Báo cáo khoa học: Differential expression of duplicated LDH-A genes during temperature acclimation of weatherfish Misgurnus fossilis Functional consequences for the enzyme

Differential Expression of Duplicated LDH-A Genes During Temperature Acclimation of Weatherfish Misgurnus Fossilis: Functional Consequences for the Enzyme

Authors: Maxim Zakhartsev, Magnus Lucassen, Liliya Kulishova, Katrin Deigweiher, Yuliya A. Smirnova, Rina D. Zinov’eva, Nikolay Mugue, Irina Baklushinskaya, Hans O. Pörtner, Nikolay D. Ozernyuk

Field: Physiology / Biochemistry / Molecular Biology

Document Summary: This research investigates how temperature acclimation affects the expression of LDH-A genes in the white skeletal muscle of weatherfish (Misgurnus fossilis). The study identifies two distinct LDH-A mRNA isoforms, a short and a long form, differing in their 3′-UTR and containing nucleotide mismatches that lead to amino acid variations in the resulting protein subunits (LDH-A and LDH-Aβ). These variations, particularly one located in the subunit contact area of the tetramer, are hypothesized to influence the enzyme’s kinetic and thermodynamic properties. The expression levels of these mRNA isoforms are found to be temperature-dependent, with the short form being predominant at higher temperatures and the long form significantly reduced at lower temperatures. This differential expression suggests a mechanism for fine-tuning the properties of skeletal LDH-A to adapt to varying environmental temperatures, potentially linked to gene duplication events in the species’ evolutionary history.

Detailed Table of Contents:

• Keywords
• Correspondence
• Abbreviations
• Temperature acclimation in poikilotherms entails metabolic rearrangements provided by variations in enzyme properties.
• LDH-A fine tuning
• Table 1. Differences identified between ‘cold’ (AT = 5 °C) and ‘warm’ (AT = 18 °C) LDH-A purified from white skeletal muscle of weatherfish Misgurnus fossilis acclimated to different temperatures (ATs) for 20-25 days.
• Fig. 1. (A) Northern hybridization of LDH-A mRNA from weatherfish Misgurnus fossilis indicates presence of two forms of LDH-A mRNA as (B) two strong signals (~1.4 kb and ~1.6 kb) at 18°C acclimation (AT=18°C), whereas (C) at 5°C acclimation (AT=5°C) the signals are weaker and moreover ~1.6 kb mRNA is almost missing.
• Fig. 2. Structure of the short (mRNAldh-a = 1332 bp) and long (mRNAdh-a = 1550 bp) forms of LDH-A mRNAs from skeletal muscle of weatherfish Misgurnus fossilis. Nucleotide mismatches are indicated outwards, whereas amino acid mismatches are indicated inwards.
• Fig. 3. Predicted quaternary structure of skeletal muscle LDH-A 24-homotetramer (LDH-Ax4) from weatherfish Misgurnus fossilis and close up view on the contact area between two neighbouring subunits. Each subunit is coloured and the corresponding mismatched amino acids are indicated.
• Table 2. Primers used in the research of mRNA of LDH-A from weatherfish Misgurnus fossilis.
• Fig. 4. Expected probabilities of iso-tetramers in overall LDH-A pool (LDH-A4, LDH-Ax3B, LDH-Ax2B2, LDH-AxB3 and LDH-AB4) in skeletal muscle of weatherfish M.fossilis under AT = 5°C and AT = 18°C due to fractional mixing of a- and ẞ-subunits correspondingly translated from mRNAidh-a and mRNAidh-a isoforms (LDH-A mRNA ratios shown in the embedded histogram), assuming similar translational activity of both mRNA isoforms and a random assembly of the tetrameric enzyme.
• Table 3. Content of a/ẞ-isoforms o mRNAldh-a in total RNA samples (1 ng total RNA per µL) from weatherfish Misgurnus fossilis acclimated to 5 °C or 18 °C for 20 days.
• Experimental procedures
• Animals and acclimation
• Karyotyping and chromosome preparation technique
• Extraction of total RNA
• Northern hybridization of mRNA
• Determination of the LDH-A mRNA sequences
• Quantification of LDH-A transcripts
• Molecular analysis and modelling
• Probabilities of tetramers
• Acknowledgements
• References