGC encoding mouse SGC is reported. The gene is divided into three exons, which are all contained within a region of approximately 13 kb. Nucleotide (nt) sequence analysis was carried out on a region of 1.2 kb upstream from the first exon. The region containing the two promoters (active in parietal yolk sac and in mast cells, respectively) was analyzed for the presence of recognition sites for known DNA-binding proteins. A number of sequences closely related to known recognition sites were found in both promoters, and one consensus octamer-binding site could be identified in the putative yolk-sac promoter. Multiple regions in the mouse genome hybridizing with DNA fragments covering the Ser-Gly repeat region have previously been described, and it has been suggested that these loci may represent other proteoglycan core proteins. Analysis of nt sequence was carried out on three out of the more than 15 of these regions present in the mouse genome. However, none of the clones analyzed was found to have any open reading frame in the region of cross-hybridization which possibly could code for a SGC protein. Instead, one of the clones was found to contain an exon encoding a highly basic protein, unrelated to SGC. Hence, no evidence was found for a multigene family of Ser-Gly-repeat-containing proteoglycan-encoding genes.

Identification and structural analysis of four serine proteases in a monotreme, the platypus, Ornithorhynchus anatinus.

Abstract. To study the emergence of the major subfamilies of serine proteases during vertebrate evolution, we present here the primary structure of four serine proteases expressed in the spleen of a monotreme, the platypus, Ornithorhynchus anatinus. Partial cDNA clones for four serine proteases were isolated by a PCR-based strategy. This strategy is based on the high level of sequence identity between various members of the large gene family of trypsin-related serine proteases, over two highly conserved regions, those of the histidine and the serine of the catalytic triad. The partial cDNA clones were used to isolate full-length or almost full-length cDNA clones for three of these proteases from a platypus spleen cDNA library. By phylogenetic analysis, these three clones were identified as being the platypus homologues of human coagulation factor X, neutrophil elastase, and a protease distantly related to the T-cell granzymes. The remaining partial clone was found to represent a close homologue of human complement factor D (adipsin). The isolation of these four clones shows that several of the major subfamilies of serine proteases had evolved as separate subfamilies long before the radiation of the major mammalian lineages of today, the monotremes, the marsupials, and the placental mammals. Upon comparison of the corresponding proteases of monotremes and eutherian mammals, the coagulation and complement proteases were shown to display a higher degree of conservation compared to the hematopoietic proteases N-elastase and the T-cell granzymes. This latter finding indicates a higher evolutionary pressure to maintain specific functions in the complement and coagulation enzymes compared to many of the hematopoietic serine proteases.