Effect Of Dscam Transmembrane Region Variation On Immune Function Output In Chinese Mitten Crab

Alternative splicing is a widespread phenomenon in eukaryotes and plays an important role in enhancing protein functional diversity and regulating gene expression and various physiological functions.Down syndrome cell adhesion molecule（Dscam）,initially known for its important role in connecting neurons,was later found in numerous studies to be involved in the arthropod immune response as a pathogenspecific recognition molecule with important regulatory roles in antimicrobial peptide expression,cell phagocytosis and hemocyte proliferation.Dscam can produce tens of thousands to hundreds of thousands of alternatively spliced isoforms,and this phenomenon only occurs in arthropods,and is a representative gene of alternative splicing.Dscam protein has two forms: transmembrane Dscam（m Dscam）and soluble Dscam（s Dscam）.The transmembrane（TM）domain of m Dscam can undergo alternative splicing to produce two transmembrane variants,namely Dscam[TM1] and Dscam[TM2].At present,the immunological difference between these two transmembrane variants is still unclear.Therefore,we took Chinese mitten crab（Eriocheir sinensis）as the research object,focused on the transmembrane domain of m Dscam,and investigated the immune functional differentiation of these two transmembrane variants.In this study,we first obtained and identified Dscam[TM1] and Dscam[TM2] from the genome of Eriocheir sinensis,named Es Dscam[TM1] and Es Dscam[TM2],and then analyzed the hydrophobicity and transmembrane mode of the transmembrane domains of the two transmembrane variants.The multiple sequence alignment indicated that the transmembrane domains of the two transmembrane variants of Dscam are highly conserved.Phylogenetic analysis showed that Dscam[TM1] and Dscam[TM2]are evolutionarily two independent branches.However,the Daphnia m Dscam with only one TM can be divided into one branch with invertebrate Dscam[TM1],which implies that there may be asymmetric evolution of Dscam[TM1] and Dscam[TM2].Tissue expression showed that Es Dscam[TM1] and Es Dscam[TM2] were highly expressed in brain and immune tissues.Following infection of hemocytes by Gram-positive and Gram-negative bacteria,Es Dscam[TM1] and Es Dscam[TM2] actively responded to pathogen invasion,and both exhibited varying degrees of upregulation at the nucleic acid level,suggesting that both transmembrane variants of Dscam were involved in the antimicrobial immune response.To investigate whether these two transmembrane variants are specifically expressed for different species of bacteria,we stimulated Eriocheir sinensis hemocytes with Vibrio parahaemolyticus（Gram-negative bacteria）and Staphylococcus aureus（Gram-positive bacteria）,respectively.The m RNA expression of Dscam[TM1] was found to be extremely significantly higher than that of Dscam[TM2] after stimulation by both bacteria by second-generation sequencing techniques targeting hemocytes,suggesting that the two transmembrane variants mentioned above may have differential functions in the antimicrobial immune response.To further clarify their immune function,we knocked down the expression of transmembrane Dscam containing TM1 or TM2 in Eriocheir sinensis hemocytes using RNA interference technology.The results indicated that the regulation of antimicrobial peptide expression by knockdown of the above genes was different,and Dscam[TM2] played a more extensive and important role than Dscam[TM1] in this process.The functional difference of the transmembrane region may also be related to the difference of the alternatively spliced exon carried by the intracellular segment,and the proportion of alternatively splice exon33 carried by Es Dscam[TM1] was found to be significantly higher than that of Es Dscam[TM2] by real-time quantitative PCR.Given the presence of endocytic motifs in the variable exon 33 of the intracellular segment of transmembrane Dscam,we analyzed the phagocytic regulatory ability of transmembrane Dscam containing TM1 or TM2 using immunofluorescence and flow cytometry,and found that Es Dscam[TM1]has a stronger regulation of hemocytes phagocytosis.The above results suggest that the two functional domains of the transmembrane region of the transmembrane Dscam in Eriocheir sinensis have differential functions in the antimicrobial immune response of the crab’s hemocytes and may be related to the alternatively splice exon of the intracellular region they carry.To a certain extent,this study fills the lack in the study of the immune function of the transmembrane functional domain of transmembrane Dscam,and provides an important basis for a comprehensive study on the molecular mechanism of immune regulation of transmembrane Dscam.