An original SERPINA3 gene cluster: Elucidation of genomic organization and gene expression in the Bos taurus 21q24 region

<p>Abstract</p> <p>Background</p> <p>The superfamily of <b><it>ser</it></b>ine <b><it>p</it></b>roteinase <b><it>in</it></b>hibitors (serpins) is involved in numerous fundamental biological processes as inflammation, blood coagulation and apoptosis. Our interest is focused on the SERPINA3 sub-family. The major human plasma protease inhibitor, α1-antichymotrypsin, encoded by the <it>SERPINA3 </it>gene, is homologous to genes organized in clusters in several mammalian species. However, although there is a similar genic organization with a high degree of sequence conservation, the reactive-centre-loop domains, which are responsible for the protease specificity, show significant divergences.</p> <p>Results</p> <p>We provide additional information by analyzing the situation of <it>SERPINA3 </it>in the bovine genome. A cluster of eight genes and one pseudogene sharing a high degree of identity and the same structural organization was characterized. Bovine <it>SERPINA3 </it>genes were localized by radiation hybrid mapping on 21q24 and only spanned over 235 Kilobases. For all these genes, we propose a new nomenclature from <it>SERPINA3-1 </it>to <it>SERPINA3-8</it>. They share approximately 70% of identity with the human <it>SERPINA3 </it>homologue. In the cluster, we described an original sub-group of six members with an unexpected high degree of conservation for the reactive-centre-loop domain, suggesting a similar peptidase inhibitory pattern. Preliminary expression analyses of these bovSERPINA3s showed different tissue-specific patterns and diverse states of glycosylation and phosphorylation. Finally, in the context of phylogenetic analyses, we improved our knowledge on mammalian SERPINAs evolution.</p> <p>Conclusion</p> <p>Our experimental results update data of the bovine genome sequencing, substantially increase the bovSERPINA3 sub-family and enrich the phylogenetic tree of serpins. We provide new opportunities for future investigations to approach the biological functions of this unusual subset of serine proteinase inhibitors.</p

A first genotyping assay of French cattle breeds based on a new allele of the extension gene encoding the melanocortin-1 receptor (Mc1r)

The seven transmembrane domain melanocortin-1 receptor (Mc1r) encoded by the coat color extension gene (E) plays a key role in the signaling pathway of melanin synthesis. Upon the binding of agonist (melanocortin hormone, α-MSH) or antagonist (Agouti protein) ligands, the melanosomal synthesis of eumelanin and/or phaeomelanin pigments is stimulated or inhibited, respectively. Different alleles of the extension gene were cloned from unrelated animals belonging to French cattle breeds and sequenced. The wild type E allele was mainly present in Normande cattle, the dominant ED allele in animals with black color (i.e. Holstein), whereas the recessive e allele was identified in homozygous animals exhibiting a more or less strong red coat color (Blonde d'Aquitaine, Charolaise, Limousine and Salers). A new allele, named E1, was found in either homozygous (E1/E1) or heterozygous (E1/E) individuals in Aubrac and Gasconne breeds. This allele displayed a 4 amino acid duplication (12 nucleotides) located within the third cytoplasmic loop of the receptor, a region known to interact with G proteins. A first genotyping assay of the main French cattle breeds is described based on these four extension alleles

Gènes bovins et qualité des viandes : présentation du programme européen GemQual.

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Gènes du développement musculaire et des qualités de la viande bovine.

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Transcriptomic monitoring of Douglas-fir heartwood formation

Abstract Objectives Molecular cues linked to heartwood formation open new (complementary) perspectives to genetic breeding programs of Douglas-fir, a tree species largely cultivated in Europe for the natural durability and civil engineering properties of its wood. Data description RNAs from a single genotype of Douglas-fir, extracted from three distinct wood zones (outer sapwood, inner sapwood and transition zone) at four vegetative seasons to generate an extensive RNA-seq dataset used to apprehend the in-wood dynamic and seasonality of heartwood formation in this hardwood model species. Previously published data collected on somatic embryos of the same genotype could be merged with the present dataset to upgrade grade the Douglas-fir reference transcriptome

Three bovine alpha-2-fucosyltransferase genes encode enzymes that preferentially transfer fucose on Gal-beta-1-3GalNAc acceptor substrates.

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GASP-1 and GASP-2, two closely structurally related proteins with a functional duality in antitrypsin inhibition specificity: a mechanistic point of view

International audienceWhile GASP-1 and GASP-2 proteins are known to regulate myogenesis by inhibiting myostatin, their structural organization suggests a putative role as multivalent protease inhibitors controlling different protease activities. In this study, we show the noncompetitive and competitive antitrypsin activities of the full-length GASP-1 and GASP-2 proteins, respectively, by using a bacterial system production and in vitro enzymatic experiments. The role of the second Kunitz domain in this functional duality is described by assessing the antitrypsin activity of GASP-1/2 chimeric proteins. Molecular dynamics simulations support the experimental data to rationalize differences in binding modes between trypsin and the GASP-1 and GASP-2 second Kunitz domains. A new inhibition mechanism was evidenced for the second Kunitz domain of GASP-2, in which the conventional cationic residue of trypsin inhibitors was substituted by the strongly interacting glutamine residue

Expression of SERPINA3s in cattle: focus on bovSERPINA3-7 reveals specific involvement in skeletal muscle

International audiencealpha(1)-Antichymotrypsin is encoded by the unique SERPINA3 gene in humans, while it is encoded by a cluster of eight closely related genes in cattle. BovSERPINA3 proteins present a high degree of similarity and significant divergences in the reactive centre loop (RCL) domains which are responsible for the antiprotease activity. In this study, we analysed their expression patterns in a range of cattle tissues. Even if their expression is ubiquitous, we showed that the expression levels of each serpin vary in different tissues of 15-month-old Charolais bulls. Our results led us to focus on bovSERPINA3- 7, one of the two most divergent members of the bovSERPINA3 family. Expression analyses showed that bovSERPINA3-7 protein presents different tissue-specific patterns with diverse degrees of N-glycosylation. Using a specific antibody raised against bovSERPINA3-7, Western blot analysis revealed a specific 96 kDa band in skeletal muscle. BovSERPINA3-7 immunoprecipitation and mass spectrometry revealed that this 96 kDa band corresponds to a complex of bovSERPINA3-7 and creatine kinase M-type. Finally, we reported that the bovSERPINA3-7 protein is present in slow-twitch skeletal myofibres. Precisely, bovSERPINA3-7 specifically colocalized with myomesin at the M-band region of sarcomeres where it could interact with other components such as creatine kinase M-type. This study opens new prospects on the bovSERPINA3-7 function in skeletal muscle and promotes opportunities for further understanding of the physiological role(s) of serpins

Mutagenesis of the bovSERPINA3-3 demonstrates the requirement of aspartate-371 for intermolecular interaction and formation of dimers

Chantier qualité GAThe family of serpins is known to fold into a metastable state that is required for the proteinase inhibition mechanism. One of the consequences of this conformational flexibility is the tendency of some mutated serpins to form polymers, which occur through the insertion of the reactive center loop of one serpin molecule into the A-sheet of another. This “A-sheet polymerization” has remained an attractive explanation for the molecular mechanism of serpinopathies. Polymerization of serpins can also take place in vitro under certain conditions (e.g., pH or temperature). Surprisingly, on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, bovSERPINA3-3 extracted from skeletal muscle or expressed in Escherichia coli was mainly observed as a homodimer. Here, in this report, by site-directed mutagenesis of recombinant bovSERPINA3-3, with substitution D371A, we demonstrate the importance of D371 for the intermolecular linkage observed in denaturing and reducing conditions. This residue influences the electrophoretic and conformational properties of bovSERPINA3-3. By structural modeling of mature bovSERPINA3-3, we propose a new “non-A-sheet swap” model of serpin homodimer in which D371 is involved at the molecular interface