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Granzymes A and K are two highly homologous serine proteases expressed by mammalian cytotoxic T cells (CTLs) and natural killer (NK) cells. The locus encoding these two proteases is the first of the hematopoietic serine protease loci to appear during vertebrate evolution. This locus is found in all jawed vertebrates including the cartilaginous fishes. Granzyme A is the most abundant of the different granzymes expressed by CTLs and NK cells and its potential function has been studied extensively for many years. However, no clear conclusions concerning its primary role in the immune defense has been obtained. In all mammals, there are only one copy each of granzyme A and K, whereas additional copies are found in both cartilaginous and ray finned fishes. In cichlids two of these copies seem to encode new members of the granzyme A/K family. These two new members appear to have changed primary specificity and to be pure chymases based on the amino acids in their active site substrate binding pockets. Interestingly, one of these gene copies is located in the middle of the granzyme A/K locus, while the other copy is present in another locus, the met-ase locus. We here present a detailed characterization of the extended cleavage specificity of one of these non-classical granzymes, a Zebra mbuna granzyme positioned in the granzyme A/K locus. This enzyme, named granzyme A2, showed a high preference for tyrosine in the P1 position of substrates, thereby being a strict chymase. We have also characterized one of the classical granzyme A/Ks of the Zebra mbuna, granzyme A1, which is a tryptase with preference for arginine in the P1 position of substrates. Based on their extended specificities, the two granzymes showed major similarities, but also some differences in preferred amino acids in positions surrounding the cleavable amino acid. Fish lack one of the hematopoietic serine protease loci of mammals, the chymase locus, where one of the major mast cell enzymes is located. An interesting question is now if cichlids have by compensatory mechanisms generated a mast cell chymase from another locus, and if similar chymotryptic enzymes have appeared also in other fish species., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Serine proteases are major granule constituents of cells from several mammalian hematopoietic cell lineages. Despite the relatively extensive knowledge about these mammalian proteases, very little is known about their bird, reptile and amphibian homologs. In order to close this gap in our understanding of the evolution of these proteases, we have characterized the extended cleavage specificity and hematopoietic expression pattern of the chicken serine protease cathepsin G-like. This protease, which clusters in a separate subfamily of serine proteases among the vertebrate hematopoietic serine proteases, has been characterized using substrate phage display and further validated by using a panel of recombinant substrates. A preference for a lysine in the P1 position of a substrate, arginines in positions P2 and P3, and the aromatic amino acid tryptophane in the P4 position was observed. Based on the sequence alignment we could identify a consensus sequence for this protease as being PGGWRRK ↓ ALSV. Mass spectrometry analysis of a peptide with the consensus sequence obtained by phage display showed that cleavage of this peptide occurred after the conserved Lys (K) residue. A screening of potential in vivo substrates based on the derived P5-P3' consensus sequence resulted in a relatively limited number of potential substrates, due to the high selectivity of this enzyme. The most interesting of these were PDGF-A, coagulation factor V and low-density lipoprotein receptor like-8. Immunohistochemical analysis of chicken white blood cells with antisera produced against chicken cathepsin G-like and chicken egg lysozyme, as a reference protein known to be expressed by hematopoietic cells, showed presence of chicken cathepsin G-like almost exclusively in thrombocytes whereas lysozyme was found at very high amounts in heterophils, and lower amounts in monocytes and thrombocytes., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Granzyme B (GzmB) is primarily expressed by mammalian cytotoxic T cells and serves as one of the key components in the defense against viral infection by the induction of apoptosis in virus infected cells. By direct cell to cell contact and delivery into target cells by perforin, cytotoxic T cells activate apoptosis through the action of GzmB by both caspase-dependent and -independent pathways. In search for early ancestors of GzmB we have in the current study identified and characterized a GzmB homologue from a reptile, the Chinese alligator. This enzyme is encoded from the same locus as the mammalian counterparts, the chymase locus. Phage display analysis of the cleavage specificity of the recombinant alligator enzyme (named MCP1A-like) shows that it is a relatively strict Glu-ase, with strong preference for glutamic acid in the P1 position of a substrate. The majority of mammalian GzmB:s are, in marked contrast to the alligator enzyme, relatively strict Asp-ases. The alligator enzyme also showed strong preference for Ala, Pro and Gly in the P2 position and Val in the P3 position indicating that it has a narrow specificity, similar to the mammalian counterparts. Analysis of the three amino acids forming the substrate binding pocket (S1 pocket) in three amphibian homologues to MCP1A-like, from the frogs Xenopus laevis and Xenopus tropicalis, shows that these amphibian enzymes have similar substrate binding pocket as their mammalian counterparts. This finding, together with the apparent lack of GzmB homologs in fish, indicates that the ancestor of GzmB did appear with the amphibians at the base of tetrapod evolution. This study is a first step in a larger effort to understand the evolutionary processes involved in shaping anti-viral immunity in non-mammalian vertebrates., (Copyright © 2021. Published by Elsevier Ltd.)

Metallo-carboxypeptidases are exopeptidases with diverse expression and function, found in all kingdoms of life from bacteria to mammals. One of them, the carboxypeptidase A3 (CPA3), has become an important component of the mammalian immune system by its expression in mast cells. Mast cells (MCs) are highly specialized sentinel cells, which store large amounts of bioactive mediators, including CPA3, in very abundant cytoplasmic granules. Clinical studies have found an increased CPA3 expression in asthma but the physiological role as well as the evolutionary origin of CPA3 remains largely unexplored. CPA3 belongs to the M14A subfamily of metallo-carboxypeptidases, which among others also includes the digestive enzymes CPA1, CPA2, CPB1 and CPO. To study the appearance of CPA3 during vertebrate evolution, we here performed bioinformatic analyses of homologous genes and gene loci from a broad panel of metazoan animals from invertebrates to mammals. The phylogenetic analysis indicated that CPA3 appeared at the base of tetrapod evolution in a branch closer to CPB1 than to other CPAs. Indeed, CPA3 and CPB1 are also located in the same locus, on chromosome 3 in humans. The presence of CPA3 only in tetrapods and not in fishes, suggested that CPA3 could have appeared by a gene duplication from CPB1 during early tetrapod evolution. However, the apparent loss of CPA3 in several tetrapod lineages, e.g. in birds and monotremes, indicates a complex evolution of the CPA3 gene. Interestingly, in the lack of CPA3 in fishes, zebrafish MCs express instead CPA5 for which the most closely related human carboxypeptidase is CPA1, which has a similar cleavage specificity as CPA3. Collectively, these findings clarify and add to our understanding of the evolution of hematopoietic proteases expressed by mast cells., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Serine proteases constitute the major protein content of mammalian mast cell granules and the selectivity for substrates by these proteases is of major importance for the role of mast cells in immunity. In order to address this subject, we present here the extended cleavage specificity of sheep mast cell protease-2 (MCP2), a chymotrypsin-type serine protease. Comparison of the extended specificity results to a panel of mammalian mast cell chymases show, in almost all aspects, the same cleavage characteristics. This includes preference for aromatic residues (Phe, Tyr, Trp) in the P1 position of substrates and a preference for aliphatic residues in most other substrate positions around the cleavage site. MCP2 also cleaved, albeit relatively low efficiency, after Leu in the P1 position. In contrast to the human, mouse, hamster and opossum chymases that show a relatively strong preference for negatively charged amino acids in the P2'position, the sheep MCP2, however, lacked that preference. Therefore, together with the rat chymase (rMCP1), sheep MCP2 can be grouped to a small subfamily of mammalian chymases that show fairly unspecific preference in the P2'position. In summary, the results here support the view of a strong evolutionary conservation of a potent chymotrypsin-type protease as a key feature of mammalian mast cells., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Here we present the extended cleavage specificity of catfish granzyme-like I, previously identified in fish NK-like cells. This protease has been characterised using substrate phage display and further validated by using a panel of recombinant substrates. A strict preference for Met in the P1 (cleavage) position, indicating metase specificity was observed. A screening of potential in vivo substrates was performed based on the derived P5-P3' consensus: Arg-Val-Thr-Gly-Met(↓)Ser-Leu-Val. Channel catfish caspase 6 was one very interesting potential target identified. This site was present in an adjacent position to the classic caspase activation site (Asp179 in human caspase 6). Cleavage of this site (hence potential activation) by the catfish granzyme-like I could reveal a novel mechanism of caspase 6 activation. This poses an interesting idea that the role of granzyme-like proteases in the activation of caspase dependent apoptosis mechanisms has been conserved for over 400 million years., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Immunoglobulin A deficiency (IgAD) is the most common primary immunodeficiency in both humans and selected breeds of domestic dogs. In both species, IgAD is associated with recurrent infections and immune mediated diseases. Previous results imply that IgAD is also common in the wild ancestor of domestic dogs, the gray wolf. Here, we report that serum IgA concentrations are significantly different in Scandinavian and Canadian wolves (p = 3.252e-15) with an increased prevalence for IgAD in Scandinavian wolves (60%), which is as high as those found in high-risk dog breeds., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Low mean concentrations of serum immunoglobulin A (IgA) and an increased frequency of overt IgA deficiency (IgAD) in certain dog breeds raises the question whether it is a breeding-enriched phenomenon or a legacy from the dog's ancestor, the gray wolf (Canis lupus). The IgA concentration in 99 serum samples from 58 free-ranging and 13 captive Scandinavian wolves, was therefore measured by capture ELISA. The concentrations were markedly lower in the wolf serum samples than in the dog controls. Potential differences in the IgA molecule between dogs and wolves were addressed by sequencing the wolf IgA heavy chain constant region encoding gene (IGHA). Complete amino acid sequence homology was found. Detection of wolf and dog IgA was ascertained by showing identity using double immunodiffusion. We suggest that the vast majority of wolves, the ancestor of the dog, are IgA deficient., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

We have recently shown that immunization against the extra domain-B (ED-B) of fibronectin, using Freund's adjuvant, reduces tumor growth in mice by 70%. In the present study we compare the immune response generated against ED-B using the non-toxic and biodegradable adjuvant Montanide ISA 720/CpG with the response elicited by Freund's adjuvant. Montanide ISA 720/CpG induced anti-ED-B antibodies with higher avidity and less variable levels between individuals than Freund's. Moreover, the duration of the immune response was longer and the generation of anti-ED-B antibodies in naïve mice was faster, when Montanide ISA 720/CpG was used. We conclude that it is possible to replace the mineral oil based adjuvant Freund's with an adjuvant acceptable for human use, which is a prerequisite for transfer of the ED-B vaccine to the clinic., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Monoclonal antibodies are used successfully in the treatment of many human disorders. However, these antibodies are expensive and have in many countries put a major strain on the health care economy. Therapeutic vaccines, directed against the same target molecules, may offer a solution to this problem. Vaccines usually involve lower amount of recombinant protein, approximately 10,000-20,000 times less, which is significantly more cost effective. Attempts to develop such therapeutic vaccines have also been made. However, their efficacy has been limited by the lack of potent immunostimulatory compounds, adjuvants, for human use. To address this problem we have conducted a broad screening for adjuvants that can enhance the efficacy of therapeutic vaccines, whilst at the same time being non-toxic and biodegradable. We have now identified adjuvants that show these desired characteristics. A combination of Montanide ISA720 and phosphorothioate stabilized CpG stimulatory DNA, induced similar or even higher anti-self-antibody titers compared to Freund's adjuvant, currently the most potent, but also toxic, adjuvant available. This finding removes one of the major limiting factors in the field and facilitates the development of a broad range of novel therapeutic vaccines.

Development of vaccines targeting important self-molecules like tumor antigens, IgE, cytokines or other regulatory molecules, brings about challenges that are not met in classical vaccine development. Tolerance inducing mechanisms reduce the levels of therapeutic antibodies in the vaccinated subject, and anti-self antibody titers are frequently more than 50-fold lower than the anti-non-self response to the carrier. In order to overcome this limitation in efficacy we have explored various methods to enhance the immunogenicity of the vaccine antigen. Vaccination with a molecule containing two IgE Cepsilon3 domains and thereby a low level of repetitiveness markedly increased the efficacy. The anti-IgE antibody titers in the animals treated with the dimeric vaccine antigen were 4.5, 5 and 8 times higher than in the animals treated with the monomer, in three independent experiments. In addition, this increase in efficacy was not masked by the use of potent adjuvants. The effect persisted even in the presence of Freunds or Montanide ISA 51, two mineral oil based adjuvants. This in contrast to most Toll-like receptor (TLR) agonists, which appear to enhance the immune response only when administrated together with weak adjuvants. This clearly shows that the introduction of a moderately repetitive structure is enough to substantially increase the efficacy of a therapeutic vaccine.

Allergic diseases are common in dogs and the involvement of IgE in the pathogenesis of canine atopic dermatitis (CAD) and flea allergy dermatitis (FAD) is documented. However, many dogs do not achieve sufficient control of their allergic disease, and there is a great need for new treatment strategies. In order to address this issue we first needed to obtain a better picture of IgE-levels in dogs and how plasma IgE-levels are affected by breed, age and health status. IgE is normally present at diminutive concentrations in sera and detection by diagnostic methods has been a technical challenge. Here, we present a new in vitro assay for determining absolute levels of total IgE in sera from dogs. In a panel of 76 adult dogs diagnosed either as healthy or suffering from atopic dermatitis, autoimmunity or infection by skin parasites, we show that levels of IgE range from 1 to 41 microg/ml. This is almost 100 times the level observed in non-atopic humans. However, these exceptionally high IgE-levels in the dogs could not be correlated to either breed or health status. To address the issue of novel treatment strategies, the possibility of reducing the IgE-levels in nine Beagle dogs by immunization with a new therapeutic vaccine was investigated. High levels of anti-IgE antibodies were induced in all dogs, and the IgE-levels were subsequently decreased by a mean of 65%. This shows that the allergy vaccine is potent enough to break the tolerance against IgE, even when the initial IgE-levels are as high as those observed in dogs. Thus, the vaccination treatment may have the potential to serve as a future therapy for dogs with atopic diseases.

TCR gamma (TRG) chain diversity in splenic gammadelta T cells was determined for an egg-laying mammal (or monotreme), the duckbill platypus. Three distinct V subgroups were found in the expressed TRG chains and these three subgroups are members of a clade not found so far in eutherian mammals or birds. Each subgroup contains approximately five V gene segments, and their overall divergence is much less than is found in eutherians and birds, consistent with their recent evolution from an ancestral V gene segment. The platypus TRG locus also contains three C region genes and many of the residues involved in TCR function, such as interactions with CD3, were conserved in the monotreme C regions. All non-eutherian mammals (monotremes and marsupials) lacked the second cysteine residue necessary to form the intradomain disulfide bond in the C region, a loss apparently due to independent mutations in marsupials and monotremes. Monotreme TRGC regions also had among the most variation in the length of the connecting peptide region described for any species due to repeated motifs.

Hematopoietic serine proteases (SPs) are stored in the granules of different leukocytes and these enzymes are important effector molecules in the immune system of mammals. However, very little is known about the presence of these proteins in lower vertebrates. Herein, the primary structures of five novel fish SPs, from the Atlantic cod (Gadus morhua) and the channel catfish (Ictalurus punctatus), are presented. One of the cod SPs is a homologue to human GzmA and K. The other fish SPs identified are termed 'Gzm-like' and are distantly related to a large heterogeneous group of hematopoietic SPs, including most of the T-cell Gzms (B-H), the mast cell chymases, the mast cell/basophil proteases of the mouse mast cell protease-8 subfamily (M8-family) and the neutrophil cathepsin G. Extensive BLAST-searches in genome and expressed sequence tag (EST) databases identified 40 additional teleost SPs related to the mammalian hematopoietic SP family. Subsequent phylogenetical analyses clearly demonstrate that the diversification into different subgroups within the GzmB/chymase/cathepsin G-related family has occurred independently in bony fishes and in mammals. In contrast, our findings suggest that the three subgroups, including (1) GzmK and the potent apoptosis-inducing GzmA, (2) the neutrophil proteases (proteinase 3, N-elastase and azurocidin), and (3) adipsin, have all evolved as distinct groups before the separation of tetrapods from the ray-finned fish approximately 420 million years ago.

In the development of a novel vaccine against atopic allergies, we have screened for adjuvants that enhance the therapeutic antibody response against self immunoglobulin E (IgE). The response against self IgE is induced by administration of a vaccine antigen, which contains both self and non-self IgE regions, together with an adjuvant. We evaluated five commonly used adjuvants; Freund's, aluminium hydroxide, ISCOMs, Montanide ISA 51 and Montanide ISA 720, and found that the mineral oil-based adjuvants; Montanide ISA 51 and Freund's induced at least 5-10-fold higher anti-self IgE titers than any of the other candidates. However, with one exception, Alum, the immune responses against the carrier, i.e. the non-self regions, were similar for all adjuvants, indicating that the ability to induce responses against self and non-self antigens differ among adjuvants. The responses against non-self IgE were more than 50-fold higher than antibody responses against self IgE in both the Freund's and Montanide 51-administered animals, indicating that the response against self molecules is markedly inhibited by tolerance-inducing mechanisms. Co-administration of Montanide ISA 51 with immuno-stimulatory substances from bacteria; muramyldipeptide (MDP), monophosphoryl-lipid A (MPL) or a formyl-methionine-containing tripeptide (fMLP), did not elevate the anti-self IgE response. Hence, adjuvants based on pure mineral oil without additional immuno-stimulatory substances appear to be the best adjuvant candidates in therapeutic vaccines aimed at regulating the in vivo levels of self-proteins.

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.

In this paper, we review data on the monotreme immune system focusing on the characterisation of lymphoid tissue and of antibody responses, as well the recent cloning of immunoglobulin genes. It is now known that monotremes utilise immunoglobulin isotypes that are structurally identical to those found in marsupials and eutherians, but which differ to those found in birds and reptiles. Monotremes utilise IgM, IgG, IgA and IgE. They do not use IgY. Their IgG and IgA constant regions contain three domains plus a hinge region. Preliminary analysis of monotreme heavy chain variable region diversity suggests that the platypus primarily uses a single VH clan, while the short-beaked echidna utilises at least 4 distinct VH families which segregate into all three mammalian VH clans. Phylogenetic analysis of the immunoglobulin heavy chain constant region gene sequences provides strong support for the Theria hypothesis. The constant region of IgM has proven to be a useful marker for estimating the time of divergence of mammalian lineages.

To further characterize genes of immunological importance from non-placental mammals, cDNAs encoding beta(2)-microglobulin (beta(2)m) were isolated from two prototherians, the platypus and an echidna, and one metatherian, a grey short-tailed opossum. In addition, a second allele of beta(2)m was identified in another metatherian species, the brushtail possum. Analysis of the deduced translations revealed conservation of key residues in these molecules over a long evolutionary history. The types of nucleotide substitutions present among the various taxa are also consistent with purifying selection at this conserved locus. An evolutionary tree of beta(2)m was constructed that supports the classic view of evolution with prototherians as the basal mammalian group.

The immunobiology of monotremes is poorly understood. In this paper, we describe the characterisation of the heavy chain of IgM from Tachyglossus aculeatus, the short-beaked echidna. The echidna heavy chain constant region of IgM (Cmu)was isolated from a spleen cDNA library using a Trichosurus vulpecula probe. It has approximately 46.5% amino acid identity to marsupial and eutherian Cmus, and approximately 30% amino acid identity with Cmu from birds and reptiles. Phylogenetic analysis of mammalian Cmu provides strong support for the Theria hypothesis, with a sister grouping of the eutherians and marsupials to the exclusion of the monotremes. Cmu sequences suggest that monotremes and therians separated approximately 170 million years ago (mya), marsupials and eutherians separated approximately 130mya, and Australian and American marsupials separated approximately 65mya.

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.