转录因子HNF1A、HNF4A和FOXA2调节肝细胞蛋白质N-糖基化

Hepatocyte nuclear factor 1 alpha(HNF1A),hepatocyte nuclear factor 4 alpha(HNF4A),and forkhead box protein A2(FOXA2)are key transcription factors that regulate a complex gene network in the liver,cre-ating a regulatory transcriptional loop.The Encode and ChIP-Atlas databases identify the recognition sites of these transcription factors in many glycosyltransferase genes.Our in silico analysis of HNF1A,HNF4A.and FOXA2 binding to the ten candidate glyco-genes studied in this work confirms a significant enrich-ment of these transcription factors specifically in the liver.Our previous studies identified HNF1A as a master regulator of fucosylation,glycan branching,and galactosylation of plasma glycoproteins.Here,we aimed to functionally validate the role of the three transcription factors on downstream glyco-gene transcriptional expression and the possible effect on glycan phenotype.We used the state-of-the-art clus-tered regularly interspaced short palindromic repeats/dead Cas9(CRISPR/dCas9)molecular tool for the downregulation of the HNF1A,HNF4A,and FOXA2 genes in HepG2 cells-a human liver cancer cell line.The results show that the downregulation of all three genes individually and in pairs affects the transcrip-tional activity of many glyco-genes,although downregulation of glyco-genes was not always followed by an unambiguous change in the corresponding glycan structures.The effect is better seen as an overall change in the total HepG2 N-glycome,primarily due to the extension of biantennary glycans.We propose an alternative way to evaluate the N-glycome composition via estimating the overall complexity of the glycome by quantifying the number of monomers in each glycan structure.We also propose a model showing feedback loops with the mutual activation of HNF1A-FOXA2 and HNF4A-FOXA2 affecting glyco-genes and protein glycosylation in HepG2 cells.

月经周期中免疫球蛋白G N-糖基化的周期性变化

Immunoglobulin G(IgG)is the most abundant plasma glycoprotein and a prominent humoral immune mediator.Glycan composition affects the affinity of IgG to ligands and consequent immune responses.The modification of IgG N-glycosylation is considered to be one of the various mechanisms by which sex hormones modulate the immune system.Although the menstrual cycle is the central sex hormonerelated physiological process in most women of reproductive age,IgG N-glycosylation dynamics during the menstrual cycle have not yet been investigated.To fill this gap,we profiled the plasma IgG Nglycans of 70 healthy premenopausal women at 12 time points during their menstrual cycles(every 7 days for 3 months)using hydrophilic interaction ultra-performance liquid chromatography(HILIC-UPLC).We observed cyclic periodic changes in the N-glycosylation of IgG in association with the menstrual cycle phase and sex hormone concentration in plasma.On the integrated cohort level,the modeled average menstrual cycle effect on the abundance of IgG N-glycosylation traits was low for each trait,with the highest being 1.1%for agalactosylated N-glycans.However,intrapersonal changes were relatively high in some cases;for example,the largest difference between the minimum and maximum values during the menstrual cycle was up to 21%for sialylated N-glycans.Across all measurements,the menstrual cycle phase could explain up to 0.72%of the variation in the abundance of a single IgG glycosylation trait of monogalactosylation.In contrast,up to 99%of the variation in the abundance of digalactosylation could be attributed to interpersonal differences in IgG N-glycosylation.In conclusion,the average extent of changes in the IgG N-glycopattern that occur during the menstrual cycle is small;thus,the IgG N-glycoprofiling of women in large sample-size studies can be performed regardless of menstrual cycle phase.

流感与COVID-19患者的免疫球蛋白G糖基化差异

The essential role of immunoglobulin G(IgG)in immune system regulation and combatting infectious diseases cannot be fully recognized without an understanding of the changes in its N-glycans attached to the asparagine 297 of the fragment crystallizable(Fc)domain that occur under such circumstances.These glycans impact the antibody stability,half-life,secretion,immunogenicity,and effector functions.Therefore,in this study,we analyzed and compared the total IgG glycome—at the level of individual glycan structures and derived glycosylation traits(sialylation,galactosylation,fucosylation,and bisecting Nacetylglucosamine(GlcNAc))—of 64 patients with influenza,77 patients with coronavirus disease 2019(COVID-19),and 56 healthy controls.Our study revealed a significant decrease in IgG galactosylation,sialylation,and bisecting GlcNAc(where the latter shows the most significant decrease)in deceased COVID19 patients,whereas IgG fucosylation was increased.On the other hand,IgG galactosylation remained stable in influenza patients and COVID-19 survivors.IgG glycosylation in influenza patients was more time-dependent:In the first seven days of the disease,sialylation increased and fucosylation and bisecting GlcNAc decreased;in the next 21 days,sialylation decreased and fucosylation increased(while bisecting GlcNAc remained stable).The similarity of IgG glycosylation changes in COVID-19 survivors and influenza patients may be the consequence of an adequate immune response to enveloped viruses,while the observed changes in deceased COVID-19 patients may indicate its deviation.