Earticle

Lysosomal acid hydrolases are crucial for degradation and recycling of various biomolecules. They are mostly produced in a form of glycoproteins with oligosaccharides containing terminal mannose-6-phosphate (M6P). Genetic deficiencies of lysosomal enzymes lead to the inability of the lysosome to digest biomolecular substrates and cause the progressive accumulation of non-degraded substrates in the lysosome. This event causes the onset of lysosomal storage disorders (LSDs). Tay-Sachs and Sandhoff diseases, a class of LSDs, are caused by deficiency of β-N-acetylhexosaminidases, thereby leading to accumulation of GM2-ganglioside in lysosomes. To remove GM2-ganglioside in diseased cells, multiple mannose-6-phosphate (M6P)-appended β-N-acetylhexosaminidases were prepared by using combined genetic code expansion and click chemistry techniques. It was found that the multiple M6P-appended β-N-acetylhexosaminidase efficiently is internalizes into cells via M6P receptor-mediated endocytosis and then reaches the lysosome to regain its enzyme activity. Furthermore, the glycoengineered enzyme was able to efficiently cleave GM2-ganglioside in primary diseased cells, indicating the restoration of its activity in cells. The present strategy using the coupled genetic code expansion and biorthogonal ligation techniques is highly attractive to construct multiple M6P-containing enzymes which can be used to study LSDs.

Cellular glycans are produced mainly in the form of glycoconjugates by the action of glycosyltransferases and glycosidases. It is known that genetic deficiencies of lysosomal glycosidases cause lysosomal storage disorders. Moreover, inhibitors of glycosidases have been developed as therapeutic agents to treat viral infections, diabetes and cancer. Because of the biological and pathological significance of glycosidases, it is of great importance to develop new methods to assess their catalytic activities. In this study glycosidase activities were rapidly assessed using carbohydrate microarrays containing fluorescent probe-conjugated glycans. The microarrays were constructed by immobilizing five glycosylated near-infrared (NIR) fluorescent probes containing hydrazide appendages on epoxy modified glass slides. The glycosidases were applied to the glycan microarrays and the fluorescence intensity were measured by using a microarray scanner. The results of microarray experiments revealed that carbohydrate microarrays immobilized by glycosylated NIR probes are highly useful for profiling glycosidase activities.

Luteolin(3’,4’,5,7-hydroxy flavone) is a flavone aglycone known to have anti-inflammatory, antioxidant and anti-cancer effects, but it is limited in pharmaceutical material due to low water-solubility. Therefore, the aim of study is to synthesize luteolin glycoside (LG) for increasing water-solubility. The LG was synthesized by the transglycosylation reaction of amylosucrase of Deinococcus geothermalis (DGAS) using luteolin and sucrose as an acceptor and a donor, respectively. The maximum yield of the LG product was established to be 89.3 % with a 1:7.16 molar ratio of donor and acceptor molecules, in 50 mM Tris-HCl buffer (pH 7) at 37°C for 24 hr and the results of HPLC showed that only one transglycosylation product was detected. The structural analyses using 13C and 1HNMR proved that the transglycosylated product was luteolin-4'-O-glucoside.

We have developed GlycoProteomeAnalyzer (GPA) for high throughput identification and quantification of N- and O-glycoproteins. GPA is a program for automatic identification of the glycan compositions and glycopeptide sequences by using three different scoring systems such as M-score for glycopeptide selection from oxonium-ions, S-score for theoretical isotope pattern match of molecular ion, and Y-score for glycopeptide identification from MS/MS fragmentation.[1] We here present the IQ-GPA Cloud which is a web based GPA application for automatic identification and quantification of site-specific N- and O-glycoproteins. IQ-GPA Cloud is a publicly accessible web application that enables users to quickly provision on-demand infrastructures for high-performance gylcoproteome analysis using Microsoft Azure cloud platforms. Users have instant access to a range of a glycan database builder as well as Nand O-GPA modules. IQ-GPA Cloud has been designed to easily handle high-throughput glycoproteomic data with graphical user interfaces. Microsoft Azure Cloud provides more than thousands of CPU to increase throughput of data analysis. Therefore, users can use glycoproteome analysis easily through IQ-GPA Cloud without any high-powered computer. The IQ-GPA Cloud is demonstrated on the website, https://cloud.igpa.kr/

Gastric cancer is one of the most common malignancy and leading cause of cancer death. Classically, CEA and CA19-9 have been used for gastric cancer detection. However, CEA and CA19-9 insufficient for cancer detection due to low specificity and sensitivity. Glycosylation is the most common post-translational modification and plays an important role in various biological processes. Whole serum based glycan profiling has been already developed and widely used in cancer biomarker study. While, targeted glycoproteomic approach is needed in clinic for better sensitivity and specificity. In previous study, we targeted serum haptoglobin for gastric cancer. We found glycosylation changes of haptoglobin on released N-glycan between gastric cancer patient and healthy control. Released glycan analysis provide compositional and isomer information while cannot give actual site of glycosylation. On the other hand, intact glycopeptide analysis provide site-specific information. Based on this actual glycosylation site and glycan heterogeneity, we monitored more detailed glycan changes and aberrant glycosylation of each site for more sensitive and specific diagnosis. In this study, we have developed the method for intact glycopeptide analysis for biomarker discovery using UHPLC MS. We successfully separated 3 glycopeptides and profiled the glycoform of 2 glycopeptides. The other one was difficult to profile glycoform according to two glycosylation sites. Therefore, this glycopeptide was fractionated and analyzed by nano LC chip Q-TOF MS. Further, cancer patient (n=40, stage IV) and healthy control (n=47) samples were applied for training set of biomarker study. A T-test based analysis was performed to identify potential biomarker (p<0.001). In the future, we will apply this platform to large clinical sample to validate potential biomarker.

Raw potato starch-based feeds controlled body weight and insulin resistance in mice. To investigate the association of the phenotypic changes with an impact on the gut microbiota, various starches-based feeds formulated based on the nutritional composition of AIN93G but substituted corn starch to wheat, rice, or potato starch, were provided ad libitum 6 week-aged C57BL/6 male mice for 16 weeks. Profiles of microbiota in the mice’s feces were compared by analyzing16s rRNA gene amplicon sequencing and QIIME pipeline with Greengenes database. It found that gut microbiota was changed according to the kinds of starches. The ratio of Firmicutes to Bacteroidetes was decreased by feeding potato starch diet and taxonomy assignment showed that potato starch diet increased the abundances of Akkermansia, Rikenellaceae and Sutterella. The large intestine of mice fed potato starch-based diet was heavier than those of other starches-fed mice. According to the literatures, it reported that Akkermansia and Sutterella are associated with improvement of obesity and especially, Akkermansia known to be correlated with the abundance of Rikenellaceae have been demonstrated to induce an increase in the large intestine weight and an increase of insulin sensitivity. Therefore, it supposes that the effects of potato starch based-diet on body weight and related insulin resistance can be elucidated from the intestine microbial changes.

Enzymatic glycosylation on solid surface is an efficient and useful tool to improve the limitation of conventional glycan synthesis and provide diverse glycan sources to use for analyzing glycan-protein interaction on surface. In this work, a novel glycan microarray platform introduced glycan-oligonucleotide conjugates was developed to prepare glycan sources for analyzing glycan-protein interactions through solid surface based-enzymatic glycan synthesis. Pasteurella multocida sialyltrnasferase (PmST), Campylobacter jejuni N-acetylgalactosaminyltransferase (CgtA), and C. jejuni galactosyltransferase (CgtB) were taken as target enzymes to synthesize GM1-related glycans such as GM1 pentasaccharide, GM2 tetrasaccharide, and GM3 trisaccharide. HPLC analysis showed that three glycosyltrnasferases expressed in Escherichia coli were produced as active forms. Synthesized glycans on this platform could be effectively obtained through DNA denaturation process under appropriate condition and identified by mass measurement. We anticipate that this novel glycan microarray platform combining enzymatic glycosylation with mass measurement can serve a solution to improve the difficulty of obtaining glycan sources and provide a new direction toward studies about glycan synthesis and glycan-related diverse interaction analysis.

Cryptococcus neoformans is a fungal pathogen causing life-threatening meningoencephalitis in immunocompromised individuals, such as AIDS patients. Several Cryptococcal mannoproteins (MPs), such as MP98, 88, and 84, are reported as key antigens stimulating host CD4 (+) T-cell response. In this study, we investigated the effect of N-glycans of Cryptococcal MPs on the interaction with host cells. The expression pattern analysis of the GPI-anchorless MPs with His (H)-tag in the wild-type (WT) and various N-/O-glycosylation mutant strains of C. neoformans indicated that MP98 is modified mainly by N-glycosylation, while MP88 and MP84 are subject to both N- and O-glycosylation. The in vitro adhesion assay using the purified MP(H)s revealed that compared to the WT-secreted MPs, the MP98(H) and MP84(H) proteins secreted from the N-glycosylation defective Cnalg3Δ mutant strain displayed a slightly increased adherence to the lung epithelial cells. Moreover, the N-glycan removed MP98(H) and MP88(H) were shown to be highly adhesive to the lung epithelial cells and macrophages. The MP98(H) and MP88(H) proteins with truncated N-glycans also stimulated more efficiently several cytokine secretion of the dendritic cells compared to the WT-secreted MP(H)s. These results indicate that highly mannosylated N-glycans seems to hide O-glycans or specific parts of proteins, which might serve as a ligand or an epitope, recognized by receptors of mammalian cells.

Fisetin (3,7,3’,4’-tetrahydroxyflavone) belongs to the flavonol subgroup of flavonoids and is found in several fruits and vegetables. Fisetin has high medicinal value and is useful natural agent against cancer and evaluated for its potential inhibitory role against cancer on cells and animal models. To efficiently diversify the therapeutic uses of fisetin, Escherichia coli was harnessed as a production factory and E. coli BL21(DE3)/ΔpgiΔzwfΔgalU mutant was engineered by overexpressing thymidine diphosphate (dTDP)-D-glucose synthase (tgs), dTDP-D-glucose 4,6-dehydratase (dh), and a sugar aminotransferase (wecE) from different sources to produce a pool of dTDP-4-amino-4,6-dideoxy-D-gal actose in the cell cytosol. To this recombinant mutant, two Arabidopsis thaliana glycosyltransferases (ArGT-3 and ArGT-4) were overexpressed to generate two glycosylation platforms (E. coli BL21(DE3)/ΔpgiΔzwfΔgalUTDW-3 and E. coli BL21(DE3)/Δ pgiΔzwfΔgalUTDW-4), which were accessed for the glycosylation of fisetin. As a result, one of the two systems, E. coli BL21(DE3)/ΔpgiΔzwfΔgalUTDW-3, was able to conjugate 4-amino-4,6-dideoxy-D-galactose sugar at the 3-OH position of fisetin, producing an unnatural fisetin 3-O-4-amino-4,6-dideoxy-D-galactoside.

Recently, it was demonstrated that glycosylation of phenolic compounds enhances their aqueous solubility while retaining biological activities. We synthesized a glycosylated derivatives (AEG) of aloe emodin (AE), which is one of phenolic compounds present in aloe plant, by enzymatic modification and studied its in vitro and in vivo anti-cancer effects against human lung cancer using A549 cells. While treatment of cells with 5 μM AE did not affect the viability of A549 cells, AEG reduced cell viability, with a 40% reduction at the same concentration. Western blot analysis showed that AEG significantly induces the activation of caspase 9 and poly (ADP-ribose) polymerase (PARP) by 3.4 and 2.1-folds, respectively, compared to those in AE-treated cells, suggesting that the cytotoxicity of AEG is associated with apoptotic events. The molecular mechanisms involved in the anti-cancer effect of AEG were investigated by western blot analysis. AEG (5 μM) decreased phosphorylation of ERK, p38 and Akt, by 35, 44, and 57%, respectively, compared to untreated control cells. In addition, whether AEG is equally active against A549 lung cancer cell xenograft tumors was examined in a mouse model. Administration of AEG through intraperitoneal injection to BALB/c-nu mice bearing A549 cells dose-dependently reduced tumor growth. The histological analysis of tumor tissues with H&E staining showed that AEG decreased the cell numbers in tumor tissues and TUNEL assay showed DNA fragmentation of tumor cells. Taken collectively, these results clearly demonstrated that glycosylated aloe emodin inhibits the tumor growth by induction of apoptotic cell death in vitro and in vivo, suggesting that it can be a good candidate as a potent anti-cancer agent against human lung cancer.

Mouse has been used as an animal model for scientific research owing to its physiological similarity to human. However, their glycosylation of glycoproteins showed many differences between mouse and human. Usually N-glycosylation is directly involved in various biological process and plays crucial role in human diseases due to their unusual biological sensitivity. Therefore, the site-specific characterization of N-glycosylation in model mouse is necessary in order to investigate progression of disease. This study provided comparison of the site-specific N-and O-glycosylation between human and mouse plasma using LC-MS/MS with GPA (GlycoProteome Analyzer) system1.Thesite-specificN-andO-glycosylationbetweenhumanandmousehasthreedifferencesinpla sma.First,thesialicacidofN-andO-glycopeptideswasalmostentirelyNeu5Gcinmouseplasma,whileinh umanplasmawasNeu5Ac.Second,O-acetylatedNeuGcofN-glycopeptidewasidentifiedinmouseplasm aonly.Third,antennalHexNAc-NeuGcofN-glycopeptidewasspecificallyidentifiedfromseveralglycop roteinsinmouseplasmaonly.Inconclusion,ourstudyhasprovidedanoverviewofthesite-specificN-and O-glycosylationofhumanandmouseinplasma.

O-GlcNAcylation is a carbohydrate post-translational sugar modification occurs on hydroxyl groups of serine or threonine residues of cytosolic and nuclear proteins. It is known that O-GlcNAc addition and removal regulate many biological events like transcription, protein degradation, proliferation. Epithelial-mesenchymal transition(EMT) is a process that epithelial cells lose its polarity and cell-cell contact, therefore get migratory capabilities to become mesenchymal stem cells. There are several reports that O-GlcNAc is associated with cancer EMT and metastasis. But there are still much to discover the role of O-GlcNAc on TGF-β-induced EMT. TGF-β is one of the powerful EMT inducer, and is primarily dependent on TGF-β/Smad signaling pathway. Here we found that Smad protein expression is regulated by global O-GlcNAc change, consequentially controls TGF-β-induced EMT. Now we’re planning to verify how O-GlcNAc controls Smad expression.

Arabinans can be synergistically degraded into L-arabinose and arabinooligosaccharides (AOS) by the actions of exo-arabinofuranosidase (AF) and endo-arabinanase (ABN). Bifidobacterium spp., the well-known probiotic bacteria, were reported to utilize linear AOS as the prebiotic carbohydrates. Two different endo-arabinanase genes were previously found and cloned from the genome of Bifidobacterium longum subsp. suis DSM 20211. In this study, the modes of action for both endo-arabinanases, BflsABN-B1 and B2, were comparatively characterized, and their gene cluster was analyzed in detail. The gene cluster analysis revealed that the corresponding contig 11 of Bf. longum subsp. suis includes several putative exo-acting AF genes, the ABC transporter-related genes, ATP-binding protein genes, as well as both ABNs-B. According to the gene cluster analysis, the extracellular BflsABN-B1 hydrolyzes arabinan polymers to the short chain AOS, which are imported into the cell membrane through the ABC transporters. Within a cell, AOS can be hydrolyzed into L-arabinose by an intracellular BflsABN-B2 and various exo-AFs. These results proposed the hypothetical enzymatic system for the arabinan utilization in Bf. longum subsp. suis.

endo-(1,5)-α-L-Arabinanase (ABN, EC 3.2.1.99) is an endo-acting arabinosyl hydrolase that can cleave α-(1,5)-L-arabinofuranosidic linkages in arabinan and arabinooligosaccharides(AOS). In the present study, a putative gene encoding ABN (LbshABN-B) has been found from the genome of Lactobacillus shenzhenensis LY-73 which is isolated from a dairy beverage in China's Shenzhen market. The open reading frame of LbshABN-B consists of total 1,242 bp nucleotides encoding 414 amino acids with a predicted signal peptide of 24 amino acids. The molecular mass of LbshABN-B without signal peptide is 46.2 kDa. It shares approximately 50% of amino acid sequence identities with the other known ABNs type-B, whereas it showed less than 29% of sequence identities with ABNs type-A. The LbshABN-B gene being fused with six-histidines at its C-terminus was constitutively expressed and purified from Escherichia coli. Although the debranched (linear) arabinan is the most preferred substrate, LbshABN-B showed the significant activity towards sugar beet (branched) arabinan as well. This enzyme showed the highest activity on debranched arabinan at 45oC and pH 6.0 in 50 mM sodium acetate buffer, respectively. LbshABN-B can be applicable to the production of various branched AOS from sugar beet arabinan.

Glycosylation of monoclonal antibody (mAb) therapeutics plays vital roles such as biological activity, stability, plasma half-life, and immunogenicity. It has been known that the glycan variations in mAbs are caused by cell-line selection and changes in culture-medium parameters. These variations can lead to changes in drug potency of therapeutic glycoproteins. Therefore, monitoring of glycosylation on protein is required to access the quality, safety, and equivalence of therapeutic antibodies. Here, in order to develop the reference data for quality by design (QbD) in therapeutic antibodies’ production, we performed the absolute quantitation and evaluation of major glycan (G0F, G1F, and G2F) on human immunoglobulin G (IgG) and five representative recombinant mAb drugs including adalimumab, bevacizumab, infliximab, rituximab, and trastuzumab using FLD response of 2-aminobenzamide (2-AB) labeled glycan standards. N-glycans on mAbs were enzymatically released and then labeled by 2-AB reagent. Followed by 2-AB labeled glycans were purified by HILIC-SPE. 2-AB labeled glycans were analyzed by UHPLC-HILIC-FLD which can provide chromatographic glycan separation and quantitation. The calibration curves of glycan standard were linear over the range of about 10 fmole to 10 amoles (3 orders of magnitude) and its linear correlation coefficients were excess of 0.999 (R2). As a result, the concentration of the glycans on commercial mAbs measured 2 to 10 times higher than that of IgG. Additionally, we confirmed that quantitative difference of glycan in each sample. This result can be directly applied to evaluate mAbs variants and/or biosimilars for both developmental and regulatory purposes.

Mesenchymal stem cells are undifferentiated stem cells that can differentiate into various cells and tissues. In this study, we confirmed the difference in expression and differentiation rate of gangliosides during the differentiation of mini-pig adipose-derived mesenchymal stem cells into neuronal cells using secretion medium from PMA-induced human macrophage-like cell line U937 (Macro SM). We showed that nm23, one of several proteins secreted in the culture medium of activated human macrophages, decreased the neuronal differentiation rate of mesenchymal stem cells derived from mini-pig adipose, and inhibited the function and the synthesis enzyme of GD3. In conclusion, one of the factors that decreased the differentiation rate of mini-pig adipose-derived mesenchymal stem cells into neuronal cells by Macro SM is the nm23 protein.

The genetic deficiency of lysosomal β-N-acetylhexosaminidases leads to incomplete digestion of GM2-ganglioside and thus causes its progressive accumulation in lysosomes. This defect causes the onset of Tay-Sachs and Sandhoff diseases, a class of lysosomal storage disorders (LSDs). To remove GM2-ganglioside accumulated in Tay-Sachs and Sandhoff diseased cells, multiple mannose-6-phosphate (M6P)-appended β -N-acetylhexosaminidases were prepared by click chemistry between multiple M6P-containing N3-peptides and alkynylated enzymes obtained by genetic code expansion. In addition, lysosome-targeting, near-infrared (NIR)-based fluorogenic probes were synthesized and applied for (real-time) monitoring of lysosomal β-N-acetylhexosaminidase activity in cells. It was found that the multiple M6P-appended β-N-acetylhexosaminidase efficiently internalizes cells via M6P receptor-mediated endocytosis and then reaches the lysosome to regain its enzyme activity. Furthermore, the glycoengineered enzyme was able to efficiently cleave GM2-ganglioside in primary diseased cells, indicating the restoration of its activity in cells. The present strategy using the coupled genetic code expansion and biorthogonal ligation techniques is highly attractive to construct multiple M6P-containing enzymes which can be used to study LSDs.

Among the animal, human uniquely contains high amount and diverse structure of soluble oligosaccharides in milk. The multiple functions for infant of human milk oligosaccharides (HMOs) have been well discussed, particularly sialic acid and fucose containing oligosaccharide. The structure and composition of HMOs structure were diverse among different mothers or breastfeeding period of a given woman as well, and nutritional conditions. The geographical origin of mothers is one of the factors that influence on the HMOs diversity. Hence, we aim to profile HMOs from mothers in Korea, China and Vietnam in the relation to their nutritional status to provide a comprehensive understanding about HMOs from maternal in these countries. The results show that oligosaccharide composition of human milk varied among countries. HMOs from Korea and China were almost similar in term of degree of polymerization (DP) whereas Vietnam HMOs has lower DP3 but high in DP4 and DP5. The ratio of non-secretor mothers that were unable to produce α-1, 2-fucosylated glycans in their secretions, following by Vietnam and lowest in Korea population. The fucosylated, and sialylated HMOs were 62-67%, 4-6% in China and Korea milk respectively while 50% and 9.2% in Vietnam maternal milk. These observed proportion were different from earlier studies that may due to the difference in geographical origin of mothers. Elucidating molecular structure of HMOs provides an important opportunities for improving diet and health care for the neonate in specific region.

Gastric cancer (GC) is one of the leading causes of cancer-related death worldwide, largely because of difficulties in early diagnosis. In spite of accumulating evidence linking aberrant glycosylation and GC, site-specific localization of the glycosylation for improving clinical specificity and sensitivity is still in analytical challenge. Here, we constructed an analytical platform with targeted glycoproteomic approach for GC biomarker discovery. Unlike conventional glycomic approach characterized by untargeted MS profiling of released glycan, developed platform can be characterized by three key features: target protein-specific, glycosylation site-specific, and structure-specific platform with one-shot non-specific enzyme digestion. Serum haptoglobin (Hp) was enriched by immunoaffinity purification and subjected to pronase to generate site-specific glycopeptides. Glycopeptides were identified and quantified by nano LC/MS and -LC/MS/MS, resulting in full characterization of glycan macro- and micro-heterogeneity. A total of 96 glycopeptides, each corresponding to a unique glycan/glycosite pairing, were tracked across all cancer and control samples. Significant alterations in site-specific glycosylation have been observed in Hp in GC patients.

Recombinant coagulation factor IX (rFIX) is an extremely heterogeneous glycoprotein having multiple N-and O-glycosylation which influence on pharmacological functions including biological activity and in vivo stability. The study of glycosylation on rFIX has mainly focused on N-glycans due to an inherent complexity of O-glycosylation and the absence of analytical platform. Here, we comprehensively characterized the O-glycosylation on recombinant coagulation factor IX (rFIX) using MS-based glycomic and glycoproteomic approach. Briefly, O-glycans were chemically released using β-elimination while O-glycopeptides were prepared by pronase digestion to determine site-specific O-glycosylation distribution. Successfully we could identify unique O-glycosylation modifications such as O-fucosylation and O-glucosylation as well as a typical O-glycosylation on rFIX using nanoLC/MS and Tandem MS. Particularly, through O-glycopeptide analysis, we demonstrated that O-glucosylation and O-fucosylation were uniformly displayed on O-sites Serine 53 and 61 of EGF domain, respectively. While core 1-type O-glycosylations were overwhelmingly distributed across other O-sites (Threonine 159, 169, 172, and 179) in activation peptide of rFIX. Site-specific profiling including micro-and macro-heterogeneity could provide macroscopic aspect to figure out O-glycosylation pattern decorated on rFIX.

Cryptococcus neoformans is an opportunistic human pathogenic yeast causing life-threatening meningoencephalitis in immunocompromised individuals. We have identified a C. neoformans ALG3 homolog (CnALG3), coding for a dolichyl-phosphate-mannose dependent α -1,3-mannosyltransferase, and constructed a null mutant strain of CnALG3 (Cnalg3Δ). The N-glycan structure analysis, carried by HPLC, exoglycosidase treatment, and MALDI-TOF, showed that Cnalg3Δ generated the truncated N-glycans carrying five mannose residues as a major species. Interestingly, most of the truncated N-glycans of Cnalg3Δ appeared to be devoid of a xylose residue. The glycoproteins produced in Cnalg3Δ, such as phospholipase PLB1, aiding fungal traversal across the blood-brain barrier, and T-cell antigen MP98, were shown to have truncated N-glycans. Cnalg3Δ displayed a moderate defect in capsule biosynthesis and in resistance to oxidative and cell wall stresses, compared to the wild-type (WT) strain. However, noticeably, the Cnalg3Δ mutant strain showed fully attenuated virulence in a mouse model of cryptococcosis, suggesting that the defect in N-glycan assembly affect considerably the pathogenicity of C. neoformans. The attachment to lung epithelial cells and the non-opsonic phagocytosis of Cnalg3Δ were shown to be comparable to those of WT during infection, indicating that the truncated N-linked glycans may not affect the early steps in interaction with host cells. However, the capacity to drive macrophage pyroptosis after phagocytosis was greatly decreased in Cnalg3Δ, indicating a critical role of cryptococcal N-glycans in inducing host cell lysis as a mechanism of host cell escape.

Fucosylation is one of the most common glycosylation and has been associated with the development of hepatocellular carcinoma (HCC). Alpha-fetoprotein (AFP) is clinically used as serological marker to diagnose HCC. Although the level of AFP increased in HCC patients, its sensitivity for diagnosis of HCC is poor because AFP levels are also increased in liver diseases, such as liver cirrhosis. Here, we introduced detection of AFP glycopeptides by parallel reaction monitoring (PRM) mass spectrometry (MS) combined with immunoprecipitation. Also, desialylation of AFP glycopeptides was used to improve MS detection limit (LOD < 2 ng/mL) and to obtain reliable signal (CV < 20%) from 0.1 μL serum. Finally, we compared serum samples from HCC with liver cirrhosis using relative percentage of fucosylated glycopeptide in AFP (AFP-fuc%). AFP-fuc% showed an area under the ROC curve (AUC = 0.949, p value < 0.0001) to discriminate between HCC and liver cirrhosis patients. These results suggest the potential of PRM-based AFP-fuc% in early diagnosis of HCC using serum samples.