Dataset Information

Cloning, expression and purification of the ?-carbonic anhydrase from the mantle of the Mediterranean mussel, Mytilus galloprovincialis.

ABSTRACT: We cloned, expressed, purified, and determined the kinetic constants of the recombinant ?-carbonic anhydrase (rec-MgaCA) identified in the mantle tissue of the bivalve Mediterranean mussel, Mytilus galloprovincialis. In metazoans, the ?-CA family is largely represented and plays a pivotal role in the deposition of calcium carbonate biominerals. Our results demonstrated that rec-MgaCA was a monomer with an apparent molecular weight of about 32?kDa. Moreover, the determined kinetic parameters for the CO2 hydration reaction were kcat?=??4.2?×?105?s-1 and kcat/Km of 3.5?×?107?M-1 ×s-1. Curiously, the rec-MgaCA showed a very similar kinetic and acetazolamide inhibition features when compared to those of the native enzyme (MgaCA), which has a molecular weight of 50?kDa. Analysing the SDS-PAGE, the protonography, and the kinetic analysis performed on the native and recombinant enzyme, we hypothesised that probably the native MgaCA is a multidomain protein with a single CA domain at the N-terminus of the protein. This hypothesis is corroborated by the existence in mollusks of multidomain proteins with a hydratase activity. Among these proteins, nacrein is an example of ?-CA multidomain proteins characterised by a single CA domain at the N-terminus part of the entire protein.

SUBMITTER: Perfetto R

PROVIDER: S-EPMC6010101 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Cloning, expression and purification of the α-carbonic anhydrase from the mantle of the Mediterranean mussel, Mytilus galloprovincialis.

Perfetto Rosa R Del Prete Sonia S Vullo Daniela D Carginale Vincenzo V Sansone Giovanni G Barone Carmela M A CMA Rossi Mosè M Alasmary Fatmah A S FAS Osman Sameh M SM AlOthman Zeid Z Supuran Claudiu T CT Capasso Clemente C

Journal of enzyme inhibition and medicinal chemistry 20171201 1

We cloned, expressed, purified, and determined the kinetic constants of the recombinant α-carbonic anhydrase (rec-MgaCA) identified in the mantle tissue of the bivalve Mediterranean mussel, Mytilus galloprovincialis. In metazoans, the α-CA family is largely represented and plays a pivotal role in the deposition of calcium carbonate biominerals. Our results demonstrated that rec-MgaCA was a monomer with an apparent molecular weight of about 32 kDa. Moreover, the determined kinetic parameters for ...[more]

PMID: 28741386

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Project description:BACKGROUND: Sessile bivalves of the genus Mytilus are suspension feeders relatively tolerant to a wide range of environmental changes, used as sentinels in ecotoxicological investigations and marketed worldwide as seafood. Mortality events caused by infective agents and parasites apparently occur less in mussels than in other bivalves but the molecular basis of such evidence is unknown. The arrangement of Mytibase, interactive catalogue of 7,112 transcripts of M. galloprovincialis, offered us the opportunity to look for gene sequences relevant to the host defences, in particular the innate immunity related genes. RESULTS: We have explored and described the Mytibase sequence clusters and singletons having a putative role in recognition, intracellular signalling, and neutralization of potential pathogens in M. galloprovincialis. Automatically assisted searches of protein signatures and manually cured sequence analysis confirmed the molecular diversity of recognition/effector molecules such as the antimicrobial peptides and many carbohydrate binding proteins. Molecular motifs identifying complement C1q, C-type lectins and fibrinogen-like transcripts emerged as the most abundant in the Mytibase collection whereas, conversely, sequence motifs denoting the regulatory cytokine MIF and cytokine-related transcripts represent singular and unexpected findings. Using a cross-search strategy, 1,820 putatively immune-related sequences were selected to design oligonucleotide probes and define a species-specific Immunochip (DNA microarray). The Immunochip performance was tested with hemolymph RNAs from mussels injected with Vibrio splendidus at 3 and 48 hours post-treatment. A total of 143 and 262 differentially expressed genes exemplify the early and late hemocyte response of the Vibrio-challenged mussels, respectively, with AMP trends confirmed by qPCR and clear modulation of interrelated signalling pathways. CONCLUSIONS: The Mytibase collection is rich in gene transcripts modulated in response to antigenic stimuli and represents an interesting window for looking at the mussel immunome (transcriptomes mediating the mussel response to non-self or abnormal antigens). On this basis, we have defined a new microarray platform, a mussel Immunochip, as a flexible tool for the experimental validation of immune-candidate sequences, and tested its performance on Vibrio-activated mussel hemocytes. The microarray platform and related expression data can be regarded as a step forward in the study of the adaptive response of the Mytilus species to an evolving microbial world.

Project description:BackgroundMussels provide many ecosystem services as habitat, food, water filtration and recreational fishing. However, mussels are vulnerable to anthropogenic pressures such as harvesting or trampling, among others. In this frame, it would be paramount to engage society in marine conservation and improving its awareness about environmental policies. The first step lies in properly assessing what is the perception and concerns of society about marine ecosystems. Our study aims to fill this gap by examining public perception of services provided by Mytilus galloprovincialis, its state of conservation and the factors (including anthropogenic activities) shaping mussel beds.MethodsThis study is based on a face-to-face survey consisting of seven open-ended and seven multiple-choice questions of 404 people conducted in 2019 at different shores in the North Portuguese coast. The influence of respondent profile in terms of age, education, gender and place of residence was also assessed.ResultsMost of the participants in our survey (74%) considered that mussels contributed to human well-being and life quality; however, only 31% considered that mussels provide us with many benefits. Regarding the perceived state of mussel services, most of the respondents asserted that mussel services (purification of seawater, habitat, food for other species) worsened in the last 10 years. In contrast, the service as human food was perceived as in an identical state and scientific and traditional knowledge was the only service perceived in a better state. Concerning the state of mussel beds, most of the participants perceived it as good (45%) but a similar percentage (41%) asserted ignoring it. When considering the influence of different factors on mussel beds, only environmental management was considered as having a positive impact by a higher percentage of respondents. The majority of the participants considered that factors included in the questionnaire contributed to worsen mussel beds, ranging between 51% for coastal erosion and 90% for pollution. Education level and age were the main socio-economic factors driving public awareness about the importance of mussel services, its state of conservation and the factors shaping mussel beds.DiscussionResults showed that perception about the importance of mussels for human well-being and the quantity of delivered benefits increased with the education level. Moreover, older people perceived human food as the most important service offered by mussels. Therefore, our results suggest that mussels are mainly known as food resource; however, most of the people ignore their relevant ecological role and the many other benefits that mussels provide. Thus, it is necessary to actively engage society about importance of mussel beds. As M. galloprovincialis is a relevant economic resource, our data could improve the diffusion of knowledge among citizens, stakeholders and scientists, contributing to its sustainability.

Dataset Information

Cloning, expression and purification of the ?-carbonic anhydrase from the mantle of the Mediterranean mussel, Mytilus galloprovincialis.

Publications

Cloning, expression and purification of the α-carbonic anhydrase from the mantle of the Mediterranean mussel, Mytilus galloprovincialis.

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