The Mechanisms Of Specific Homophilic Binding Of SDscam Isoforms In Mesobuthus Martensii

In the assembly of neural circuits,the dendrites or axons of the same neuron repel each other to produce the phenomenon of “self-avoidance”,and the neurites of different neurons can be identified as “non-self” with each other and then crossed together.This process requires cells to express a large number of recognition molecules,which endow neurons with unique identities,so that neurites can accurately distinguish self/non-self components.Down syndrome cell adhesion molecule(Dscam1)and clustered protocadherin(Pcdh)play an important role on neuron selfrecognition of Drosophila and vertebrate respectively.It is known that Dscam1 encodes 38,016 proteins through alternative splicing.And in vertebrates,clustered Pcdh generate ?60 clustered protocadherins(Pcdh)through promoter choice.Studies show that Dscam1 and clustered Pcdh generate extensive isoform diversity by strict homophilic specificity.However,it is not clear whether there is a molecule with similar homologous diversity in other species.Recently,our team discovered a “hybrid” gene family in the subphylum Chelicerata that is particularly relevant to the remarkable functional convergence of Drosophila Dscam1 and vertebrate clustered Pcdh.This molecule is shorter than Dscam1,we therefore refer to this type of Dscam as shortened Dscam(sDscam)to distinguish it from classic Dscam.The member of this gene family has a high sequence similarity with fly Dscam1 gene,and share obvious organizational resemblance with the 5' variable repeated tandem sequence of vertebrate clustered Pcdh.Studies have shown that the sDscam gene encode ?50 to 100 isoforms each with alternative promoters for the number of isoforms varying across Chelicerata species(Based on their different variable 5'cassettes,these sDscams can be subdivided into sDscam? and sDscam? subfamilies,respectively).However,whether these isoforms have a specific homophilic binding mechanism similar to Dscam1 and clustered Pcdh is currently unclear.Therefore,this paper mainly explores the interaction mechanism of Mesobuthus martensii sDscam.The research content is mainly divided into four parts.First part,to explore the trans(cellcell)interaction and related domain of M.martensii sDscam.Firstly,sDscam was heterologously expressed in Sf9 cells,its homophilic binding ability were explored through quantitative analysis of cell aggregation.Then,domain deletion and shuffling experiments are used to explore the relevant domains that affect the trans-interaction of sDscam;Second part,to analyze the specificity of trans interactions between different sDscams.Firstly,cell aggregates were assessed via mixing of two fluorescently labeled cell population to explore binding specificity.Then,domain shuffling to identify the specificity-determining domain.Finally,To gain insight into how the domain mediates specific homophilic binding and which residues affect specific homophilic binding,homology modeling was carried out;Third part,analysis of the cis(within the same cell)interaction of the M.martensii sDscam.Through co-immunoprecipitation(Co-IP)and Western-blot experiments on the overexpressed sDscam isoforms in vitro,to explore whether sDscam exhibit cis-multimers,and further analyze the key domain mediating multimers;Fourth part,coexpression of multiple sDscam isoforms to simulate the combined expression and explore its homophilic specificity diversity in vitro,and propose a molecular model of sDscam-mediated cell recognition.Based on the above content,the following research results are obainted:(1)Among the 86 tested sDscam isoforms,all of the 34 sDscam?s formed homophilic aggregates.In contrast,only 15 of the 52 sDscam? isoforms tested formed homophilic aggregates;2 and 8 of these belonged to the ?6 and ?5 cluster,respectively.The size of the cell aggregates varied markedly across the sDscam subfamilies.Generally,aggregates of the cells expressing sDscam? isoforms were largely smaller than those expressing sDscam?s.There is no correlation between isoform membrane location and expression level and cell aggregation outcome among individual isoforms;(2)N-terminal deletion experiments show that when the variable Ig1 domain is deleted,sDscam cannot mediate cell adhesion.Similarly,the C-terminal series deletion and transmembrane(TM)domain replacement experiments show that the constitutive fibronectin(FNIII)domain and TM domain also have an effect on cell aggregation;(3)Binding specificity assay shown that except for the sDscam?20&36 isoform pair,other sDscam? isoforms exhibited exclusively homophilic interaction specificity with no observed heterophilic interactions.And sDscam? and sDscam? also have strict homophilic specific binding;(4)Domain shuffling experiments show that the variable Ig1 domain determines the binding specificity of sDscam? and sDscam?,and the variable Ig2 domain of sDscam? has nothing to do with its specificity;(5)Protein modeling and point mutation analysis shown that sDscams interact in trans via antiparallel Ig1 self-binding,and its interaction interface was mainly concentrated on the ABED strand;(6)The sDscam? pairs with high sequence similarity were analyzed for structural modeling,point mutation and cell binding specificity assay,and determined that the amino acid residues that affect the shape complementarity and charge complementarity of Ig1 homodimers are related to the specificity of sDscam;(7)Co-IP and Western-blot experiments show that sDscam can form cis-multimers without trans-interaction,and the formation of multimers is related to the constitutive FNIII domain;(8)Coexpression of multiple sDscam isoforms to simulate cell recognition shows that,the mismatch of a single sDscam isoform with binding ability can lead to cell-specific recognition,and the trans and cis interaction of sDscam is the molecular basis of cell recognition.In summary,this article propose a molecular model of sDscam-mediated cell recognition in M.martensii.That is,sDscam form a “zipper-like” connection through trans and cis interaction in the surface of cell membrane.This structure greatly expands the diversity of sDscam isoforms,enough to provide a specific identification tag for a single cell,and then provide a structural foundation for the self-recognition of M.martensii neurons.