Blood Metabolite Profiling of Antarctic Expedition Members: An 1H NMR Spectroscopy-Based Study.

Serum samples from eight participants during the XV winter-over at Concordia base (Antarctic expedition) collected at defined time points, including predeparture, constituted the key substrates for a specific metabolomics study. To ascertain acute changes and chronic adaptation to hypoxia, the metabolic profiles of the serum samples were analyzed using NMR spectroscopy, with principal components analysis (PCA) followed by partial least squares and orthogonal partial least squares discriminant analyses (PLS-DA and OPLS-DA) used as supervised classification methods. Multivariate data analyses clearly highlighted an adaptation period characterized by an increase in the levels of circulating glutamine and lipids, mobilized to supply the body energy needs. At the same time, a reduction in the circulating levels of glutamate and N-acetyl glycoproteins, stress condition indicators, and proinflammatory markers were also found in the NMR data investigation. Subsequent pathway analysis showed possible perturbations in metabolic processes, potentially related to the physiological adaptation, predominantly found by comparing the baseline (at sea level, before mission onset), the base arrival, and the mission ending collected values.

Comparative Analysis of the pIgR Gene from the Antarctic Teleost Trematomus bernacchii Reveals Distinctive Features of Cold-Adapted Notothenioidei.

The IgM and IgT classes were previously identified and characterized in the Antarctic teleost Trematomus bernacchii, a species belonging to the Perciform suborder Notothenoidei. Herein, we characterized the gene encoding the polymeric immunoglobulin receptor (pIgR) in the same species and compared it to the pIgR of multiple teleost species belonging to five perciform suborders, including 11 Antarctic and 1 non-Antarctic (Cottoperca gobio) notothenioid species, the latter living in the less-cold peri-Antarctic sea. Antarctic pIgR genes displayed particularly long introns marked by sites of transposable elements and transcription factors. Furthermore, analysis of T. bernacchii pIgR cDNA unveiled multiple amino acid substitutions unique to the Antarctic species, all introducing adaptive features, including N-glycosylation sequons. Interestingly, C. gobio shared most features with the other perciforms rather than with the cold-adapted relatives. T. bernacchii pIgR transcripts were predominantly expressed in mucosal tissues, as indicated by q-PCR and in situ hybridization analysis. These results suggest that in cold-adapted species, pIgR preserved its fundamental role in mucosal immune defense, although remarkable gene structure modifications occurred.

The evolutionary puzzle solution for the origins of the partial loss of the Cτ2 exon in notothenioid fishes.

Cryonotothenioidea is the main group of fishes that thrive in the extremely cold Antarctic environment, thanks to the acquisition of peculiar morphological, physiological and molecular adaptations. We have previously disclosed that IgM, the main immunoglobulin isotype in teleosts, display typical cold-adapted features. Recently, we have analyzed the gene encoding the heavy chain constant region (CH) of the IgT isotype from the Antarctic teleost Trematomus bernacchii (family Nototheniidae), characterized by the near-complete deletion of the CH2 domain. Here, we aimed to track the loss of the CH2 domain along notothenioid phylogeny and to identify its ancestral origins. To this end, we obtained the IgT gene sequences from several species belonging to the Antarctic families Nototheniidae, Bathydraconidae and Artedidraconidae. All species display a CH2 remnant of variable size, encoded by a short Cτ2 exon, which retains functional splicing sites and therefore is included in the mature transcript. We also considered representative species from the three non-Antarctic families: Eleginopsioidea (Eleginops maclovinus), Pseudaphritioidea (Pseudaphritis urvillii) and Bovichtidae (Bovichtus diacanthus and Cottoperca gobio). Even though only E. maclovinus, the sister taxa of Cryonotothenioidea, shared the partial loss of Cτ2, the other non-Antarctic notothenioid species displayed early molecular signatures of this event. These results shed light on the evolutionary path that underlies the origins of this remarkable gene structural modification.

Cold Adaptation in Antarctic Notothenioids: Comparative Transcriptomics Reveals Novel Insights in the Peculiar Role of Gills and Highlights Signatures of Cobalamin Deficiency.

Far from being devoid of life, Antarctic waters are home to Cryonotothenioidea, which represent one of the fascinating cases of evolutionary adaptation to extreme environmental conditions in vertebrates. Thanks to a series of unique morphological and physiological peculiarities, which include the paradigmatic case of loss of hemoglobin in the family Channichthyidae, these fish survive and thrive at sub-zero temperatures. While some of the distinctive features of such adaptations have been known for decades, our knowledge of their genetic and molecular bases is still limited. We generated a reference de novo assembly of the icefish Chionodraco hamatus transcriptome and used this resource for a large-scale comparative analysis among five red-blooded Cryonotothenioidea, the sub-Antarctic notothenioid Eleginops maclovinus and seven temperate teleost species. Our investigation targeted the gills, a tissue of primary importance for gaseous exchange, osmoregulation, ammonia excretion, and its role in fish immunity. One hundred and twenty genes were identified as significantly up-regulated in Antarctic species and surprisingly shared by red- and white-blooded notothenioids, unveiling several previously unreported molecular players that might have contributed to the evolutionary success of Cryonotothenioidea in Antarctica. In particular, we detected cobalamin deficiency signatures and discussed the possible biological implications of this condition concerning hematological alterations and the heavy parasitic loads typically observed in all Cryonotothenioidea.

Immune response of the Antarctic teleost Trematomus bernacchii to immunization with Psychrobacter sp. (TAD1).

Adult Trematomus bernacchii have been immunized intraperitoneally with heat-killed cells of the Antarctic marine bacterium Psychrobacter sp. (TAD1) up to 60 days. After immunizations and sampling at various times, fish sera were tested for specific IgM by ELISA, and different tissues (head kidney and spleen) were investigated for transcription of master genes of the acquired immune response (IgM, IgT, TRß, TRγ). Results from ELISA assays showed a time-dependent induction of specific serum anti-TAD1 IgM, and western blot analysis of TAD1 lysates probed with fish sera revealed enhanced immunoreactivity in immunized animals compared to controls. Quantitative PCR analysis of transcripts coding for IgM, IgT, TRß, TRγ was performed in T. bernacchii tissues to assess basal expression, and then on cDNAs of cells from head kidney and spleen of fish injected for 8, 24, and 72 h with inactivated TAD1. The results showed a differential basal expression of transcripts in the examined tissues, and a time-dependent strong up-regulation of IgT, TRß, TRγ genes upon in vivo stimulation with TAD1. These results represent a first in vivo study on the mounting of a specific immune response in an Antarctic teleost species.

The structure of the CD3 ζζ transmembrane dimer in POPC and raft-like lipid bilayer: a molecular dynamics study.

Plasma membrane lipids significantly affect assembly and activity of many signaling networks. The present work is aimed at analyzing, by molecular dynamics simulations, the structure and dynamics of the CD3 ζζ dimer in palmitoyl-oleoyl-phosphatidylcholine bilayer (POPC) and in POPC/cholesterol/sphingomyelin bilayer, which resembles the raft membrane microdomain supposed to be the site of the signal transducing machinery. Both POPC and raft-like environment produce significant alterations in structure and flexibility of the CD3 ζζ with respect to nuclear magnetic resonance (NMR) model: the dimer is more compact, its secondary structure is slightly less ordered, the arrangement of the Asp6 pair, which is important for binding to the Arg residue in the alpha chain of the T cell receptor (TCR), is stabilized by water molecules. Different interactions of charged residues with lipids at the lipid-cytoplasm boundary occur when the two environments are compared. Furthermore, in contrast to what is observed in POPC, in the raft-like environment correlated motions between transmembrane and cytoplasmic regions are observed. Altogether the data suggest that when the TCR complex resides in the raft domains, the CD3 ζζ dimer assumes a specific conformation probably necessary to the correct signal transduction.

Investigation of immunoglobulins in skin of the Antarctic teleost Trematomus bernacchii.

The presence and production of IgM in the skin of the Antarctic teleost Trematomus bernacchii were investigated in this study. Immunoglobulins purified from cutaneous mucus and analysed by SDS-PAGE run under non-reducing and reducing conditions, were composed of heavy and light chains of 78 kDa and 25 kDa respectively, with a relative molecular mass of 830 kDa indicating that mucus IgM are tetramers as the serum IgM. Mature transcripts encoding the constant domains of both the secretory and membrane-bound Igµ chain were seen in T. bernacchii skin using a PCR strategy and the expression of the secretory Igµ chain in the skin was compared with that in other tissues by Real-time PCR. Cytological investigations revealed the presence of either immunoglobulins or their transcripts in occasional lymphocytes distributed close to the basal membrane. IgM once produced here, enters the filament-containing cells and is released into the mucus when these cells degenerate and detach from the epidermis. Our findings indicate that a cutaneous defence mechanism, functioning as anatomical and physiological barrier under subzero conditions, is present in this Antarctic species as an important component of the immune system.

A structural peculiarity of Antarctic fish IgM drives the generation of an engineered mAb by CRISPR/Cas9.

IgM is the major circulating Ig isotype in teleost fish, showing in Antarctic fish unique features such as an extraordinary long hinge region, which plays a crucial role in antibody structure and function. In this work, we describe the replacement of the hinge region of a murine monoclonal antibody (mAb) with the peculiar hinge from Antarctic fish IgM. We use the CRISPR/Cas9 system as a powerful tool for generating the engineered mAb. Then, we assessed its functionality by using an innovative plasmonic substrate based on bimetallic nanoislands (AgAuNIs). The affinity constant of the modified mAb was 2.5-fold higher than that obtained from wild-type mAb against the specific antigen. Here, we show the suitability of the CRISPR/Cas9 method for modifying a precise region in immunoglobulin gene loci. The overall results could open a frontier in further structural modifications of mAbs for biomedical and diagnostic purposes.

Evolutionary analysis of Antarctic teleost Toll-like receptor 2.

In the present study we address the investigation of TLR2 evolutionary selection in two Antarctic teleosts, Trematomus bernacchii (Nototheniidae) and Chionodraco hamatus (Channichthyidae). The nucleotide sequence of TLR2 has been determined in both species, encoding 20 leucine-rich repeats (LRRs) in the extracellular region and a classical Toll/IL-1R (TIR) domain in the intracellular region. High expression level of T. bernacchii TLR2 was found in spleen and skin. Using different methods we identified six codons that underwent Darwinian selection while 20 were found to be negatively selected. Molecular models of C. hamatus and T. bernacchii TLR2 ectodomain as well as of the TIR domain were built by Homology Modeling. Molecular Dynamics simulations were performed in water for 15 ns. The sites under positive selection were residing on the convex side of the solenoid, four out of six were in a 35-residue-long region including the central/N-terminal domain boundary: two in the external loop of LRR11 and the other two in the LRR12 loop. This region has been demonstrated to be the functional site of ligand interaction in human TLR2 structure. Antarctic TLR2 models showed more flexibility than TLR2 from the temperate species Gasterosteus aculeatus. These results suggest that the selective pressure has shaped TLR2 molecule in such a way that increased its activity under the peculiar Antarctic environmental conditions.

Production of a Chimeric Mouse-Fish Monoclonal Antibody by the CRISPR/Cas9 Technology.

The CRISPR/Cas9 system, a defense mechanism naturally occurring in prokaryotes, has been recently repurposed as an RNA-guided DNA targeting platform and widely used as a powerful tool for genome editing. Here we describe how to modify the carboxy-terminal region, called Fragment crystallizable (Fc) region, of a murine monoclonal antibody by replacing the heavy chain constant exons with those from a teleost fish antibody by the CRISPR/Cas9 system. We outline optimal conditions for knockout and knockin mechanisms to edit the Immunoglobulin heavy chain (IgH) constant region gene locus in a murine hybridoma cell line. A chimeric mouse-fish monoclonal antibody can be successfully produced by hybridoma cell lines engineered according to this protocol.

Identification, Characterization, and Expression Analysis of Immunoglobulin Genes from Antarctic Fish by PCR Methods.

We describe different applications of the polymerase chain reaction (PCR) method to amplify up to 2.5-kb DNA regions of the immunoglobulin genes from Antarctic teleost fishes and to evaluate their expression in different tissues. To enhance amplification of poorly expressed transcripts or difficult to amplify, for example, products generated by 5' or 3' RACE, we have set up optimal primer annealing conditions based on a three-step cycle strategy.The protocols reported here along with materials required and general tips are modifications of conventional PCR.

Antarctic teleost immunoglobulins: more extreme, more interesting.

We have investigated the immunoglobulin molecule and the genes encoding it in teleosts living in the Antarctic seas at the constant temperature of -1.86 °C. The majority of Antarctic teleosts belong to the suborder Notothenioidei (Perciformes), which includes only a few non-Antarctic species. Twenty-one Antarctic and two non-Antarctic Notothenioid species were included in our studies. We sequenced immunoglobulin light chains in two species and µ heavy chains, partially or totally, in twenty species. In the case of heavy chain, genomic DNA and the cDNA encoding the secreted and the membrane form were analyzed. From one species, Trematomus bernacchii, a spleen cDNA library was constructed to evaluate the diversity of VH gene segments. T. bernacchii IgM, purified from the serum and bile, was characterized. Homology Modelling and Molecular Dynamics were used to determine the molecular structure of T. bernacchii and Chionodraco hamatus immunoglobulin domains. This paper sums up the previous results and broadens them with the addition of unpublished data.

On Origin and Evolution of the Antibody Molecule.

The vertebrate immune system provides a powerful defense because of the ability to potentially recognize an unlimited number of pathogens. The antibody molecule, also termed immunoglobulin (Ig) is one of the major mediators of the immune response. It is built up from two types of Ig domains: the variable domain, which provides the capability to recognize and bind a potentially infinite range of foreign substances, and the constant domains, which exert the effector functions. In the last 20 years, advances in our understanding of the molecular mechanisms and structural features of antibody in mammals and in a variety of other organisms have uncovered the underlying principles and complexity of this fundamental molecule. One notable evolutionary topic is the origin and evolution of antibody. Many aspects have been clearly stated, but some others remain limited or obscure. By considering a wide range of prokaryotic and eukaryotic organisms through a literature survey about the topic, we have provided an integrated view of the emergence of antibodies in evolution and underlined the very ancient origins.

An evolutionary conserved motif is responsible for immunoglobulin heavy chain packing in the B cell membrane.

All species of vertebrates synthesize immunoglobulin molecules, which differ in an number of aspects but also share a few common features responsible for their function, such as the presence of a transmembrane domain in the membrane bound form of the immunoglobulin heavy chain (IgTMD) that ensures communication with the signal transducing Igα-Igß peptides. We have analyzed the gene sequence encoding the IgTMD of different heavy chain isotypes of very distant species, from shark to mammals. The IgTMD sequences show a high degree of sequence identity and their encoding nucleotide sequences were shown to be subject to purifying selection at most sites. We have built molecular models of seven IgTMDs from different vertebrate species and have investigated the formation of homodimer in a palmitoyl oleoyl phosphatidylcholine (POPC) lipid bilayer by molecular dynamics simulations. We found that the conserved FXXXFXXS/TXXXS motif, never observed to date in protein transmembrane chains, is responsible for the two heavy chains association through two pairs of Phe-Phe hydrophobic interactions and two pairs of Ser/Thr-Ser/Ser hydrogen bonds. This interaction pattern, which stabilizes the dimer conformation in the lipid bilayer, was unique, being different from any other pattern identified in transmembrane helices to date.

Evolution of the Antarctic teleost immunoglobulin heavy chain gene.

Notothenioid teleosts underwent major modifications of their genome to adapt to the cooling of the Antarctic environment. In order to identify specific features of the Antarctic teleost immunoglobulin, transcripts encoding the constant region of the IgM heavy chain from 13 Antarctic and non-Antarctic notothenioid species were sequenced. The primary mRNA splicing for the membrane form was found to be atypical in the majority of Antarctic species, because it led to exclusion of two entire constant exons, and to inclusion of 39-nucleotide exons encoding an unusually long Extracellular Membrane-Proximal Domain (EMPD). Genomic DNA analysis revealed that each 39-nucleotide exon fell within a long sequence that was the reverse complement of an upstream region. Deduced amino acid sequence analysis lead to the identification of cysteine encoding codons in the 39-nucleotide exons, but not in the respective sequence counterpart, suggesting that these residues might play an important role in the folding of the EMPD.

Immunoglobulin light chain isotypes in the teleost Trematomus bernacchii.

Three immunoglobulin light chain (IgL) isotypes TrbeL1, TrbeL2, and TrbeL3 were identified in the Antarctic teleost Trematomus bernacchii by immunoscreening a cDNA expression library, and using RT-PCR, and 5' RACE. One of them was distinguished in two subisotypes TrbeL1A and TrbeL1B. Real-time PCR experiments showed that the different isotypes were expressed in similar ratios in the various tissues analyzed. Interestingly, the expression level of TrbeL1A isotype was very high in all tissues. Molecular models of the CH1-CL domain pairings were built and minimized for the different isotypes. Several differences were identified in the superimposable structures mainly in the loops. In addition, the isotype-specific residues determined a different distribution of the charges on the external CL domain surface. Phylogenetic trees of 43 isotype representative sequences of CL domain from teleost species, built by different methods, indicated that all teleost light chain isotypes are distributed into three groups. Furthermore, the split of the group IgL1 into two subgroups, one of them carrying a micro-satellite DNA insertion, may have occurred in the Acanthopterygean ancestor.

Evidence for hepato-biliary transport of immunoglobulin in the antarctic teleost fish Trematomus bernacchii.

Purified Trematomus bernacchii bile IgM analysed by SDS-PAGE under reducing and non-reducing conditions consisted essentially of tetramers of the basic structure H2L2. The relative molecular mass of the glycosilated H chain was 76 kDa, while that of L chain was 25 kDa. In addition, the presence in the liver of IgM and mu chain-specific mRNA was demonstrated. Immunohistochemistry detected IgH- and IgL-reactivity in perisinusoidal cells, bile canaliculi and pre-ductules. In the anterior intestine, the intraluminal mucus retained a significant Ig-immunoreactivity, while the mucosa housed a limited density of Ig-producing cells. These findings strongly indicate that Ig could be transported across the hepatocytes to be secreted into the bile and protect the intestinal epithelium. In addition, extravasated plasma cells accumulated within liver portal tracts and close to the capsule that, in turn, was evenly coated by Ig molecules at the peritoneal surface.

New insights into evolution of IgT genes coming from Antarctic teleosts.

Cloning and characterization of IgT heavy chain genes were performed in the Antarctic Notothenioid teleost Trematomus bernacchii and in a non-Antarctic Notothenioid species, Bovichtus diacanthus, belonging to a phyletically basal lineage of Notothenioids. Compared to IgT from other non-Antarctic teleost species, including B. diacanthus, T. bernacchii IgT lacked most of the second constant domain but maintained only a few amino acid residues, which could be aligned to B. diacanthus CH2 domain. By analyzing several cDNA clones from a single specimen, three differently sized IgT transcript variants, named Long, Short and Shortest, were identified. Genomic analysis of T. bernacchii and B. diacanthus IgH loci revealed that, in the case of T. bernacchii, within the intron between the exons coding for the entire first and second constant domains a reminiscence of the ancestral second exon was present. The Long and Short variants were found to be encoded by indel alleles, whereas the Shortest variant was generated by alternative splicing that led to the CH2 exonic remnant skipping. Through comparison between genomic and cDNA sequences we hypothesized the presence of three different copies of the IgT heavy chain gene, one of which being considered the functional gene since the corresponding transcripts were identified. Moreover, either Long or Short exonic variants were found to be used in IgT heavy chain membrane form in an unbiased manner, as seen for the secretory form. Phylogenetic analysis was performed on the constant region from all teleost IgT available to date, including IgT from another Antarctic Notothenioid species, Notothenia coriiceps, identified by searching the transcriptome. The loss of almost an entire domain together with the conservation of some amino acids such as proline, glycine and cysteine in the CH2 domain remnant, could be interpreted as another distinctive feature of the Antarctic fish genome evolution, providing also new insights into the structural variation of teleost immunoglobulin genes.

Evolution of the complement system C3 gene in Antarctic teleosts.

Notothenioidei are typical Antarctic teleosts evolved to adapt to the very low temperatures of the Antarctic seas. Aim of the present paper is to investigate sequence and structure of C3, the third component of the complement system of the notothenioid Trematomus bernacchii and Chionodraco hamatus. We determined the complete nucleotide sequence of two C3 isoforms of T. bernacchii and a single C3 isoform of C. hamatus. These sequences were aligned against other homologous teleost sequences to check for the presence of diversifying selection. Evidence for positive selection was observed in the evolutionary lineage of Antarctic teleost C3 sequences, especially in that of C. hamatus, the most recently diverged species. Adaptive selection affected numerous amino acid positions including three residues located in the anaphylatoxin domain. In an attempt to evaluate the link between sequence variants and specific structural features, we constructed molecular models of Antarctic teleost C3s, of their proteolytic fragments C3b and C3a, and of the corresponding molecules of the phylogenetically related temperate species Epinephelus coioides, using human crystallographic structures as templates. Subsequently, we compared dynamic features of these models by molecular dynamics simulations and found that the Antarctic C3s models show higher flexibility, which likely allows for more pronounced movements of both the TED domain in C3b and the carboxyl-terminal region of C3a. As such dynamic features are associated to positively selected sites, it appears that Antarctic teleost C3 molecules positively evolved toward an increased flexibility, to cope with low kinetic energy levels of the Antarctic marine environment.

Characterization of serum immunoglobulin M of the Antarctic teleost Trematomus bernacchii.

Trematomus bernacchii immunoglobulin M concentration was determined in the serum by ELISA; the mean concentration value was 2.7 mg/ml corresponding to 9.6% of the total serum proteins. Purified IgM was analyzed by SDS-polyacrylamide gel electrophoresis, isoelectrofocusing and 2D electrophoresis. The relative molecular mass of the polymeric form was 830 kDa; that of separated H and L chains was, respectively, 78 and 25 kDa. The isoelectric points of the entire molecule ranged from 4.4 to 6.5, that of isolated H chains was between 4.0 and 6.0. Separated H chains were shown to reaggregate in tetrameric form. The cleavage site of trypsin was at the end of the CH1 domain, as confirmed by the N-terminal amino acid sequence of one of the resultant peptides. Immunoblotting was used to detect carbohydrates in the H and L chains labeled with digoxigenin. Glycosyl residues were detected only in the H chain. The carbohydrate content was evaluated to be 12.8% of the entire chain. Purified Igs were hydrolyzed by N-glycosidase F at different conditions and at least four different hydrolytic sites were revealed by limited deglycosylation. T. bernacchii IgM was also compared to those of five other polar fish species.