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1.
Mol Immunol ; 59(2): 194-9, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24667069

RESUMO

Single-domain antibodies (sdAb) from camelids and sharks represent the smallest immunoglobulin-based functional binding domains, and are known for their thermal stability and ability to refold after denaturation. Whereas target-binding sdAb have been derived from both immunized and naïve sharks and camelids, the stability of camelid-derived sdAb have been evaluated much more extensively. To address this disparity we characterized 20 sdAb derived from spiny dogfish shark and smooth dogfish shark in terms of their protein production, melting temperature and ability to refold after heat denaturation. Using the same expression system and protocol as we follow to produce camelid sdAb, production of the shark sdAb was quite poor, often resulting in less than a tenth of the typical yield for camelid sdAb. We measured the melting temperature of each of the sdAb. Similar to camelid sdAb, the shark-derived sdAb, showed a range of melting temperature values from 42°C to 77°C. Also similar to what has been observed in camelids, the sdAb from both shark species showed a range of ability to refold after heat denaturation. This work demonstrated that although shark sdAb can possess high melting temperatures and refolding ability, no clear advantage over sdAb derived from camelids in terms of thermostability and renaturation was obtained.


Assuntos
Dobramento de Proteína , Anticorpos de Domínio Único/química , Squalus acanthias/imunologia , Temperatura de Transição , Sequência de Aminoácidos , Animais , Camelídeos Americanos/imunologia , Temperatura Alta , Dados de Sequência Molecular , Biblioteca de Peptídeos , Estrutura Terciária de Proteína
2.
Fish Shellfish Immunol ; 34(6): 1404-15, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23454429

RESUMO

CD79α (also known as Igα) is a component of the B cell antigen receptor complex and plays an important role in B cell signalling. The CD79α protein is present on the surface of B cells throughout their life cycle, and is absent on all other healthy cells, making it a highly reliable marker for B cells in mammals. In this study the spiny dogfish (Squalus acanthias) CD79α (SaCD79α) is described and its expression studied under constitutive and stimulated conditions. The spiny dogfish CD79α cDNA contains an open reading frame of 618 bp, encoding a protein of 205 amino acids. Comparison of the SaCD79α gene with that of other species shows that the gross structure (number of exons, exon/intron boundaries, etc.) is highly conserved across phylogeny. Additionally, analysis of the 5' flanking region shows SaCD79α lacks a TATA box and possesses binding sites for multiple transcription factors implicated in its B cell-specific gene transcription in other species. Spiny dogfish CD79α is most highly expressed in immune tissues, such as spleen, epigonal and Leydig organ, and its transcript level significantly correlates with those of spiny dogfish immunoglobulin heavy chains. Additionally, CD79α transcription is up-regulated, to a small but significant degree, in peripheral blood cells following stimulation with pokeweed mitogen. These results strongly indicate that, as in mammals, spiny dogfish CD79α is expressed by shark B cells where it associates with surface-bound immunoglobulin to form a fully functional BCR, and thus may serve as a pan-B cell marker in future shark immunological studies.


Assuntos
Adjuvantes Imunológicos/metabolismo , Antígenos CD79/genética , Proteínas de Peixes/genética , Regulação da Expressão Gênica , Squalus acanthias/genética , Squalus acanthias/imunologia , Região 5'-Flanqueadora , Sequência de Aminoácidos , Animais , Sequência de Bases , Antígenos CD79/química , Antígenos CD79/metabolismo , Clonagem Molecular , Proteínas de Peixes/química , Proteínas de Peixes/metabolismo , Dados de Sequência Molecular , Especificidade de Órgãos , Filogenia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Alinhamento de Sequência , Squalus acanthias/metabolismo
3.
Fish Shellfish Immunol ; 34(5): 1158-69, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23439398

RESUMO

Cartilaginous fishes are the oldest group in which an adaptive immune system based on immunoglobulin-superfamily members is found. This manuscript compares humoral immune function in small-spotted catshark (Scyliorhinus canicula) with that described for spiny dogfish (Squalus acanthias), another member of the Squalomorphi superorder, and nurse shark, the model for humoral immunity in elasmobranchs and a member of the Galeomorphi superorder. Although small-spotted catshark and nurse shark are separated by over 200 million years we found that immunoglobulin isoforms are well conserved between the two species. However, the plasma protein profile of small-spotted catshark was most similar to that of spiny dogfish, with low levels of pentameric IgM, and IgNAR present as a multimer in plasma rather than a monomer. We show that an antigen-specific monomeric IgM response, with a profile similar to that described previously for nurse sharks, can be raised in small-spotted catshark. Lacking polyclonal or monoclonal antibody reagents for detecting catshark IgNAR we investigated phage-display and recombinant Fc-fusion protein expression as alternative methods to look for an antigen-specific response for this isotype. However, we could find no evidence of an antigen-specific IgNAR in the animals tested using either of these techniques. Thus, unlike nurse sharks where antigen-specific monomeric IgM and IgNAR appear together, it seems there may be a temporal or complete 'uncoupling' of these isotypes during a humoral response in the small-spotted catshark.


Assuntos
Imunidade Humoral , Imunoglobulinas/genética , Tubarões/genética , Tubarões/imunologia , Sequência de Aminoácidos , Animais , Southern Blotting , DNA Complementar/genética , DNA Complementar/metabolismo , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Isotipos de Imunoglobulinas/sangue , Isotipos de Imunoglobulinas/genética , Imunoglobulinas/sangue , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Escócia , Alinhamento de Sequência , Análise de Sequência de DNA , Tubarões/metabolismo , Especificidade da Espécie , Squalus acanthias/genética , Squalus acanthias/imunologia , Squalus acanthias/metabolismo
4.
Dev Comp Immunol ; 36(4): 665-79, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22040740

RESUMO

The cartilaginous fish (chimeras, sharks, skates and rays) are the oldest group relative to mammals in which an adaptive immune system founded upon immunoglobulins has been found. In this manuscript we characterize the immunoglobulins of the spiny dogfish (Squalus acanthias) at both the molecular and expressed protein levels. Despite the presence of hundreds of IgM clusters in this species the serum levels of this isotype are comparatively low. However, analysis of cDNA sequences and serum protein suggests microheterogeneity in the IgM heavy chains and supports the proposal that different clusters are preferentially used in the two forms (monomer or pentamer) of this isotype. We also found that the IgNAR isotype in this species exists in a previously unknown multimeric format in serum. Finally, we identified a new form of the IgW isotype (the shark IgD orthologue), in which the leader is spliced directly to the first constant domain, resulting in a molecule lacking an antigen-binding domain.


Assuntos
Imunoglobulinas/química , Imunoglobulinas/imunologia , Squalus acanthias/imunologia , Sequência de Aminoácidos , Animais , Imunoglobulinas/genética , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência , Tubarões/genética , Tubarões/imunologia , Squalus acanthias/genética
5.
Dev Comp Immunol ; 36(4): 707-17, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22155638

RESUMO

B-cell activating factor (BAFF), also known as tumour necrosis factor (TNF) ligand superfamily member 13B, is an important immune regulator with critical roles in B-cell survival, proliferation, differentiation and immunoglobulin secretion. A BAFF gene has been cloned from spiny dogfish (Squalus acanthias) and its expression studied. The dogfish BAFF encodes for an anchored type-II transmembrane protein of 288 aa with a putative furin protease cleavage site and TNF family signature as seen in BAFFs from other species. The identity of dogfish BAFF has also been confirmed by conserved cysteine residues, and phylogenetic tree analysis. The dogfish BAFF gene has an extra exon not seen in teleost fish, birds and mammals that encodes for 29 aa and may impact on receptor binding. The dogfish BAFF is highly expressed in immune tissues, such as spleen, and is up-regulated by PWM in peripheral blood leucocytes, suggesting a potentially important role in the immune system.


Assuntos
Fator Ativador de Células B/imunologia , Squalus acanthias/imunologia , Sequência de Aminoácidos , Animais , Fator Ativador de Células B/química , Fator Ativador de Células B/genética , Fator Ativador de Células B/metabolismo , Sequência de Bases , Células Sanguíneas/imunologia , Éxons , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Proteínas de Peixes/metabolismo , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência , Squalus acanthias/metabolismo
6.
Brain Behav Evol ; 72(1): 48-58, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18635929

RESUMO

Myelin, the insulating sheath made by extensive plasma membrane wrapping, is dependent on the presence of highly adhesive molecules that keep the two sides of the membrane in tight contact. The Po glycoprotein (Po) is the major component of the peripheral nervous system (PNS) myelin of mammals. The exact role that Po protein has played in the evolution of myelin is still unclear, but several phylogenetic observations suggest that it is a crucial component in the development of myelin as a multi-lamellar membrane structure. Sharks, which appeared in the fossil record about 400 million years ago, are the first fully myelinated organisms. In this study we investigated the expression pattern of shark myelin Po to suggest a way it might have played a role in the evolution of myelin in the central nervous system. We found that sharks have more than two isoforms (32, 28 and 25 kD), and that some of these might not be fully functional because they lack the domains known for Po homophilic adhesion.


Assuntos
Proteína P0 da Mielina/química , Tubarões/metabolismo , Squalus acanthias/metabolismo , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos , Western Blotting , Sistema Nervoso Central/química , Sistema Nervoso Central/embriologia , Epitopos/imunologia , Evolução Molecular , Glicosilação , Humanos , Dados de Sequência Molecular , Peso Molecular , Proteína P0 da Mielina/análise , Proteína P0 da Mielina/imunologia , Proteína P0 da Mielina/isolamento & purificação , Sistema Nervoso Periférico/química , Sistema Nervoso Periférico/embriologia , Isoformas de Proteínas/análise , Isoformas de Proteínas/imunologia , Isoformas de Proteínas/isolamento & purificação , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Coelhos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Tubarões/embriologia , Tubarões/imunologia , Especificidade da Espécie , Squalus acanthias/imunologia , Vertebrados/metabolismo
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