Alteration Of Protein Glycosylation In Zebrafish Fin Regeneration And The Effect Of O-GlcNAcylation On Regeneration

Background:Zebrafish is a sort of small vertebrate which is commonly used as a model animal for scientific research because of its advantages of strong reproductive ability and rapid developmental capacity,as well as the facility of operating its transparent embryos.Besides,the homology of more than 80%of its total genes is similar comparing with that of human's.The fin of zebrafish has strong regenerative capacity.Studing the process and mechanism of fin regeneration might bring us some enlightenment to the development of regenerative medicine.Post-translational modification of protein glycosylation plays an important role in cell adhesion,recognition and interaction,etc.It is closely related to animal embryo development and various disease progression.The role of glycosylation modification in the regeneration of zebrafish fin is currently unclear Therefore,this study intends to study the glycosylation pattern in the process of zebrafish fin regeneration and to explore its mechanism,which may provides useful reference to further clarify the limb regeneration mechanismMethods:The study is divided into five parts 1.Using lectin microarray to detect the expression profiles of protein glycosylation in regenerating zebrafish fin,analyze and compare the differences.Furthermore,using lectin histochemistry and lectin blot to verify results2.Using MALDI-TOF-MS technology to identify the N-and O-glycans of regenerating fin tissue protein,obtaining fingerprints of N-and O-glycans3.Using chemical enzyme labeling method to enrich O-GlcNAc glycoprotein in regenerating zebrafish fin and identifications of these glycoproteins by LC-MS/MS4.Analyzing the differences of transcription levels of ogt and oga genes by RT-qPCR.Verifying the spatio-temporal expression of ogt and oga by in situ hybridization.Analyzing the expression and localization of OGT and O-GlcNAcylation in fin tissue by WB and IF staining.5.Using microinjection method to inject OGT/OGA inhibitors and the O-GlcNAcylation substrate Glucose into the zebrafish fin.Observing the phenotype of zebrafish fin with O-GlcNAcylation level variationsResults:1.The glycosylation alteration in the process of zebrafish fin regeneration Ctrl,0.5dpa,ldpa,2dpa,4dpa and 6dpa tissue proteins were extracted and screened using lectin microarray.There are 8 glycans which are specifically recognized by lectins,(WGA,STL,DSA,ConA,BS-I,UEA-I,EEL,PHA-E+L)were found to be up-regulated while other 8 glycans(MAL-II,SNA,NPA,SB A,MPL,RCA120,PTL-II,LTL)were considered to be down-regulated in the process of fin regeneration.GlcNAc was significantly up-regulated at all the time points in the regeneration process.Lectin histochemistry was used to further validate the lectin binding profiles and assessed the distribution of glycosidic residues.The results showed that some glycans were concentrated in epidermal cells while others were concentrated in osteoblasts.The expression trend of WGA,DSA,STL,EEL was basically consistent with the results of lectin microarray.The expression level of WGA-recognized O-GlcNAc and MAL-II-recognized a-2,3 sialic showed similar expression trend compared with lectin blotting results2.Identification of the N-and O-glycans in the process of zebrafish fin regeneration by using MALDI-TOF-MS:13,17,22,11,18,20 N-glycans were identified by MALDI-TOF-MS in Ctrl,0.5dpa,ldpa,2dpa,4dpa and 6dpa groups,respectively.A total of five N-glycans were identified in all of the six groups(eg m/z 1345.401,m/z 1972.937,m/z 2458.539).Only one glycan was identified in 0.5dpa,ldpa,2dpa,4dpa and 6dpa groups(m/z 1211.868).3,8,3,5,7 N-glycans were particularly indentified in Ctrl,0.5dpa,2dpa,4dpa and 6dpa groups.29,37,32,31,51 and 51 O-glycans were identified in the Ctrl,0.5dpa,ldpa,2dpa,4dpa and 6dpa groups.A total of 8 O-glycans were identified in 6 groups of samples(eg m/z 584.442,m/z 643.508,m/z 868.700)and only one O-glycan was identified in 0.5dpa,ldpa,2dpa,4dpa and 6dpa groups(m/z 651.473).2,9,9,16,and 11 O-glycans were particularly indentified in Ctrl,0.5dpa,1dpa,4dpa and 6dpa groups3.Isolation and identification of O-GlcNAc glycoprotein by chemical labeling method and LC-MS/MS:1595,1525,1445,1892,1678,1778 O-GlcNAc glycoproteins were identified in the Ctrl,0.5dpa,ldpa,2dpa,4dpa,6dpa groups with GO annotation.784 proteins were coexisted in 6 groups.Moreover,each group had its own specific identification glycoproteins.Through the WEGO function annotation,we found that the some of the identified proteins were similar in cell components,molecular functions,biological processes while others had different biological functions,such as antioxidant activity f,rhythm process,etc.The number of genes involved in the fin regeneration is different.Through Funrich GO enrichment of O-GlcNAc glycoprotein,we found that the fin-regeneration-involved genes were highly up-regulated.More O-GlcNAc glycoproteins were involved in the regulation of the fin regeneration.They are related in stem cell differentiation,TGF-? receptor signaling pathway,tissue remodeling and tissue regeneration process.The genes involved in these biology process were highly correlated with the process of fin regeneration.The proteins involved in these biological processes were screened out and analyzed by KEGG pathway.We found that 13 proteins were involved in the splice pathway and 26 proteins had strong interaction among each other.4.Analysis of gene and protein expression levels of O-GlcNAcylation-related carbohydrase in the process of fin regeneration The transcription level of ogt and oga during fin regeneration showed similar expression profiles.Both of the genes were up-regulated in ldpa,2dpa,4dpa,6dpa groups compared with that of in Ctrl group.At ldpa and 2dpa,the expression of ogt and oga was significantly up-regulated and reached the peak level at 2dpa.The results of in situ hybridization were consistent with that of qRT-PCR.WB showed that the expression level of OGT was up-regulated during the whole regenerationprocess.At 4dpa,the expression level of OGT reached the peak level.The change of O-GlcNAcylation level during fin regeneration was similar to that of OGT.The expression of OGT in the fin tissue showed that it was gradually increased during the regeneration process and was concentrated at the top of the blastema tissue at ldpa and 2dpa.The expression level of O-GlcNAcylation is similarly to the expression level of OGT,O-GlcNAcylation-modified proteins are mainly distributed in the cytoplasm,cell membrane of epidermal cells and fibroblasts5.Effect of O-GlcNAcylation on the regeneration process of zebrafish fin As the activity of OGT was affected by either of the inhibitor Alloxan or OSMI-1,the level of O-GlcNAcylation was significantly decreased,the fin regeneration was delayed and the process of regeneration was inhibited due to the injection treatment.By injection Thiamet-G and O-GlcNAcylation substrate,glucose,the level of O-GlcNAcylation was increased and the process of regeneration was promoted thus accelerated fin regenerationConlusion:Our results showed that the glycosylation modification was changed during the process of zebrafish fin regeneration.O-GlcNAc glycoprotein played an important role in the regulation of regeneration.The expression of O-GlcNAcylation was significantly up-regulated in the fin regeneration process.OGT promoted fin regeneration by up-regulating the O-GlcNAcylation level during the regeneration of zebrafish fin.