The Role Of The O-GalNAc Transferase PpGalNAc-T13 In Neuronal Differentiation

Purpose:O-GalNAc glycosylation has been reportedly involved in cell proliferation and apoptosis,cell migration and invasion,organ and individual development,as well as the progression of multiple diseases;however,there is still little information on its roles in neural development.pp GalNAc-T13 is a neural-specific member of polypeptide N-acetylgalactosaminyltransferases(pp GalNAc-Ts),which could initiate the O-GalNAc glycosylation by transferring GalNAc to the hydroxyl group of serine or threonine.In the first part of this study,we sought to explore the potential roles of pp GalNAc-T13 in neural development using the in vitro and in vivo models of neuronal differentiation.In the second part,we systematically analyzed the effects of pp GalNAc-T13 depletion on the expression of the different secreted proteins during neuronal differentiation using the comparative glycoproteomics technology.In the third part,we constructed the Galnt13 conditional knockout mice and investigated the associated phenotypes.Experimental design:P1)The distribution and expression pattern of pp GalNAc-T13 were examined by western blotting and immunofluorescence.The influences of pp GalNAc-T13knockout on the expression of its potential substrates were investigated.HPLC and MALDI-TOF-MS analysis were performed to elucidate the molecular mechanisms of pp GalNAc-T13 in neuronal differentiation.P2)The wildtype and pp GalNAc-T13knockout cells were labeled with azido sugar analog Ac₄Gal NAz during neuronal differentiation.The culture medium was collected,and the glycoproteins were enriched by click chemistry reaction.The obtained glycoproteins were then on-beads digested and subjected to Orbitrap LC-MS analysis.Bioinformatic analysis was performed to prediect the biological functions and to screen the specific substrates of pp GalNAc-T13 during neuronal differentiation.3)“Cre-Lox P”recombinant enzyme System was employed to construct the Galnt13 conditional knockout mice.The knockout efficiency was confirmed by genotyping and western blotting.The numbers of offsprings and their weights were recorded and analyzed.Results:P1)ppGalNAc-T13 was dramatically up-regulated during the neuronal differentiation and specifically distributed in neurons and neural stem cells.Knockout of pp GalNAc-T13 inhibited the aggregation and neuronal differentiation,and decreased the expression level of its potential substrate PDPN(podoplanin).These effects could not be restored by the overexpression of pp GalNAc-T1 in pp GalNAc-T13 knockout cells,indicating the distinct roles of pp GalNAc-T13 in neuronal differentiation.Further evidence demonstrated that pp GalNAc-T13-mediated O-glycosylation on PDPN contributes to its stability and secretion,and promotes the neuronal differentiation.P2)Based on the comparative glycoproteomic results,we totally identified 1590 proteins.Bioinformatic analysis indicated that?90%of them were glycoproteins and over 30%of them(511)were secreted or transmembrane proteins.Importantly,260 proteins were not included in the published O-GalNAc protein database.GO(Gene Ontology)analysis for the differentially expressed secreted and transmembrane proteins upon the pp GalNAc-T13 knockout suggested that pp GalNAc-T13 was implicated in the neurogenesis and axonogenesis,which was highly likely through the semaphoring,Notch,Wnt and integrin signaling pathways.P3)The genotyping and western blotting results indicated the Galnt13 general knockout and neural-specific knockout mice were successfully constructed.The neural-specific Galnt13 knockout mice were fertile,and the genotypic ratios were consistent with Mendel's law of inheritance.Interestingly,the growth retardation was observed in Galnt13 neural-specific knockout mice,which was more significant among the female ones.The phenotypes of Galnt13 general knockout mice need to be further explored.Conclusion:In conclusion,we reported for the first time that the O-GalNAc glycosyltransferase pp GalNAc-T13 plays important role in neuronal differentiation in mammalians,it contributes to neuronal differentiation by glycosylating and stabilizing the classical mucin-type O-GalNAc protein PDPN.Also,we provided a list of potential protein substrates of pp GalNAc-T13 that may be associated with its function in neuronal differentiaon.Last but not least,we constructed Galnt13 knockout mice,which could be a good model for the investigation ofpp GalNAc-T13 function.This study may contribute to the functional study on O-GalNAc glycosylation in nervous system,and serve as an important model for the comprehensive analysis of the biological function of pp GalNAc-T13.