Global Mapping Of Glycosylation Pathways In Human-derived Cells

Carbohydrates,lipids,proteins,and nucleic acids are known as four biological macromolecules.Glycosylation in the human body participated in various physiological activities including signal transduction.The metabolism of glycans is closely related to biological processes such as fertilization,cell differentiation,tissue development,immunization,nervous system formation and cell carcinogenesis.It is of great importance to study the cellular glycan structures for understanding the biological metabolic processes related to glycans.Compared with proteins and nucleic acids,the complexity and diversity of the glycans make it even more difficult to identify the structures.Although previous studies have reduced the difficulty of identification about glycan structures through prediction of the glycan structures.However,they are still limited.For example,they only focus on a specific type of glycosylation pathway such as N-glycosylation,in this case lacking a comprehensive evaluation strategy for the complex glycosylation relative physiological activities in the whole cell so far.Additionally,these studies have not been applied to analyze the glycosylation of human cells.Therefore,it is desired to explore an easier analytical tool for the systematical prediction of the glycan structures in human cells.Recently RNA-sequence technology is well developed,which makes the prediction of the glycosylation level in human cells based on gene transcription data to be possible.In order to solve the problems in glycosylation analysis,transcriptional information was integrated into the glycosylation metabolic pathway to visualize the glycosylation process with HEK293 as a model cell in this study.Besides we explored a new strategy to predict the glycan structures using transcriptional data.The purpose of this study is to develop an understood visualization tool for glycosylating metabolic pathways and to comprehensively predict the influence of glyco-relative gene transcriptional changes on the glycan structures by a bird 's-eye view in human-derived cells.The main findings of this study are followed:(1)Based on the latest literature reports,this study included 951 genes related to glycosylation,which were divided into 30 categories according to their biological functions involved in glycosylation,and 19 categories of glycosylation metabolic map were draw.We further developed an automatic drawing network tool.The tool integrated the glycosylationrelative gene expression profile into the silico metabolic pathways,in which arrows were utilized to represent the detailed information of genes involved in glycosylation to visualize the metabolic process of glycosylation and describe the synthetic potential of glycan structures.By using this analytical tool,we found that the glycosylation pathway of HEK293 cells was consistent with the glycosylation pattern of cells in the published researches.(2)HEK293 cells were confirmed to experienced a variety of glycosylation modifications by mass spectrometry and lectin staining,Through the study of terminal glycan epitopes,it was confirmed that the glycosyltransferase genes with the lowest transcriptional level in the metabolic pathway played a critical role in the determination of the final glycan structures.In the O-glycosylation pathway studies,we found the predicted results highly matched the glycan structures identified by MS with statistical analysis.It meant this tool could accurately predict the potential of glycan synthesis.Further analysis of glycosylation relative gene expression levels in the database of various human-derived cells and reported identified glycan structures showed that this predictive tool in this study can be widely used for glycosylation prediction in human-derived cells when the threshold of TPM is 1.(3)Lectin staining was used to detect the glycan structures of the 40 N-glycan synthetic defective cell lines.It was confirmed that lectin staining analysis could reveal the changes of the cell surface glycan structures of knockout cells.It was found that the surface glycan of the knockout cell lines which expressed high mannose type of N-glycans showed high clustering,and only the polylactosamine modification was abnormally up-regulated in the terminal modification structures.Mass spectrometry analysis of seven typical N-glycan relative genedeficient cell lines showed that the glycolipid composition of T-KO and MGAT1-KO cell lines,which mainly produced high mannose type of N-glycans,was significantly changed.By the analysis of the transcriptome data,we found in T-KO cells CERS1,HAS2 and RENBP increased significantly.The up-regulated transcription of the above genes in T-KO cells was verified by RT-q PCR.Furthermore,we found that CERS1 increased the synthesis of corresponding lipid substrates,HAS2 enhanced the release of hyaluronan into the extracellular matrix and RENBP promoted the isomerization of Glc NAc into Man NAc.Combined with the feature that in the T-KO cells the terminal Glc NAc of N-glycans cannot be further modified,we found that the accumulated UDP-Glc NAc which could not be utilized in the N-glycosylation,later led to cell stress.By triggering an adaptive transcriptional regulation,cells turned up CERS1,HAS2 and RENBP,to reduce the intracellular UDP-Glc NAc or Glc NAc content by changing the consumption strategy of sugar nucleotide substrates.(4)We analyzed the transcription data from the liver and small intestine in Human Protein Atlas(HPA)database by Glyco Maple.a large number of critical O-glycosyltransferases of low expression level in the liver,which led to the O-glycans are simple with Core 1 or sialic acid modified Core 1 structures.On the contrary,most glycosyltransferases involved in Oglycosylation are expressed vigorously in digestive organs like the small intestine with a strong mucin expression ability,which explained the variety of O-glycan structures.The expression profiles of O-glycan-relative genes in 37 human tissues showed that similar transcription patterns in the organs of similar functions,such as digestive organs,revealing that the high expression of critical glyco-relative genes in the glycosylation pathways contributed to the richness and complexity of glycan structures in O-glycosylation.In The analysis of transcriptome data from human colon samples from The Cancer Genome Atlas Program(TCGA)database and The Genotype-Tissue Expression Project(GTEX),changes in glycan structures during cancerization were successfully predicted.In this work,we collected genes involved in glycosylation and use metabolic profiling to map the glycosylation pathways in human-derived cells,demonstrating that transcriptome information can be utilized to predict glycan structures.Transcriptional analysis can keenly capture the glyco-relative genes which are transcriptionally regulated in cells and explain the changes in the glycan structures in cells.Also,the network tool of this study can be used to predict the glycosylation level of human-derived cells.It reveals that the organ formed the function-relative glycosylation changes due to the changes in the expression profile of glycosylation genes during cell differentiation.On the other hand,it can predict the structural changes of glycan during pathological processes such as carcinogenesis.This research is expected to provide new ideas for disease treatment and biomarker discovery in the future.