Extended Cleavage Specificity of two Hematopoietic Serine Proteases from a Ray-Finned Fish, the Spotted Gar (Lepisosteus oculatus).

The extended cleavage specificities of two hematopoietic serine proteases originating from the ray-finned fish, the spotted gar (Lepisosteus oculatus), have been characterized using substrate phage display. The preference for particular amino acids at and surrounding the cleavage site was further validated using a panel of recombinant substrates. For one of the enzymes, the gar granzyme G, a strict preference for the aromatic amino acid Tyr was observed at the cleavable P1 position. Using a set of recombinant substrates showed that the gar granzyme G had a high selectivity for Tyr but a lower activity for cleaving after Phe but not after Trp. Instead, the second enzyme, gar DDN1, showed a high preference for Leu in the P1 position of substrates. This latter enzyme also showed a high preference for Pro in the P2 position and Arg in both P4 and P5 positions. The selectivity for the two Arg residues in positions P4 and P5 suggests a highly specific substrate selectivity of this enzyme. The screening of the gar proteome with the consensus sequences obtained by substrate phage display for these two proteases resulted in a very diverse set of potential targets. Due to this diversity, a clear candidate for a specific immune function of these two enzymes cannot yet be identified. Antisera developed against the recombinant gar enzymes were used to study their tissue distribution. Tissue sections from juvenile fish showed the expression of both proteases in cells in Peyer's patch-like structures in the intestinal region, indicating they may be expressed in T or NK cells. However, due to the lack of antibodies to specific surface markers in the gar, it has not been possible to specify the exact cellular origin. A marked difference in abundance was observed for the two proteases where gar DDN1 was expressed at higher levels than gar granzyme G. However, both appear to be expressed in the same or similar cells, having a lymphocyte-like appearance.

Phenotypic and Functional Heterogeneity of Monocytes and Macrophages.

Macrophages are likely to be the first immune cells to have appeared during the evolution of multicellular organisms [...].

The Extended Cleavage Specificity of Channel Catfish Granzyme-like II, A Highly Specific Elastase, Expressed by Natural Killer-like Cells.

The extended cleavage specificity of catfish granzyme-like II has been characterized using substrate phage display. The preference for particular amino acids at and surrounding the cleavage site was further validated by using a panel of recombinant substrates. This serine protease, which has previously been isolated as cDNA from a catfish natural killer-like cell line showed a preference for Ala in the P1 position of the substrate, and for multiple basic amino acids N-terminally of the cleavage site. A closely related zebrafish serine protease (zebrafish esterase-like) showed a very similar cleavage specificity, indicating an evolutionary conservation of this protease specificity among various fish species. Two catfish serine proteases, originating from NK-like cells, have now been isolated and characterized. One of them is highly specific met-ase with similar characteristics as the mammalian granzyme M. This enzyme may be involved in the induction of apoptosis in virus-infected cells, with a potential target in (catfish) caspase 6. In contrast to catfish granzyme-like I, the second enzyme analyzed here does not seem to have a direct counterpart in mammalian NK cells, and its role in the immune function of catfish NK cells is, therefore, still not known. However, this enzyme seems to be able to cleave a number of cytoskeletal proteins, indicating a separate strategy to induce apoptosis in target cells. Both of these enzymes are very interesting targets for further studies of their roles in catfish immunity, as enzymes with similar specificities have also been identified in zebrafish.

Identification of the Major Protein Components of Human and Cow Saliva.

Cows produce saliva in very large quantities to lubricate and facilitate food processing. Estimates indicate an amount of 50-150 L per day. Human saliva has previously been found to contain numerous antibacterial components, such as lysozyme, histatins, members of the S-100 family and lactoferrin, to limit pathogen colonization. Cows depend on a complex microbial community in their digestive system for food digestion. Our aim here was to analyze how this would influence the content of their saliva. We therefore sampled saliva from five humans and both nose secretions and saliva from six cows and separated the saliva on SDS-PAGE gradient gels and analyzed the major protein bands with LC-MS/MS. The cow saliva was found to be dominated by a few major proteins only, carbonic anhydrase 6, a pH-stabilizing enzyme and the short palate, lung and nasal epithelium carcinoma-associated protein 2A (SPLUNC2A), also named bovine salivary protein 30 kDa (BSP30) or BPIFA2B. This latter protein has been proposed to play a role in local antibacterial response by binding bacterial lipopolysaccharides (LPSs) and inhibiting bacterial growth but may instead, according to more recent data, primarily have surfactant activity. Numerous peptide fragments of mucin-5B were also detected in different regions of the gel in the MS analysis. Interestingly, no major band on gel was detected representing any of the antibacterial proteins, indicating that cows may produce them at very low levels that do not harm the microbial flora of their digestive system. The nose secretions of the cows primarily contained the odorant protein, a protein thought to be involved in enhancing the sense of smell of the olfactory receptors and the possibility of quickly sensing potential poisonous food components. High levels of secretory IgA were also found in one sample of cow mouth drippings, indicating a strong upregulation during an infection. The human saliva was more complex, containing secretory IgA, amylase, carbonic anhydrase 6, lysozyme, histatins and a number of other less abundant proteins, indicating a major difference to the saliva of cows that show very low levels of antibacterial components, most likely to not harm the microbial flora of the rumen.

Vaccination against galectin-1 promotes cytotoxic T-cell infiltration in melanoma and reduces tumor burden.

Galectin-1 (Gal1) is a glycan-binding protein that promotes tumor progression by several distinct mechanisms. Through direct binding to vascular endothelial growth factor (VEGF)-receptor 2, Gal1 is able to induce VEGF-like signaling, which contributes to tumor angiogenesis. Furthermore, several studies have demonstrated an immunosuppressive function of Gal1 through effects on both effector and regulatory T cells. Elevated Gal1 expression and secretion have been shown in many tumor types, and high Gal1 serum levels have been connected to poor prognosis in cancer patients. These findings suggest that therapeutic strategies directed against Gal1 would enable simultaneous targeting of angiogenesis, immune evasion and metastasis. In the current study, we have analyzed the potential of Gal1 as a cancer vaccine target. We show that it is possible to generate high anti-Gal1 antibody levels in mice immunized with a recombinant vaccine protein consisting of bacterial sequences fused to Gal1. Growth of Gal1 expressing melanomas was significantly impaired in the immunized mice compared to the control group. This was associated with improved perfusion of the tumor vasculature, as well as increased infiltration of macrophages and cytotoxic T cells (CTLs). The level of granzyme B, mainly originating from CTLs in our model, was significantly elevated in Gal1 vaccinated mice and correlated with a decrease in tumor burden. We conclude that vaccination against Gal1 is a promising pro-immunogenic approach for cancer therapy that could potentially enhance the effect of other immunotherapeutic strategies due to its ability to promote CTL influx in tumors.

Quantitative Analysis of the Transcriptome of Two Commonly Used Human Monocytic Cell Lines-THP-1 and Mono Mac 6-Reveals Their Arrest during Early Monocyte/Neutrophil Differentiation.

Cell lines of monocyte/macrophage origin are often used as model systems to study monocyte/macrophage biology. A relevant question is how similar these cell lines are to their in vivo counterparts? To address this issue, we performed a detailed analysis of the transcriptome of two commonly used human monocyte/macrophage cell lines, Mono Mac 6 and THP-1. Both of these cell lines originate from leukemic cells with myelo-monocytic characteristics. We found that both Mono Mac 6 and THP-1 represent cells of very immature origin. Their transcriptomes show more similarities to immature neutrophils than cells of the monocyte/macrophage lineage. They express significant levels of N-elastase, proteinase 3, cathepsin G, and azurocidin but very low levels of CD14, ficolin, and complement factor P. All major MHC class II genes are also expressed at low levels. They show high levels of lysozyme and low levels of one of the immunoglobulin Fc receptors, FCGRIIA, which is characteristic of both neutrophils and monocytes. THP-1, but not Mono Mac 6, also expresses the high-affinity receptor for IgG, FCGRIA. Both cell lines lack the expression of the connective tissue components fibronectin, proteoglycan 4, and syndecan 3, which are characteristics of tissue macrophages but are absent in blood monocytes, indicating that they originate from bone marrow precursors and not yolk sac-derived hematopoietic cells. Both of these cell lines seem, therefore, to represent cells arrested during early myelo-monocytic development, at a branch point between neutrophil and monocyte differentiation. Their very immature phenotype indicates that great care should be taken when using these cell lines as models for normal monocyte/macrophage biology.

The Human Monocyte-A Circulating Sensor of Infection and a Potent and Rapid Inducer of Inflammation.

Monocytes were previously thought to be the precursors of all tissue macrophages but have recently been found to represent a unique population of cells, distinct from the majority of tissue macrophages. Monocytes and intestinal macrophages seem now to be the only monocyte/macrophage populations that originate primarily from adult bone marrow. To obtain a better view of the biological function of monocytes and how they differ from tissue macrophages, we have performed a quantitative analysis of its transcriptome in vivo and after in vitro stimulation with E. coli LPS. The monocytes rapidly responded to LPS by producing extremely high amounts of mRNA for the classical inflammatory cytokines, IL-1α, IL-1ß, IL-6 and TNF-α, but almost undetectable amounts of other cytokines. IL-6 was upregulated 58,000 times, from almost undetectable levels at baseline to become one of the major transcripts already after a few hours of cultivation. The cells also showed very strong upregulation of a number of chemokines, primarily IL-8, Ccl2, Ccl3, Ccl3L3, Ccl20, Cxcl2, Cxcl3 and Cxcl4. IL-8 became the most highly expressed transcript in the monocytes already after four hours of in vitro culture in the presence of LPS. A high baseline level of MHC class II chains and marked upregulation of super oxide dismutase (SOD2), complement factor B, complement factor C3 and coagulation factor 3 (F3; tissue factor) at four hours of in vitro culture were also observed. This indicates a rapid protective response to high production of oxygen radicals, to increase complement activation and possibly also be an inducer of local coagulation. Overall, these findings give strong support for monocytes acting primarily as potent mobile sensors of infection and rapid activators of a strong inflammatory response.

Quantitative In-Depth Transcriptome Analysis Implicates Peritoneal Macrophages as Important Players in the Complement and Coagulation Systems.

To obtain a more detailed picture of macrophage (MΦ) biology, in the current study, we analyzed the transcriptome of mouse peritoneal MΦs by RNA-seq and PCR-based transcriptomics. The results show that peritoneal MΦs, based on mRNA content, under non-inflammatory conditions produce large amounts of a number of antimicrobial proteins such as lysozyme and several complement components. They were also found to be potent producers of several chemokines, including platelet factor 4 (PF4), Ccl6, Ccl9, Cxcl13, and Ccl24, and to express high levels of both TGF-ß1 and TGF-ß2. The liver is considered to be the main producer of most complement and coagulation components. However, we can now show that MΦs are also important sources of such compounds including C1qA, C1qB, C1qC, properdin, C4a, factor H, ficolin, and coagulation factor FV. In addition, FX, FVII, and complement factor B were expressed by the MΦs, altogether indicating that MΦs are important local players in both the complement and coagulation systems. For comparison, we analyzed human peripheral blood monocytes. We show that the human monocytes shared many characteristics with the mouse peritoneal MΦs but that there were also many major differences. Similar to the mouse peritoneal MΦs, the most highly expressed transcript in the monocytes was lysozyme, and high levels of both properdin and ficolin were observed. However, with regard to connective tissue components, such as fibronectin, lubricin, syndecan 3, and extracellular matrix protein 1, which were highly expressed by the peritoneal MΦs, the monocytes almost totally lacked transcripts. In contrast, monocytes expressed high levels of MHC Class II, whereas the peritoneal MΦs showed very low levels of these antigen-presenting molecules. Altogether, the present study provides a novel view of the phenotype of the major MΦ subpopulation in the mouse peritoneum and the large peritoneal MΦs and places the transcriptome profile of the peritoneal MΦs in a broader context, including a comparison of the peritoneal MΦ transcriptome with that of human peripheral blood monocytes and the liver.

Quantitative Transcriptome Analysis of Purified Equine Mast Cells Identifies a Dominant Mucosal Mast Cell Population with Possible Inflammatory Functions in Airways of Asthmatic Horses.

Asthma is a chronic inflammatory airway disease and a serious health problem in horses as well as in humans. In humans and mice, mast cells (MCs) are known to be directly involved in asthma pathology and subtypes of MCs accumulate in different lung and airway compartments. The role and phenotype of MCs in equine asthma has not been well documented, although an accumulation of MCs in bronchoalveolar lavage fluid (BALF) is frequently seen. To characterize the phenotype of airway MCs in equine asthma we here developed a protocol, based on MACS Tyto sorting, resulting in the isolation of 92.9% pure MCs from horse BALF. We then used quantitative transcriptome analyses to determine the gene expression profile of the purified MCs compared with total BALF cells. We found that the MCs exhibited a protease profile typical for the classical mucosal MC subtype, as demonstrated by the expression of tryptase (TPSB2) alone, with no expression of chymase (CMA1) or carboxypeptidase A3 (CPA3). Moreover, the expression of genes involved in antigen presentation and complement activation strongly implicates an inflammatory role for these MCs. This study provides a first insight into the phenotype of equine MCs in BALF and their potential role in the airways of asthmatic horses.

Marked difference in efficiency of the digestive enzymes pepsin, trypsin, chymotrypsin, and pancreatic elastase to cleave tightly folded proteins.

In order for the intestinal mucosa to absorb dietary proteins they have to be digested into single amino acids or very short peptides of a length of not more than four amino acids. In order to study the efficiency of the digestive endopeptidases to digest folded proteins we have analyzed several target proteins under different conditions, native proteins, heat denatured and acid treated. The three pancreatic serine proteases, trypsin, chymotrypsin, and pancreatic elastase, were found to be remarkable inefficient in cleaving native folded proteins whereas pepsin, which acts at a very low pH (pH 1.2) was much more efficient, possibly due to the denaturing conditions and thereby better accessibility to internal cleavage sites at the low pH. Heat treatment improved the cleavage considerably by all three pancreatic enzymes, but acid treatment followed by return to neutral pH did not have any major effect. Cleavage at the low pH when the protein is in a denatured state, is apparently very efficient. This indicates that pepsin is the prime enzyme cleaving the properly folded native proteins and that the pancreatic enzymes primarily are involved in generating single amino acids or very short peptides for efficient uptake by the intestinal mucosa.

The Evolutionary History of the Chymase Locus -a Locus Encoding Several of the Major Hematopoietic Serine Proteases.

Several hematopoietic cells of the immune system store large amounts of proteases in cytoplasmic granules. The absolute majority of these proteases belong to the large family of chymotrypsin-related serine proteases. The chymase locus is one of four loci encoding these granule-associated serine proteases in mammals. The chymase locus encodes only four genes in primates, (1) the gene for a mast-cell-specific chymotryptic enzyme, the chymase; (2) a T-cell-expressed asp-ase, granzyme B; (3) a neutrophil-expressed chymotryptic enzyme, cathepsin G; and (4) a T-cell-expressed chymotryptic enzyme named granzyme H. Interestingly, this locus has experienced a number of quite dramatic expansions during mammalian evolution. This is illustrated by the very large number of functional protease genes found in the chymase locus of mice (15 genes) and rats (18 genes). A separate expansion has also occurred in ruminants, where we find a new class of protease genes, the duodenases, which are expressed in the intestinal region. In contrast, the opossum has only two functional genes in this locus, the mast cell (MC) chymase and granzyme B. This low number of genes may be the result of an inversion, which may have hindered unequal crossing over, a mechanism which may have been a major factor in the expansion within the rodent lineage. The chymase locus can be traced back to early tetrapods as genes that cluster with the mammalian genes in phylogenetic trees can be found in frogs, alligators and turtles, but appear to have been lost in birds. We here present the collected data concerning the evolution of this rapidly evolving locus, and how these changes in gene numbers and specificities may have affected the immune functions in the various tetrapod species.

Mast Cells and Basophils in the Defense against Ectoparasites: Efficient Degradation of Parasite Anticoagulants by the Connective Tissue Mast Cell Chymases.

Ticks, lice, flees, mosquitos, leeches and vampire bats need to prevent the host's blood coagulation during their feeding process. This is primarily achieved by injecting potent anticoagulant proteins. Basophils frequently accumulate at the site of tick feeding. However, this occurs only after the second encounter with the parasite involving an adaptive immune response and IgE. To study the potential role of basophils and mast cells in the defense against ticks and other ectoparasites, we produced anticoagulant proteins from three blood-feeding animals; tick, mosquito, and leech. We tested these anticoagulant proteins for their sensitivity to inactivation by a panel of hematopoietic serine proteases. The majority of the connective tissue mast cell proteases tested, originating from humans, dogs, rats, hamsters, and opossums, efficiently cleaved these anticoagulant proteins. Interestingly, the mucosal mast cell proteases that contain closely similar cleavage specificity, had little effect on these anticoagulant proteins. Ticks have been shown to produce serpins, serine protease inhibitors, upon a blood meal that efficiently inhibit the human mast cell chymase and cathepsin G, indicating that ticks have developed a strategy to inactivate these proteases. We show here that one of these tick serpins (IRS-2) shows broad activity against the majority of the mast cell chymotryptic enzymes and the neutrophil proteases from human to opossum. However, it had no effect on the mast cell tryptases or the basophil specific protease mMCP-8. The production of anticoagulants, proteases and anti-proteases by the parasite and the host presents a fascinating example of an arms race between the blood-feeding animals and the mammalian immune system with an apparent and potent role of the connective tissue mast cell chymases in the host defense.

Extended Cleavage Specificity of the Rat Vascular Chymase, a Potential Blood Pressure Regulating Enzyme Expressed by Rat Vascular Smooth Muscle Cells.

Serine proteases constitute the major protein content of the cytoplasmic granules of several hematopoietic cell lineages. These proteases are encoded from four different loci in mammals. One of these loci, the chymase locus, has in rats experienced a massive expansion in the number of functional genes. The human chymase locus encodes 4 proteases, whereas the corresponding locus in rats contains 28 such genes. One of these new genes has changed tissue specificity and has been found to be expressed primarily in vascular smooth muscle cells, and therefore been named rat vascular chymase (RVC). This ß-chymase has been claimed to be a potent angiotensin-converting enzyme by cleaving angiotensin (Ang) I into Ang II and thereby having the potential to regulate blood pressure. To further characterize this enzyme, we have used substrate phage display and a panel of recombinant substrates to obtain a detailed quantitative view of its extended cleavage specificity. RVC was found to show a strong preference for Phe and Tyr in the P1 position, but also to accept Leu and Trp in this position. A strong preference for Ser or Arg in the P1' position, just C-terminally of the cleavage site, and a preference for aliphatic amino acids in most other positions surrounding the cleavage site was also seen. Interesting also was a relatively strict preference for Gly in positions P3' and P4'. RVC thereby shares similarity in its specificity to the mouse mucosal mast cell chymase mMCP-1, which efficiently converts Ang I to Ang II. This similarity adds support for the role of ß-chymases as potent angiotensin converters in rodents, as their α-chymases, which have the capacity to efficiently convert Ang I into Ang II in other mammalian lineages, have become elastases. However, interestingly we found that RVC cleaved both after Arg2 and Phe8 in Ang I. Furthermore this cleavage was more than two hundred times less efficient than the consensus site obtained from the phage display analysis, indicating that RVC has a very low ability to cleave Ang I, raising serious doubts about its role in Ang I conversion.

Potent and Broad but not Unselective Cleavage of Cytokines and Chemokines by Human Neutrophil Elastase and Proteinase 3.

In two recent studies we have shown that three of the most abundant human hematopoietic serine proteases-mast cell chymase, mast cell tryptase and neutrophil cathepsin G-show a highly selective cleavage of cytokines and chemokines with a strong preference for a few alarmins, including IL-18, TSLP and IL-33. To determine if this is a general pattern for many of the hematopoietic serine proteases we have analyzed the human neutrophil elastase (hNE) and human proteinase 3 (hPR-3) for their cleavage of a panel of 69 different human cytokines and chemokines. Our results showed that these two latter enzymes, in sharp contrast to the two previous, had a very potent and relatively unrestrictive cleavage on this panel of targets. Almost all of these proteins were cleaved and many of them were fully degraded. In light of the proteases abundance and their colocalization, it is likely that together they have a very potent degrading activity on almost any protein in the area of neutrophil activation and granule release, including both foreign bacterial or viral proteins as well as various self-proteins in the area of inflammation/infection. However, a few very interesting exceptions to this pattern were found indicating a high resistance to degradation of some cytokines and chemokines, including TNF-α, IL-5, M-CSF, Rantes, IL-8 and MCP-1. All of these are either important for monocyte-macrophage, neutrophil or eosinophil proliferation, recruitment and activation, suggesting that cytokines/chemokines and proteases may have coevolved to not block the recruitment of monocytes-macrophages, neutrophils and possibly eosinophils during an inflammatory response involving neutrophil activation.

Extended cleavage specificities of two mast cell chymase-related proteases and one granzyme B-like protease from the platypus, a monotreme.

Mast cells (MCs) are inflammatory cells primarily found in tissues in close contact with the external environment, such as the skin and the intestinal mucosa. They store large amounts of active components in cytoplasmic granules, ready for rapid release. The major protein content of these granules is proteases, which can account for up to 35 % of the total cellular protein. Depending on their primary cleavage specificity, they can generally be subdivided into chymases and tryptases. Here we present the extended cleavage specificities of two such proteases from the platypus. Both of them show an extended chymotrypsin-like specificity almost identical to other mammalian MC chymases. This suggests that MC chymotryptic enzymes have been conserved, both in structure and extended cleavage specificity, for more than 200 million years, indicating major functions in MC-dependent physiological processes. We have also studied a third closely related protease, originating from the same chymase locus whose cleavage specificity is closely related to the apoptosis-inducing protease from cytotoxic T cells, granzyme B. The presence of both a chymase and granzyme B in all studied mammals indicates that these two proteases bordering the locus are the founding members of this locus.

Highly Selective Cleavage of Cytokines and Chemokines by the Human Mast Cell Chymase and Neutrophil Cathepsin G.

Human mast cell chymase (HC) and human neutrophil cathepsin G (hCG) show relatively similar cleavage specificities: they both have chymotryptic activity but can also cleave efficiently after leucine. Their relatively broad specificity suggests that they may cleave almost any substrate if present in high enough concentrations or for a sufficiently long time. A number of potential substrates have been identified for these enzymes and, recently, these enzymes have also been implicated in regulating cytokine activity by cleaving numerous cytokines and chemokines. To obtain a better understanding of their selectivity for various potential in vivo substrates, we analyzed the cleavage of a panel of 51 active recombinant cytokines and chemokines. Surprisingly, our results showed a high selectivity of HC; only 4 of 51 of these proteins were substantially cleaved. hCG cleaved a few additional proteins, although this occurred after adding almost equimolar amounts of enzyme to target. The explanation for this wide difference in activity against peptides or other linear substrates compared with native proteins is most likely related to the reduced accessibility of the enzymes to potential cleavage sites in folded proteins. In this article, we present evidence that sites not exposed on the surface of the protein are not cleaved by the enzyme. Interestingly, both enzymes readily cleaved IL-18 and IL-33, two IL-1-related alarmins, as well as the cytokine IL-15, which is important for T cell and NK cell homeostasis. Cleavage of the alarmins by HC and hCG suggests a function in regulating excessive inflammation.

Highly Selective Cleavage of TH2-Promoting Cytokines by the Human and the Mouse Mast Cell Tryptases, Indicating a Potent Negative Feedback Loop on TH2 Immunity.

Mast cells (MC) are resident tissue cells found primarily at the interphase between tissues and the environment. These evolutionary old cells store large amounts of proteases within cytoplasmic granules, and one of the most abundant of these proteases is tryptase. To look deeper into the question of their in vivo targets, we have analyzed the activity of the human MC tryptase on 69 different human cytokines and chemokines, and the activity of the mouse tryptase (mMCP-6) on 56 mouse cytokines and chemokines. These enzymes were found to be remarkably restrictive in their cleavage of these potential targets. Only five were efficiently cleaved by the human tryptase: TSLP, IL-21, MCP3, MIP-3b, and eotaxin. This strict specificity indicates a regulatory function of these proteases and not primarily as unspecific degrading enzymes. We recently showed that the human MC chymase also had a relatively strict specificity, indicating that both of these proteases have regulatory functions. One of the most interesting regulatory functions may involve controlling excessive TH2-mediated inflammation by cleaving several of the most important TH2-promoting inflammatory cytokines, including IL-18, IL-33, TSLP, IL-15, and IL-21, indicating a potent negative feedback loop on TH2 immunity.

Extended Cleavage Specificities of Rabbit and Guinea Pig Mast Cell Chymases: Two Highly Specific Leu-Ases.

Serine proteases constitute the major protein content of mast cell (MC) secretory granules. These proteases can generally be subdivided into chymases and tryptases based on their primary cleavage specificity. Here, we presented the extended cleavage specificities of a rabbit ß-chymase and a guinea pig α-chymase. Analyses by phage display screening and a panel of recombinant substrates showed a marked similarity in catalytic activity between the enzymes, both being strict Leu-ases (cleaving on the carboxyl side of Leu). Amino acid sequence alignment of a panel of mammalian chymotryptic MC proteases and 3D structural modeling identified an unusual residue in the rabbit enzyme at position 216 (Thr instead of more common Gly), which is most likely critical for the Leu-ase specificity. Almost all mammals studied, except rabbit and guinea pig, express classical chymotryptic enzymes with similarly extended specificities, indicating an important role of chymase in MC biology. The rabbit and guinea pig are the only two mammalian species currently known to lack a classical MC chymase. Key questions are now how this major difference affects their MC function, and if genes of other loci can rescue the loss of a chymotryptic activity in MCs of these two species.

The Appearance and Diversification of Receptors for IgM During Vertebrate Evolution.

Three different receptors that interact with the constant domains of IgM have been identified: the polymeric immunoglobulin (Ig) receptor (PIGR), the dual receptor for IgA/IgM (FcαµR) and the IgM receptor (FcµR). All of them are related in structure and located in the same chromosomal region in mammals. The functions of the PIGRs are to transport IgM and IgA into the intestinal lumen and to saliva and tears, whereas the FcαµRs enhance uptake of immune complexes and antibody coated bacteria and viruses by B220+ B cells and phagocytes, as well as dampening the Ig response to thymus-independent antigens. The FcµRs have broad-spectrum effects on B-cell development including effects on IgM homeostasis, B-cell survival, humoral immune responses and also in autoantibody formation. The PIGR is the first of these receptors to appear during vertebrate evolution and is found in bony fish and all tetrapods but not in cartilaginous fish. The FcµR is present in all extant mammalian lineages and also in the Chinese and American alligators, suggesting its appearance with early reptiles. Currently the FcαµR has only been found in mammals and is most likely the evolutionary youngest of the three receptors. In bony fish, the PIGR has either 2, 3, 4, 5 or 6 extracellular Ig-like domains, whereas in amphibians, reptiles and birds it has 4 domains, and 5 in all mammals. The increase in domain number from 4 to 5 in mammals has been proposed to enhance the interaction with IgA. Both the FcαµRs and the FcµRs contain only one Ig domain; the domain that confers Ig binding. In both of these receptors this domain shows the highest degree of sequence similarity to domain 1 of the PIGR. All Ig domains of these three receptors are V type domains, indicating they all have the same origin although they have diversified extensively in function during vertebrate evolution by changing expression patterns and cytoplasmic signaling motifs.

Development of a novel therapeutic vaccine carrier that sustains high antibody titers against several targets simultaneously.

With the aim to improve the efficacy of therapeutic vaccines that target self-antigens, we have developed a novel fusion protein vaccine on the basis of the C-terminal multimerizing end of the variable lymphocyte receptor B (VLRB), the Ig equivalent in jawless fishes. Recombinant vaccines were produced in Escherichia coli by fusing the VLRB sequence to 4 different cancer-associated target molecules. The anti-self-immune response generated in mice that were vaccinated with VLRB vaccines was compared with the response in mice that received vaccines that contained bacterial thioredoxin (TRX), previously identified as an efficient carrier. The anti-self-Abs were analyzed with respect to titers, binding properties, and duration of response. VLRB-vaccinated mice displayed a 2- to 10-fold increase in anti-self-Ab titers and a substantial decrease in Abs against the foreign part of the fusion protein compared with the response in TRX-vaccinated mice (P < 0.01). VLRB-generated Ab response had duration similar to the corresponding TRX-generated Abs, but displayed a higher diversity in binding characteristics. Of importance, VLRB vaccines could sustain an immune response against several targets simultaneously. VLRB vaccines fulfill several key criteria for an efficient therapeutic vaccine that targets self-antigens as a result of its small size, its multimerizing capacity, and nonexposed foreign sequences in the fusion protein.-Saupe, F., Reichel, M., Huijbers, E. J. M., Femel, J., Markgren, P.-O., Andersson, C. E., Deindl, S., Danielson, U. H., Hellman, L. T., Olsson, A.-K. Development of a novel therapeutic vaccine carrier that sustains high antibody titers against several targets simultaneously.