Functional Exploration Of Two CRP Genes Involved In The Stress Response Of Macrobrachium Nipponense

Cysteine-rich protein（CRP）is a highly conserved small protein that widely exists in animals and has a LIM domain,which is the basic member of the LIM protein family.Studies have found that mammalian CRP has zinc ion transport activity and is involved in physiological processes such as growth and development and immune regulation of organisms,and is closely related to cancer and other diseases,but the molecular mechanism of their action is still unclear.In our laboratory,two related genes（MnMLP/MnCRP3 and MnCRIP）were previously identified from Macrobrachium nipponense.Transcriptome analysis showed that both were up-regulated under bacterial stimulation,suggesting that MnMLP and MnCRIP may be related to the antibacterial immunity and even the anti-stress response of M.nipponense.To explore the functional activity and molecular mechanism of MnMLP and MnCRIP in the immune and stress resistance processes of M.nipponense,this study firstly analyzed the molecular characteristics of the two by bioinformatics method,and then used q RT-PCR method to detect the stimuli of MnMLP and MnCRIP such as viruses,bacteria and heavy metals.The subcellular localization of MnMLP and its transport under stress conditions were analyzed in293T cells.Finally,the Pull-down technology was used to screen the interacting proteins of MnMLP.The results are as follows:1.Gene sequence analysis confirmed that MnCRIP and MnMLP are small proteins with LIM domains,and their molecular weights are 8742 Da and 10064 Da.Exist between MnCRIP and MnMLP amino acid sequences 38.33%similarity（identity）,the similarity between the two and human Hs CRIP and Hs MLP,respectively 68.83%and 53.33%.2.Quantitative analysis of genes found that the expression of MnCRIP and MnMLP in various tissues of M.nipponense were quite different,but both had high levels of expression in the intestine.The intestine of M.nipponense was selected as the research focus in subsequent studies.Stimulation of White spot syndrome virus（WSSV）,Aeromonas veronii and heavy metal ion Cd²⁺can cause up-regulation of both.3.The recombinant protein rMnCRIP and rMnMLP were obtained by prokaryotic expression,and the thermodynamic parameters of the two and Zn²⁺were measured by microcalorimetric isothermal titration calorimeter（ITC）,which confirmed that both rMnCRIP and rMnMLP had zinc ion binding activity.4.After transformation of 293T cells,immunofluorescence assay showed that MnMLP was mainly expressed in the cytoplasm;after cells were treated with lipopolysaccharide（LPS）,Cd²⁺or H₂O₂,part of MnMLP could be observed to transfer to the nucleus,suggesting that under various stress conditions,this protein may enter the nucleus to function.5.The proteins that may interact with MnMLP in the intestinal cells of M.nipponense were detected by pull-down and LC/MS,and a total of 33 sequences were identified;one of the sequences named Mn RNase H1 was selected,and co-immunoprecipitation（Co-IP）technology was used.The results showed that MnMLP and Mn RNase H1 can interact in cells.Subcellular localization showed that Mn RNase H1 was mainly expressed in the nucleus.The comet assay showed that the DNA damage was weakened after the cells transformed with MnMLP were treated with H₂O₂.It is speculated that after cells are stressed,MnMLP translocates into the nucleus and interacts with Mn RNase H1 to participate in DNA damage repair.In conclusion,this study found that MnCRIP and MnMLP of M.nipponense are involved in host immunity,antioxidant and other stress-resistant processes,and MnMLP may play a role in protecting the integrity of genomic DNA by promoting DNA damage repair.This study provides an important reference for exploring the molecular mechanism of CRP in animal physiology and human disease.