Dietary eubiotics of microbial muramidase and glycan improve intestinal villi,ileum microbiota composition and production trait of broiler

Background Optimal gut health is important to maximize growth performance and feed efficiency in broiler chickens.A total of 1,365 one-day-old male Ross 308 broiler chickens were randomly divided into 5 treatments groups with 21 replicates,13 birds per replicate.The present research investigated effects of microbial muramidase or a precision glycan alone or in combination on growth performance,apparent total tract digestibility,total blood carotenoid content,intestinal villus length,meat quality and gut microbiota in broiler chickens.Treatments included:NC:negative control(basal diet group);PC:positive control(basal diet+0.02%probiotics);MR:basal diet+0.035%microbial muramidase;PG:basal diet+0.1%precision glycan;and MRPG:basal diet+0.025%MR+0.1%PG,respectively.Results MRPG group increased the body weight gain and feed intake(P<0.05)compared with NC group.Moreover,it significantly increased total serum carotenoid(P<0.05)and MRPG altered the microbial diversity in ileum contents.The MRPG treatment group increased the abundance of the phylum Firmicutes,and family Lachnospiraceae,Ruminococcaceae,Oscillospiraceae,Lactobacillaceae,Peptostreptococcaceae and decreased the abundance of the phylum Campilobacterota,Bacteroidota and family Bacteroidaceae.Compared with the NC group,the chickens fed MRPG showed significantly increased in duodenum villus length at end the trial.Conclusion In this study,overall results showed that the synergetic effects of MR and PG showed enhancing growth performance,total serum carotenoid level and altering gut microbiota composition of broilers.The current research indicates that co-supplementation of MR and PG in broiler diets enhances intestinal health,consequently leading to an increased broiler production.

Comparing different domains of analysis for the characterisation of N-glycans on monoclonal antibodies

With the size of the biopharmaceutical market exponentially increasing,there is an aligned growth in the importance of data-rich analyses,not only to assess drug product safety but also to assist drug development driven by the deeper understanding of structure/function relationships.In monoclonal antibodies,many functions are regulated by N-glycans present in the constant region of the heavy chains and their mechanisms of action are not completely known.The importance of their function focuses analytical research efforts on the development of robust,accurate and fast methods to support drug development and quality control.Released N-glycan analysis is considered as the gold standard for glycosylation characterisation;however,it is not the only method for quantitative analysis of glycoform heterogeneity.In this study,ten different analytical workflows for N-glycan analysis were compared using four monoclonal antibodies.While observing good comparability between the quantitative results generated,it was possible to appreciate the advantages and disadvantages of each technique and to summarise all the observations to guide the choice of the most appropriate analytical workflow according to application and the desired depth of data generated.

卵巢上皮性癌中inhibin及其受体β-glycan的表达

目的探讨α-inhibin及其受体β-glycan在恶性卵巢上皮性肿瘤中的表达,评估两种多肽信号对卵巢癌生长的调控作用,探讨卵巢癌的发病机制。方法应用EliVision方法检测30例正常卵巢组织、30例卵巢囊腺瘤和80例卵巢囊腺癌中α-in-hibin及其受体β-glycan的表达。结果α-inhibin在正常卵巢、卵巢囊腺瘤和卵巢囊腺癌中的总阳性表达率分别为93.3%、60.0%、36.3%,差异均有显著性(P<0.01);β-glycan在正常卵巢、卵巢囊腺瘤、卵巢囊腺癌的阳性表达率呈逐渐下降的趋势分别为100%、80.0%、75.0%,卵巢囊腺瘤、囊腺癌与正常卵巢组比较,差异有显著性(P<0.05),但卵巢囊腺瘤与囊腺癌比较,差异无显著性(P>0.05)。结论α-inhibin蛋白在卵巢囊腺癌中表达明显下降,而β-glycan在卵巢肿瘤中的表达仍然很高,提示β-glycan对增强α-inhibin抑制卵巢囊腺癌的形成可能起着一定的作用。

Recent progress in targeting the sialylated glycan-SIGLEC axis in cancer immunotherapy

Malignant tumors are complex structures composed of cancer cells and tumor microenvironmental cells.In this complex structure,cells cross-talk and interact,thus jointly promoting cancer development and metastasis.Recently,immunoregulatory molecule-based cancer immunotherapy has greatly improved treatment efficacy for solid cancers,thus enabling some patients to achieve persistent responses or cure.However,owing to the development of drug-resistance and the low response rate,immunotherapy against the available targets PD-1/PD-L1 or CTLA-4 has limited benefits.Although combination therapies have been proposed to enhance the response rate,severe adverse effects are observed.Thus,alternative immune checkpoints must be identified.The SIGLECs are a family of immunoregulatory receptors(known as glyco-immune checkpoints)discovered in recent years.This review systematically describes the molecular characteristics of the SIGLECs,and discusses recent progress in areas including synthetic ligands,monoclonal antibody inhibitors,and Chimeric antigen receptor T(CAR-T)cells,with a focus on available strategies for blocking the sialylated glycan-SIGLEC axis.Targeting glyco-immune checkpoints can expand the scope of immune checkpoints and provide multiple options for new drug development.

Serum N-glycan markers for diagnosing liver fibrosis induced by hepatitis B virus

BACKGROUND Hepatitis B virus (HBV) infection is the primary cause of hepatitis with chronic HBV infection,which may develop into liver fibrosis,cirrhosis and hepatocellular carcinoma.Detection of early-stage fibrosis related to HBV infection is of great clinical significance to block the progression of liver lesion.Direct liver biopsy is regarded as the gold standard to detect and assess fibrosis;however,this method is invasive and prone to clinical sampling error.In order to address these issues,we attempted to find more convenient and effective serum markers for detecting HBV-induced early-stage liver fibrosis.AIM To investigate serum N-glycan profiling related to HBV-induced liver fibrosis and verify multiparameter diagnostic models related to serum N-glycan changes.METHODS N-glycan profiles from the sera of 432 HBV-infected patients with liver fibrosis were analyzed.Significant changed N-glycan levels (peaks)(P <0.05) in differentfibrosis stages were selected in the modeling group,and multiparameter diagnostic models were established based on changed N-glycan levels by logistic regression analysis.The receiver operating characteristic (ROC) curve analysis was performed to evaluate diagnostic efficacy of N-glycans models.These models were then compared with the aspartate aminotransferase to platelet ratio index (APRI),fibrosis index based on the four factors (FIB-4),glutamyltranspeptidase platelet albumin index (S index),GlycoCirrho-test,and GlycoFibro-test.Furthermore,we combined multiparameter diagnostic models with alanine aminotransferase (ALT) and platelet (PLT) tests and compared their diagnostic power.In addition,the diagnostic accuracy of N-glycan models was also verified in the validation group of patients.RESULTS Multiparameter diagnostic models constructed based on N-glycan peak 1,3,4and 8 could distinguish between different stages of liver fibrosis.The area under ROC curves (AUROCs) of Model A and Model B were 0.890 and 0.752,respectively differentiating fibrosis F0-F1 from F2-F4,and F0-F2 from F3-F4,and surpassing other serum panels.However,AUROC (0.747) in Model C used for the diagnosis of F4 from F0-F3 was lower than AUROC (0.795) in FIB-4.In combination with ALT and PLT,the multiparameter models showed better diagnostic power (AUROC=0.912,0.829,0.885,respectively) when compared with other models.In the validation group,the AUROCs of the three combined models (0.929,0.858,and 0.867,respectively) were still satisfactory.We also applied the combined models to distinguish adjacent fibrosis stages of 432patients (F0-F1/F2/F3/F4),and the AUROCs were 0.917,0.720 and 0.785.CONCLUSION Multiparameter models based on serum N-glycans are effective supplementary markers to distinguish between adjacent fibrosis stages of patients caused by HBV,especially in combination with ALT and PLT.

Antitumor Active Protein-containing Glycans from the Body of Ganoderma tsugae

To explore the effects of traditional herbal medicine Ganoderrna tsugae（G, tsugae） on immunomodulatory and antitumor activities, the crude polysaccharides of G. tsugae were purified by filtration, diethylaminoethyl（DEAE） sepharose-fast flow chromatography and sephadex G-100 size-exclusion chromatography. Two main fractions, pro- tein-containing glyeans CSSLP-1 and CSSLP-2, were obtained via the gradient elution. The protein content, molecu- lar weight, and monosaccharide composition of the two fractions were analyzed. Furthermore, the influence of the protein-containing glycans from G. tsugae on the activation of human acute monocytic leukemia cell line（THP-l） and their antitumor activities to the human hepatocellular liver carcinoma cell（HepG-2） in vitro were evaluated. The re- sults indicate that CSSLP-1 and CSSLP-2 could increase the pinocytie activity of THP-1 cells and induce THP-1 cells to produce the eytokines of TNFa and IL-2, significantly. CSSLP-1 and CSSLP-2 also played an inhibiting effect on the cancer cell（HepG-2）. Moreover, the anti-proliferation activity of CSSLP-1 and CSSLP-2 increased with the par- ticipation of TNFa and IL-2 or other antitumor factors induced from THP-1 cells by G. tsugae protein-containing glycan fractions.

N-glycans released from glycoproteins using a commercial kit and comprehensively analyzed with a hypothetical database

The glycosylation of proteins is responsible for their structural and functional roles in many cellular activities.This work describes a strategy that combines an efficient release, labeling and liquid chromatography–mass spectral analysis with the use of a comprehensive database to analyze N-glycans. The analytical method described relies on a recently commercialized kit in which quick deglycosylation is followed by rapid labeling and cleanup of labeled glycans. This greatly improves the separation, mass spectrometry（MS） analysis and fluorescence detection of N-glycans. A hypothetical database, constructed using Glyc Resoft, provides all compositional possibilities of N-glycans based on the common sugar residues found in N-glycans. In the initial version this database contains ＆gt; 8,700 N-glycans, and is compatible with MS instrument software and expandable. N-glycans from four different well-studied glycoproteins were analyzed by this strategy. The results provided much more accurate and comprehensive data than that had been previously reported. This strategy was then used to analyze the N-glycans present on the membrane glycoproteins of gastric carcinoma cells with different degrees of differentiation. Accurate and comprehensive N-glycan data from those cells was obtained efficiently and their differences were compared corresponding to their differentiation states. Thus, the novel strategy developed greatly improves accuracy, efficiency and comprehensiveness of N-glycan analysis.

Chemoselective labeling-based spermatozoa glycan imaging reveals abnormal glycosylation in oligoasthenotspermia

Spermatogenesis, maturation, capacitation and fertilization are precisely regulated by glycosylation. However, the relationship between altered glycosylation patterns and the onset and development of reproductive disorders is unclear, mainly limited by the lack of in situ imaging techniques for spermatozoa glycosylation. We developed an efficient and highly specific spermatozoa glycan imaging technique based on the robust chemoselective labeling of sialic acid(Sia) and N-acetyl-D-galactosamine(Gal/GalNAc). We further proposed a “tandem glycan chemoselective labeling” strategy to achieve simultaneous imaging of two types of glycans on spermatozoa. We applied the developed method to the spermatozoa from oligozoospermic patients and diabetic mice and found that these spermatozoa showed higher levels of Sia and Gal/Gal NAc expression than the normal groups. Moreover, spermatozoa from diabetic mice showed a severe decrease in number, viability, and forward motility, suggesting that in vivo glucose metabolism disorders may lead to an elevated level of spermatozoa glycosylation and have a correlation with the development of oligoasthenotspermia. Our work provides a research tool to reveal the relationship between glycosylation modification and spermatozoa quality, and a promising clue for the development of glycan-based reproductive markers.

Immune regulatory networks coordinated by glycans and glycan-binding proteins in autoimmunity and infection

The immune system is coordinated by an intricate network of stimulatory and inhibitory circuits that regulate host responses against endogenous and exogenous insults.Disruption of these safeguard and homeostatic mechanisms can lead to unpredictable inflammatory and autoimmune responses,whereas deficiency of immune stimulatory pathways may orchestrate immunosuppressive programs that contribute to perpetuate chronic infections,but also influence cancer development and progression.Glycans have emerged as essential components of homeostatic circuits,acting as fine-tuners of immunological responses and potential molecular targets for manipulation of immune tolerance and activation in a wide range of pathologic settings.Cell surface glycans,present in cells,tissues and the extracellular matrix,have been proposed to serve as“self-associated molecular patterns”that store structurally relevant biological data.The responsibility of deciphering this information relies on different families of glycan-binding proteins(including galectins,siglecs and C-type lectins)which,upon recognition of specific carbohydrate structures,can recalibrate the magnitude,nature and fate of immune responses.This process is tightly regulated by the diversity of glycan structures and the establishment of multivalent interactions on cell surface receptors and the extracellular matrix.Here we review the spatiotemporal regulation of selected glycan-modifying processes including mannosylation,complex N-glycan branching,core 2 O-glycan elongation,LacNAc extension,as well as terminal sialylation and fucosylation.Moreover,we illustrate examples that highlight the contribution of these processes to the control of immune responses and their integration with canonical tolerogenic pathways.Finally,we discuss the power of glycans and glycan-binding proteins as a source of immunomodulatory signals that could be leveraged for the treatment of autoimmune inflammation and chronic infection.

Binding of the SARS-CoV-2 spike protein to glycans

The pandemic of the severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)has caused a high number of deaths in the world.To combat it,it is necessary to develop a better understanding of how the virus infects host cells.Infection normally starts with the attachment of the virus to cell-surface glycans like heparan sulfate(HS)and sialic acid-containing glycolipids/glycoproteins.In this study,we examined and compared the binding of the subunits and spike(S)proteins of SARS-CoV-2,SARS-Co V,and Middle East respiratory disease(MERS)-Co V to these glycans.Our results revealed that the S proteins and subunits can bind to HS in a sulfation-dependent manner and no binding with sialic acid residues was detected.Overall,this work suggests that HS binding may be a general mechanism for the attachment of these coronaviruses to host cells,and supports the potential importance of HS in infection and in the development of antiviral agents against these viruses.

Trimming of N-Glycans by the Golgi-Localized α-1,2-Mannosidases, MNS1 and MNS2, Is Crucial for Maintaining RSW2 Protein Abundance during Salt Stress in Arabidopsis

Asparagine （Asn/N）-Iinked glycans are important for protein folding, trafficking, and endoplasmic reticulum-associated degradation in eukaryotes. The maturation of glycoproteins involves the trimming of mannosyl residues by mannosidases and addition of other sugar molecules to three-branched N-glycans in the Golgi. However, the biological importance of Golgi-mediated mannose trimming is not fully understood. Here, we show that abolishment of two functionally redundant mannosidases, MNS1 and MNS2, responsible for α-1,2-mannose trimming on the A and C branches of plant N-glycans lead to severe root growth inhibition under salt stress conditions in Arabidopsis. In contrast, mutants with defects in the biosynthesis of the oligosaccharide precursor displayed enhanced salt tolerance in the absence of mannose trimming. However, mutation in EBS3, which is required for the formation of the branched N-glycan precursor, suppressed the salt-sensitive phenotype of mnsl mns2 double mutant. Interestingly, we observed that cellulose biosynthesis was compromised in mnsl mns2 roots under high salinity. Consistently, abundance of a membrane anchored endo-13-1,4-endoglucanase （RSW2/KOR） that plays a key role in cellulose biosynthesis and its mutant variant rsw2-1 were modulated by α-1,2-mannose trimming under salt stress. Overexpression of RSW2 could partially rescue the salt-sensitive phenotype of mnsl mns2. Taken together, these results suggest that MNS1/2-mediated mannose trimming of N-glycans is crucial in modulating glycoprotein abundance to withstand salt stress in plants.

Structural Basis of Glycan Recognition in Globally Predominant Human P[8]Rotavirus

Rotavirus(RV)causes acute gastroenteritis in infants and children worldwide.Recent studies showed that glycans such as histo-blood group antigens(HBGAs)function as cell attachment factors affecting RV host susceptibility and prevalence.P[8]is the predominant RV genotype in humans,but the structural basis of how P[8]RVs interact with glycan ligands remains elusive.In this study,we characterized the interactions between P[8]VP8~*s and glycans which showed that VP8~*,the RV glycan binding domain,recognized both mucin core 2 and H type 1 antigens according to the ELISA-based oligosaccharide binding assays.Importantly,we determined the structural basis of P[8]RV-glycans interaction from the crystal structures of a Rotateq P[8]VP8~*in complex with core 2 and H type 1 glycans at 1.82.3?,respectively,revealing a common binding pocket and similar binding mode.Structural and sequence analysis demonstrated that the glycan binding site is conserved among RVs in the P[Ⅱ]genogroup,while genotype-specific amino acid variations determined different glycan binding preference.Our data elucidated the detailed structural basis of the interactions between human P[8]RVs and different host glycan factors,shedding light on RV infection,epidemiology,and development of anti-viral agents.

Growth phase-dependent expression of proteins with decreased plant-specific N-glycans and immunogenicity in tobacco BY2 cells

Plants possess some desirable characteristics to synthesize recombinant glycoproteins for pharma-ceutical application.However,the mammalian glycoproteins produced in plants are somewhat different from their natural counterparts in terms of N-glycoforms.The immunogenicity of plant-specific glyco-epitopes is the major concern in human therapy.Here,the distribution of N-glycans in different growth phases of tobacco BY2 cells and their immunogenicity in mice were determined.It was ob-served that the percentage of β1,2-xylose and α1,3-fucose in proteins of growing cells increased and the corresponding protein extracts caused accelerated immune response in mice.Based on this observation,the recombinant erythropoietin in BY2 cells was expressed and characterized,and Western blot analysis showed that the recombinant erythropoietin contained a relatively small amount of plant-specific glyco-epitopes in the early phase of culture growth.This study may provide a simple but effective strategy for the production of therapeutic glycoproteins with human-like N-glycan structures in plant hosts to avoid a great allergenic risk.

Glycan targeting nanoparticle for photodynamic immunotherapy of melanoma

Interaction between tumour cells and macrophages enables cancer cells to evade immune detection and clearance by interfering with macrophage phagocytosis.The anti-phagocytic signals regulated by anti-phagocytic proteins are termed"don't eat me"signals;these signals include sialic acidbinding immunoglobulin-type lectin-10(Siglec-10)and the recently revealed CD24 immune checkpoint(ICP).In this study,we demonstrate that targeting a specific glycan on CD24 exhibits the potential to inhibit ICP.Sambucus nigra agglutinin(SNA),a sialic acid-binding lectin,was employed to block CD24 and to enhance phagocytosis in melanoma tumours.In addition,we prepared SNA-conjugated hollow gold-iron oxide nanoparticles for photothermal therapy of tumours.Our findings show that the combination treatment of SNA-conjugated photothermal nanoparticles and near-infrared exposure successfully augments tumour cell phagocytosis both in vitro and in vivo models.

Effects of N-Linked Glycan on Lassa Virus Envelope Glycoprotein Cleavage,Infectivity,and Immune Response

Lassa virus(LASV)belongs to the Mammarenavirus genus(family Arenaviridae)and causes severe hemorrhagic fever in humans.The glycoprotein complex(GPC)contains eleven N-linked glycans that play essential roles in GPC functionalities such as cleavage,transport,receptor recognition,epitope shielding,and immune response.We used three mutagenesis strategies(asparagine to glutamine,asparagine to alanine,and serine/tyrosine to alanine mutants)to abolish individual glycan chain on GPC and found that all the three strategies led to cleavage inefficiency on the 2nd(N89),5th(N119),or 8th(N365)glycosylation motif.To evaluate N to Q mutagenesis for further research,it was found that deletion of the 2nd(N89Q)or 8th(N365Q)glycan completely inhibited the transduction efficiency of pseudotyped particles.We further investigated the role of individual glycan on GPC-mediated immune response by DNA immunization of mice.Deletion of the individual 1st(N79Q),3rd(N99Q),5th(N119Q),or 6th(N167Q)glycan significantly enhanced the proportion of effector CD4+cells,whereas deletion of the 1st(N79Q),2nd(N89Q),3rd(N99Q),4th(N109Q),5th(N119Q),6th(N167Q),or 9th(N373Q)glycan enhanced the proportion of CD8+effector T cells.Deletion of specific glycan improves the Th1-type immune response,and abolishment of glycan on GPC generally increases the antibody titer to the glycan-deficient GPC.However,the antibodies from either the mutant or WT GPC-immunized mice show little neutralization effect on wild-type LASV.The glycan residues on GPC provide an immune shield for the virus,and thus represent a target for the design and development of a vaccine.

Two-step derivatization and mass spectral distinction of α2,3 and α2,6sialic acid linkages on N-glycans by MALDI-TOF

Sialylation is one of important glycosylation in human beings and plays an important role in cancer development. α2,3-Linked and α2,6-linked sialic acids are normally observed on the end of N-glycans and have different functions. Derivatization on sialic acid was designed to detect the different linkages by MALDI-TOF MS. In this study, a two-step derivatization by dimethylamine and ammonium hydroxide was improved to modify the sialic acid and made it easier to detect the different linkages of sialic acids on MALDI-TOF MS. Using this derivatization method, specific sialic acids linkages on N-glycans of protein samples such as fetuin and lactoferrin were detected. For complex cell samples, increased a2,3-linked and a2,6-linked sialic acids on bi-antennary and tri-antennary N-glycans were observed in A549 cells induced by hypoxia environment. Taken together, our two-step derivatization of sialic acids offers a simple and accurate way to detect specific linkages on N-glycans with MALDI-TOF mass spectrometer.

Automated Chemical Solid-Phase Synthesis of Glycans

Automated chemical solid-phase synthesis is an automation platform for rapid and reliable synthesis of glycans.Since the seminal work of Automated Glycan Assembly(AGA)disclosed by Seeberger in 2001,AGA has evolved from a proof-of-concept to a robust and reliable technology for streamlined production of various types of glycans.Through more than 20 years of unceasing efforts,the major breakthroughs in AGA including linkers,approved building blocks,and synthesizers have been acquired,and numerous influential achievements have been made in complex glycan synthesis.In addition,the HPLC-assisted automated synthesis emerges as a promising automation platform to access glycans.In this review,we highlight the key advances in the field of automated chemical solid-phase synthesis,especially in AGA.The synthesis of representative glycans based on AGA is also described.

Fluorescent glycan nanoparticle-based FACS assays for the identification of genuine drug-resistant cancer cells with differentiation potential

Herein we develop a unique differentiated-uptake strategy capable of efficient and high-purity isolation of genuine drug-resistant(DR)cells from three types of drug-surviving cancer cells,which include paclitaxel-surviving human ovarian OVCAR-3 cancer cells and human lung carcinoma A549/Taxol cells,and doxorubicin-surviving human immortalized myelogenous leukemia K562/ADR cells.By using this strategy which relies on fluorescent glycan nanoparticle(FGNP)-based fluorescence-activated cell sorting(FACS)assays,two subpopulations with distinct fluorescences existing in drug-surviving OVCAR-3 cells were separated,and we found that the lower fluorescence(LF)subpopulation consisted of DR cells,while the higher fluorescence(HF)subpopulation was comprised of non-DR cells.Besides,the DR cells and their progenies were found distinct in their increased expression of drug-resistant genes.More intriguingly,by using the FGNP-based FACS assay to detect DR/non-DR phenotypes,we found that the DR phenotype had a potential to differentiate into the non-DR progeny,which demonstrates the differentiation feature of stem-like cancer cells.Further research disclosed that the assay can quantitatively detect the degree of drug resistance in DR cells,as well as the reversal of drug resistance that are tackled by various therapeutic methods.The strategy thus paves the way to develop theranostic approaches associated with chemotherapy-resistance and cancer stemness.

O-glycan profiling of serum glycan for potential renal cancer biomarkers

Serum was obtained from 25 male renal cell carcinoma(RCC) patients and 21 healthy males.O-glycans were released by a-elimination reaction and purified by graphitized carbon cartridge solid phase extraction,then profiled by matrix-assisted laser desorption/ionisation-time of flight mass spectrometry.After noise removal and peak alignment,1372 peaks were extracted from 200000 data points.Feature peaks were analyzed by calculation of differential sensitivity and specificity.The combination of two feature peaks was chosen as a biomarker and could clearly differentiate RCC and normal samples in our study group.

Chemical approaches towards installation of rare functional groups in bacterial surface glycans

Bacterial surface glycans perform a diverse and important set of biological roles,and have been widely used in the treatment of bacterial infectious diseases.The majority of bacterial surface glycans are decorated with diverse rare functional groups,including amido,acetamidino,carboxamido and pyruvate groups.These functional groups are thought to be important constituents for the biological activities of glycans.Chemical synthesis of glycans bearing these functional groups or their variants is essential for the investigation of structure-activity relationships by a medicinal chemistry approach.To date,a broad choice of synthetic methods is available for targeting the different rare functional groups in bacterial surface glycans.This article reviews the structures of naturally occurring rare functional groups in bacterial surface glycans,and the chemical methods used for installation of these groups.