Molecular Mechanism Of Antifreeze Protein Gene Ld4 Improving Cold Resistance Of Fish

In the coastal waters of Antarctica,marine fishes experience annual environmental temperatures near the freezing point of seawater(-1.86°C)for much of the year with only rare warming periods during the austral summer.Evolution in these subzero waters has resulted in a series of adaptations allowing survival in this frigid,ice-filled environment including the evolution of antifreeze proteins,the loss of heat shock response,the loss of hemoglobin,and metabolic compensation.Currently,five types of antifreeze proteins have been found in fish,namely antifreeze glycoprotein(AGFP),type? antifreeze protein(AFP?),type ? antifreeze protein(AFP ?),type ? antifreeze protein.(AFP?)and type ? antifreeze protein(AFP?).Even though the AFP types are fundamentally different in terms of their primary sequences and three-dimensional structures,they all have equivalent properties allowing them to bind to ice and depress the freezing point of the solutions.Lycodichthys dearborni is a species of the genus Aphididae,which is distributed in the Southern Ocean in the Ross Sea.It is a benthic fish and capable of synthesizing type ? antifreeze protein(AFP ?).The three-dimensional structure of AFPIII is globular and has no amino acid compositional bias.Previously,our laboratory identified a type ? antifreeze protein containing 12 repeat domains from Lycodichthys dearborni and named ld12.In this experiment,ld12 was used as a template to clone the antifreeze genes ld1,ld2,ld3 and ld4 containing one,two,three and four domains,respectively.Traditionally,their antifreeze function is a physical adsorption and inhibition function,without the biochemical function of cell physiology.On the other hand,there is increasing evidence that many antifreeze proteins have the ability to inhibit the growth of ice crystals,and also have the ability to plants to protect plants and animals from the biochemical damage caused by low temperature.The latter function does not depend on the inhibition of ice crystal growth and is a function of the physiological level of cells.Therefore,in this experiment,the anti-freezing protein genes ld1,ld2,ld3,and ld4 were simultaneously studied at the physiological level of cells in vivo and in vitro.In vitro,the ZF4 cell line that stably inherited ld1,ld2,ld3,and ld4 was constructed,and the corresponding antifreeze protein was successfully expressed.After10°C low temperature stress on the transgenic cell line,the cytotoxicity of the cell line transfected with antifreeze protein genes was significantly lower than that of the control,and the viability of the cell was significantly higher than that of the control,indicating that the antifreeze protein gene can enhance the resistance ability of ZF4 cells to cold stress.In vivo,the transgenic zebrafish embryos transiently expressing the antifreeze protein genes were constructed by microinjection,and the transgenic zebrafish embryos were observed and counted after 24 h of low temperature stress at 12°C.It can be seen from the results that compared with the control group,the developmental state of the embryos transgenic with the antifreeze protein genes is relatively good,and the developmental state of the embryo is positively correlated with the number of AFP? functional domains of the antifreeze protein.Both in vivo and in vitro experiments have demonstrated that these antifreeze genes ld1,ld2,ld3 and ld4 have the biological function of protecting cells and organisms against low temperature injury at non-freezing temperatures,and can play a role in both cells and individuals,which affects may be the basic life activity process of the cell.It was also found that the more repeated the AFP? domain,the stronger the cold resistance,which is consistent with the previous conclusion that the antifreeze activity and molecular weight of the antifreeze protein are positively correlated.In order to explore the anti-cold mechanism of antifreeze proteins,we chose ld4,which has the strongest cold resistance,as a research object.The ZF4 cells transfected with ld4 were used as the experimental group,and the ZF4 cells transfected with empty vector were used as the control group.The transcriptome and proteome were sequenced after 2 days of stress at 10°C,respectively.The cold resistance mechanism of anti-freeze protein LD4 was explored by bioinformatics.By transcriptome and protein analysis,there was a significant difference between the experimental group and the control group.By analyzing and comparing the differentially expressed genes and differentially expressed proteins of the two groups,and correlation analysis between the two groups,it was found that the main enriched pathways of the experimental group were Protein processing in endoplasmic reticulum,Cellular senescence,Regulation actin cytoskeleton,Calcium signaling Pathway and Apelin signaling pathway which differ greatly from the signal pathways enriched by the transcriptome,but are similar to the signaling pathways of the proteome.The differentially expressed genes in the transcriptome are mainly involved in Rbosome,Oxidative phosphorylation,Cellular senescence,FoxO signaling pathway,p53signaling pathway and Spliceosome.The differential proteins of the proteome are mainly involved in Protein processiong in endoplasmic reticulum,Regulation actincytoskeleton and Necroptosis.This indicates that under low temperature stress,the cells are finally stressed by Protein processiong in endoplasmic reticulum,Cellular senescence,Regulation actincytoskeleton,Apelin signaling pathway,Calcium signaling pathway,p53signaling pathway and Necroptosis,while Rbosome,Oxidative phosphorylation,Cellular senescence,FoxO signaling pathway and Spliceosome are processes experienced in cellular stress responses.Combined with the analysis results of transcriptome and proteome,we experimentally verified the possible cold resistance mechanism of LD4.One of the breakthroughs in this experiment was to find proteins that interact with LD4 protein.By using co-immunoprecipitation and tandem mass spectrometry,we successfully explored that the interacting protein of LD4 is Bip protein(also known as GRP78).It is the marker protein of the endoplasmic reticulum and is mainly involved in endoplasmic reticulum stress.Combined with omics analysis,we hypothesized that LD4 is involved in the endoplasmic reticulum stress by interacting with Bip protein to reduce cell apoptosis,thereby increasing cell survival,and this endoplasmic reticulum stress may be calcium-dependent.Therefore,by monitoring the intracellular calcium ion and detecting the level of apoptosis,it was found that LD4 protein can reduce the intracellular calcium ion concentration and reduce cell apoptosis.But the specific mechanism needs further exploration.Previous studies in our laboratory found that calmodulin(CaM)is highly expressed in Antarctic fish(Dissostichus mawsoni)(0.23%),and the transfer of the Antarctic fish CaM gene into tobacco could significantly increase cold resistance ability to the low temperature.Also found that only the two Ca²⁺binding domains(CaMm)retaining the N-terminus have the same cold-resistance effect as the intact domain(CaM).CaM,as a sensor of Ca²⁺,participates in the low temperature stress response by regulating the Ca²⁺signaling pathway.In order to explore whether the Antarctic fish CaM gene has the same function on zebrafish cells,and whether co-expression of CaM and ld4 can further improve the tolerance of zebrafish cells to low temperature,ZF4 cells were used to construct transgenic cell lines for investigation.The results showed that the CaM and CaMm genes of Antarctic fish could enhance the ability of ZF4 cells to resist cold stress in short-term stress,but the CaM and CaMm genes of Antarctic fish could not continue to function with the prolongation of stress time.However,ld4 was co-transfected into ZF4 cells with CaM and CaMm,respectively,and the cold tolerance of the cells was improved.With the prolongation of stress time,the cells co-transfected with ld4 and CaMm showed stronger cold tolerance.The above results indicated that the CaM and CaMm genes of Antarctic fish can improve the cold tolerance of ZF4 cells,and ld4 can further enhance the cold tolerance.This may be the result of LD4 and CaM co-regulating Ca²⁺signaling pathway,and further explain that Ca²⁺signaling pathway is an important role in the cold resistance process in LD4.In summary,we first explored and confirmed a new function of LD4 antifreeze protein-cold resistance,and this function is positively correlated with molecular weight,that is,cold resistance LD4>LD3>LD3>LD1.This cold-resistant function of antifreeze proteins differs from the biophysical adsorption-suppressing function,but is a physiological function of cells.This function may be achieved by calcium-dependent endoplasmic reticulum stress,which ultimately reduces the level of apoptosis in cells under low temperature stress..