Recent studies demonstrated that mutations in B3GNT1, an enzyme proposed to be involved in poly-N-acetyllactosamine synthesis, were causal for congenital muscular dystrophy with hypoglycosylation of α-dystroglycan (secondary dystroglycanopathies). Since defects in the O-mannosylation protein glycosylation pathway are primarily responsible for dystroglycanopathies and with no established O-mannose initiated structures containing a β3 linked GlcNAc known, we biochemically interrogated this human enzyme. Here we report this enzyme is not a β-1,3-N-acetylglucosaminyltransferase with catalytic activity towards β-galactose but rather a β-1,4-glucuronyltransferase, designated B4GAT1, towards both α- and β-anomers of xylose. The dual-activity LARGE enzyme is capable of extending products of B4GAT1 and we provide experimental evidence that B4GAT1 is the priming enzyme for LARGE. Our results further define the functional O-mannosylated glycan structure and indicate that B4GAT1 is involved in the initiation of the LARGE-dependent repeating disaccharide that is necessary for extracellular matrix protein binding to O-mannosylated α-dystroglycan that is lacking in secondary dystroglycanopathies.
Dystroglycan is a protein that is essential for muscles to function correctly, and helps to connect the interior framework of muscle cells with the external matrix of molecules that hold the cells together in the tissue. As is the case for many proteins, dystroglycan must have particular carbohydrate molecules joined to it in order to work correctly. Enzymes called glycosyltransferases assist with the reactions that build the carbohydrates on a protein.
Mutations in multiple glycosyltransferases that add carbohydrates to dystroglycan can cause a group of diseases that are characterized by a progressive loss of muscle function, known as congenital muscular dystrophies. Praissman et al. use biochemical experimentation to investigate the role of one of these enzymes, known as B3GNT1. The enzyme's name is based on a code that describes which carbohydrate it helps to bind to proteins. However, Praissman et al. (and independently, Willer et al.) discovered that this enzyme actually works with a different donor and acceptor than previously thought, and so should be called B4GAT1 instead.
Praissman et al. propose that the B4GAT1 enzyme starts the process of forming the carbohydrate structures that help muscle cells bind to the muscle tissue matrix. B4GAT1 forms short carbohydrates on the surface of the part of dystroglycan that sits on the surface of cells. These carbohydrates are then extended into longer chains by another glycosyltransferase called LARGE. The results of Praissman et al. suggest that another enzyme is also involved in this process, which will require further studies to identify. Understanding the role of B4GAT1 and other glycosyltransferases that build functionally glycosylated dystroglycan could help to develop treatments for diseases such as muscular dystrophies.
congenital muscular dystrophy; O-mannosylation; glycosylation; alpha-dystroglycan; B4GAT1; B3GNT1; human
A simple two-step protocol for modification of atomic force microscopy tip and substrate by using a “click reaction” has been developed. The modified tip and substrate were applied to detect trace amounts of ricin by using single molecule recognition force microscopy (SMRFM1). A key feature of the approach is the use of a PEG derivative functionalized with a thiol and azide group end group. This compound could be attached to a gold coated AFM tip by a strong Au-thiol bond. The azide of the immobilized PEG was used for the attachment of an anti-ricin antibody modified with an alkyne group using a ‘click reaction’. The latter reaction is highly efficient, compatible with the presence of many functional groups and proceeds under mild reaction conditions. In a separate step, ricin was immobilized on the gold substrate surface that was modified by active esters. For this process, a novel bifunctional reagent was employed containing an active ester and a thioctic acid moiety. The SMRFM results showed a sub fg/mL level of detection sensitivity. The unbinding force between the anti-ricin antibody and ricin was studied by the force-distance curves (F-D). The unbinding force between the ricin and the antibody was determined to be 64.89±1.67 pN by constructing a force histogram.
Studies of post-translational modification by β-N-acetyl-D-glucosamine (O-GlcNAc) are hampered by a lack of efficient tools such as O-GlcNAc specific antibodies that can be employed for detection, isolation, and site localization. We have obtained a large panel of O-GlcNAc-specific IgG MAbs having a broad spectrum of binding partners by combining three-component immunogen methodology with hybridoma technology. Immunoprecipitation followed by large-scale shotgun proteomics led to the identification of more than 200 mammalian O-GlcNAc modified proteins, including a large number of novel glycoproteins. A substantial number of the glycoproteins were only enriched by one of the antibodies and this observation combined with results of inhibition ELISAs suggests that the antibodies in addition to their O-GlcNAc-dependence also appear to have different, but overlapping, local peptide determinants. The MAbs made it possible to delineate differentially modified proteins of liver in response to trauma-hemorrhage and resuscitation in a rat model.
O-GlcNAc; Immunogen; Glycopeptide; Proteomics; Post-translational Modification
The sugar nucleotide dTDP‐L‐rhamnose is critical for the biosynthesis of the Group A Carbohydrate, the molecular signature and virulence determinant of the human pathogen Group A S
treptococcus (GAS). The final step of the four‐step dTDP‐L‐rhamnose biosynthesis pathway is catalyzed by dTDP‐4‐dehydrorhamnose reductases (RmlD). RmlD from the Gram‐negative bacterium S
almonella is the only structurally characterized family member and requires metal‐dependent homo‐dimerization for enzymatic activity. Using a biochemical and structural biology approach, we demonstrate that the only RmlD homologue from GAS, previously renamed GacA, functions in a novel monomeric manner. Sequence analysis of 213 Gram‐negative and Gram‐positive RmlD homologues predicts that enzymes from all Gram‐positive species lack a dimerization motif and function as monomers. The enzymatic function of GacA was confirmed through heterologous expression of gac
A in a S. mutans rml
D knockout, which restored attenuated growth and aberrant cell division. Finally, analysis of a saturated mutant GAS library using Tn‐sequencing and generation of a conditional‐expression mutant identified gac
A as an essential gene for GAS. In conclusion, GacA is an essential monomeric enzyme in GAS and representative of monomeric RmlD enzymes in Gram‐positive bacteria and a subset of Gram‐negative bacteria. These results will help future screens for novel inhibitors of dTDP‐L‐rhamnose biosynthesis.
The chemical synthesis of an orthogonally protected trisaccharide derived from the polysaccharide of Staphylococcus aureus Type 5, which is an attractive candidate for the development of immunotherapies, is described. The challenging α-fucosylation and β-mannosylation are addressed through the careful choice of protecting groups. Lactamization of a β-d-ManpNAcA moiety during deprotection was avoided by a late stage oxidation approach. Versatility of the trisaccharide was demonstrated by its transformation into a spacer-containing repeating unit suitable for immunological investigations.
It remains challenging to produce decisive vaccines against MUC1, a tumor-associated antigen widely expressed by pancreas, breast and other tumors. Employing clinically relevant mouse models, we ruled out such causes as irreversible T-cell tolerance, inadequate avidity, and failure of T-cells to recognize aberrantly glycosylated tumor MUC1. Instead, every tested MUC1 preparation, even non-glycosylated synthetic 9mer peptides, induced interferon gamma-producing CD4+ and CD8+ T-cells that recognized glycosylated variants including tumor-associated MUC1. Vaccination with synthetic peptides conferred protection as long as vaccination was repeated post tumor challenge. Failure to revaccinate post challenge was associated with down-regulated tumor MUC1 and MHC molecules. Surprisingly, direct admixture of MUC1-expressing tumor with MUC1-hyperimmune T-cells could not prevent tumor outgrowth or MUC1 immunoediting, whereas ex vivo activation of the hyperimmune T-cells prior to tumor admixture rendered them curative. Therefore, surrogate T-cell preactivation outside the tumor bed, either in culture or by repetitive vaccination, can overcome tumor escape.
Heparan sulfate (HS) 3-O-sulfation determines
the binding specificity of HS/heparin for antithrombin III and plays
a key role in herpes simplex virus (HSV) infection. However, the low
natural abundance of HS 3-O-sulfation poses a serious
challenge for functional studies other than the two cases mentioned
above. By contrast, multiple distinct isoforms of 3-O-sulfotranserases exist in mammals (up to seven isoenzymes). Here
we describe a novel peeling reaction that specifically degrades HS
chains with 3-O-sulfated glucosamine at the reducing-end.
When HS/heparin is enzymatically depolymerized for compositional analysis,
3-O-sulfated glucosamine at the reducing ends appears
to be susceptible to degradation under mildly basic conditions. We
propose a 3-O-desulfation initiated peeling reaction
mechanism based on the intermediate and side-reaction products observed.
Our discovery calls for the re-evaluation of the natural abundance
and functions of HS 3-O-sulfation by taking into
consideration the negative impact of this novel peeling reaction.
A new anomeric linker has been developed that facilitates the purification of glycans prepared by chemoenzymatic approaches and can readily give compounds that are appropriately modified for microarray development or glycan derivatives with a free reducing end that are needed as standards for the development of analytical protocols.
The mucin MUC1 is overexpressed and aberrantly glycosylated by many epithelial cancer cells manifested by truncated O-linked saccharides. Although tumor-associated MUC1 has generated considerable attention because of its potential for the development of a therapeutic cancer vaccine, it has been difficult to design constructs that consistently induce cytotoxic T-lymphocytes (CTLs) and ADCC-mediating antibodies specific for the tumor form of MUC1. We have designed, chemically synthesized, and immunologically examined vaccine candidates each composed of a glycopeptide derived from MUC1, a promiscuous Thelper peptide, and a TLR2 (Pam3CysSK4) or TLR9 (CpG-ODN 1826) agonist. It was found that the Pam3CysSK4-containing compound elicits more potent antigenic and cellular immune responses, resulting in a therapeutic effect in a mouse model of mammary cancer. It is thus shown, for the first time, that the nature of an inbuilt adjuvant of a tripartite vaccine can significantly impact the quality of immune responses elicited against a tumor-associated glycopeptide. The unique adjuvant properties of Pam3CysSK4, which can reduce the suppressive function of regulatory T cells and enhance the cytotoxicity of tumor-specific CTLs, are likely responsible for the superior properties of the vaccine candidate 1.
adjuvants; cancer; carbohydrates; mucins; peptides; vaccines
Strain-promoted 1,3-dipolar cycloaddition of cyclooctynes with 1,3-dipoles such as azides, nitrones, and nitrile oxides, are of interest for the functionalization of polymers. In this study, we have explored the use of a 4-dibenzocyclooctynol (DIBO)-containing chain transfer agent in reversible addition–fragmentation chain transfer polymerizations. The controlled radical polymerization resulted in well-defined DIBO-terminating polymers that could be modified by 1,3-dipolar cycloadditions using nitrones, nitrile oxides, and azides having a hydrophilic moiety. The self-assembly properties of the resulting block copolymers have been examined. The versatility of the methodology was further demonstrated by the controlled preparation of gold nanoparticles coated with the DIBO-containing polymers to produce materials that can be further modified by strain-promoted cyclo-additions.
alkynes; cycloaddition; gold; nanoparticles; polymerization
click chemistry; glycosyltransferase; sialic acid; glycan remodeling; antibody
Although hundreds of heparan sulfate (HS) binding proteins have been implicated in a myriad of physiological and pathological processes, very little information is known about ligand requirements for binding and mediating biological activities by these proteins. We report here a streamlined approach for the preparation of modular disaccharide building blocks that will facilitate the assembly of libraries of HS oligosaccharides for structure-activity relationship studies. In particular, we have found that glucuronic acid donors, which usually perform poorly in glycosylations, can give high yields of coupling product when the C-2 hydroxyl is protected with a permanent 4-acetoxy-2,2-dimethyl butanoyl- (PivOAc) or temporary levulinoyl (Lev) ester and the C-4 hydroxyl modified with a selectively removable 2-methylnaphthyl (Nap) ether. It has been shown that the PivOAc ester can be removed without affecting sulfate esters making it an ideal protecting group for HS oligosaccharide assembly. Iduronic acid donors exhibit more favorable glycosyl donating properties and a compound protected with a Lev ester at C-2 and an Fmoc function at the C-4 hydroxyl gave coupling products in high yield. The new donors avoid post-glycosylation oxidation and therefore allow the facile preparation of modular disaccharide building blocks.
Selective derivatization of solvent-exposed cysteine residues in peptides and proteins is achieved by brief irradiation of an aqueous solution containing 3-(hydroxymethyl)-2-naphthol derivatives (NQMPs) with 350 nm fluorescent lamp. NQMP can be conjugated with various moieties, such as PEG, dyes, carbohydrates, or possess a fragment for further selective derivatization, e.g., biotin, azide, alkyne, etc. Attractive features of this labeling approach include an exceptionally fast rate of the reaction and a requirement for low equivalence of the reagent. The NQMP-thioether linkage is stable under ambient conditions, survives protein digestion and MS analysis. Irradiation of NQMP-labeled protein in a dilute solution (<40 μM) or in the presence of a vinyl ether results in a traceless release of the substrate. The reversible biotinylation of bovine serum albumin, as well as capture and release of this protein using NeutrAvidin Agarose resin beads has been demonstrated.
Dystroglycanopathies are a subgroup of muscular dystrophies that arise from defects in the enzymes implicated in the recently elucidated O-mannosylation pathway, resulting in underglycosylation of α-dystroglycan. The emerging identification of additional brain proteins modified by O-mannosylation provides a broader context for interpreting the range of neurological consequences associated with dystroglycanopathies. This form of glycosylation is associated with protein mucin-like domains which present numerous serine and threonine residues as possible sites for modification. Further, the O-Man glycans coexist in this region with O-GalNAc glycans, conventionally associated with such protein sequences, resulting in a complex glycoconjugate landscape. Sorting out the relationships between the various molecular defects in glycosylation and the modes of disease presentation, as well as the regulatory interplay among the O-Man glycans, and the effects on other modes of glycosylation in the same domain is challenging. Here we provide a perspective on chemical biology approaches employing synthetic and analytical methods to address these questions.
Glycopeptides; Carbohydrates; Muscular Dystrophy; α-Dystroglycan; Dystroglycanopathy; Protein O-Mannosylation
SEEL cell surface glycans
N-Glycans of living cells could be selectively tagged by exogenously administered recombinant ST6Gal I sialyltransferase and azido-modified CMP-Neu5Ac followed by a strain promoted cycloaddition using biotin modified dibenzylcyclooctynol (DIBO). The methodology will make it possible to dissect the mechanisms that underlie altered glycoconjugate recycling and storage in disease and identify the glycoconjugates whose cell surface localization or secretion are affected.
click chemistry; glycosyltransferase; N-glycans; sialic acid; trafficking
Major histocompatibility complex (MHC) class I and II are glycoproteins that can present antigenic peptides at the cell surface for recognition and activation of circulating T lymphocytes. Here, the importance of the modification of protein antigens by glycans on cellular uptake, proteolytic processing, presentation by MHC and subsequent T-cell priming is reviewed. Antigen glycosylation is important for a number of diseases and vaccine design. All of the key proteins involved in antigen recognition and the orchestration of downstream effector functions are glycosylated. The influence of protein glycosylation on immune function and disease is covered.
We report here a synthetic route to oxime, azide and nitrone-bearing copolymers via reversible addition-fragmentation chain transfer copolymerization of 4-vinylbenzaldehyde and 1-(chloromethyl)-4-vinylbenzene with styrene. The azide and nitrone moieties could be employed in strain-promoted 1,3-dipolar cycloadditions with various functionalized dibenzocyclooctynols (DIBO) for metal-free post-functionalization of the polymers. In situ oxidation of the oximes with hypervalent iodine gave nitrile oxides, which could also be employed as 1,3-dipoles for facile cycloadditions with DIBO derivatives. Kinetic measurements demonstrated that the pendant nitrile oxides reacted approximately twenty times faster compared to similar cycloadditions with azides. A block copolymer, containing azide and oxime groups in segregated blocks, served as a scaffold for attachment of hydrophobic and hydrophilic moieties by sequential strain-promoted alkyne-azide and strain-promoted alkyne-nitrile oxide cycloadditions. This sequential bi-functionalization approach made it possible to prepare in a controlled manner multi-functional polymers that could self-assemble into well-defined nanostructures.
grafting; click chemistry; SPAAC; SPANOC; SPANC
Glycoproteins expressed by Cryptosporidium parvum are
immunogenic in infected individuals but the nature of the epitopes recognised in
C. parvum glycoproteins is poorly understood. Since a known
immunodominant antigen of Cryptosporidium, the 17 kDa
glycoprotein, has previously been shown to bind to lectins that recognise the Tn
antigen (GalNAcα1-Ser/Thr-R), a large number of glycopeptides with
different Tn valency and presentation were prepared. In addition, glycopeptides
were synthesised based on a 40 kDa cryptosporidial antigen, a polymorphic
surface glycoprotein with varying numbers of serine residues, to determine the
reactivity with sera from C. parvum-infected humans. These
glycopeptides and non-glycosylated peptides were used to generate a glycopeptide
microarray to allow screening of sera from C. parvum-infected
individuals for the presence of IgM and IgG antibodies. IgG but not IgM in sera
from C. parvum-infected individuals bound to multivalent Tn
antigen epitopes presented on glycopep-tides, suggesting that glycoproteins from
C. parvum that contain the Tn antigen induce immune
responses upon infection. In addition, molecular differences in glycosylated
peptides (e.g. substituting Ser for Thr) as well as the site of glycosylation
had a pronounced effect on reactivity. Lastly, pooled sera from individuals
infected with either Toxoplasma or Plasmodium
were also tested against the modified Cryptosporidium peptides
and some sera showed specific binding to glycopeptide epitopes. These studies
reveal that specific anti-glycopeptide antibodies that recognise the Tn antigen
may be useful diagnostically and in defining the roles of parasite
glycoconjugates in infections.
Cryptosporidium parvum; Tn antigen; Glycopeptide microarrays; Toxoplasma; Plasmodium
A systematic, efficient means of producing diverse libraries of
asymmetrically branched N-glycans is needed to investigate the
specificities and biology of glycan binding proteins. To that end, we describe a
core pentasaccharide that at potential branching positions is modified by
orthogonal protecting groups to allow selective attachment of unique saccharide
moieties by chemical glycosylation. The appendages were selected in such a way
that the antenna of the resulting deprotected compounds could be selectively
extended by glycosyltransferases to give libraries of asymmetrical
multi-antennary glycans. The power of the methodology was demonstrated by the
preparation of a series of complex oligosaccharides that were printed as
microarrays and screened for binding to lectins and influenza-virus
hemagglutinins, which showed that recognition is modulated by presentation of
minimal epitopes in the context of complex N-glycans.
Inhibition of Golgi α-mannosidase II (GMII), which acts late in the N-glycan processing pathway, provides a route to blocking cancer-induced changes in cell surface oligosaccharide structures. To probe the substrate requirements of GMII, oligosaccharides were synthesized that contained an α(1,3)- or α(1,6)-linked 1-thiomannoside. Surprisingly, these oligosaccharides were not observed in X-ray crystal structures of native Drosophila GMII (dGMII). However, a mutant enzyme in which the catalytic nucleophilic aspartate was changed to alanine (D204A) allowed visualization of soaked oligosaccharides and led to the identification of the binding site for the α(1,3)-linked mannoside of the natural substrate. These studies also indicate that the conformational change of the bound mannoside to a high-energy B2,5 conformation is facilitated by steric hindrance from, and the formation of strong hydrogen bonds to, Asp204. The observation that 1-thio-linked mannosides are not well tolerated by the catalytic site of dGMII led to the synthesis of a pentasaccharide containing the α(1,6)-linked Man of the natural substrate and the β(1,2)-linked GlcNAc moiety proposed to be accommodated by the extended binding site of the enzyme. A cocrystal structure of this compound with the D204A enzyme revealed the molecular interactions with the β(1,2)-linked GlcNAc. The structure is consistent with the ~80-fold preference of dGMII for the cleavage of substrates containing a nonreducing β(1,2)-linked GlcNAc. By contrast, the lysosomal mannosidase lacks an equivalent GlcNAc binding site and kinetic analysis indicates oligomannoside substrates without non-reducing-terminal GlcNAc modifications are preferred, suggesting that selective inhibitors for GMII could exploit the additional binding specificity of the GlcNAc binding site.
Heparan sulfate (HS) glucosaminyl 3-O-sulfotranferases sulfate the C3-hydroxyl group of certain glucosamine residues on heparan sulfate. Six different 3-OST isoforms exist, each of which can sulfate very distinct glucosamine residues within the HS chain. Among these isoforms, 3-OST1 has been shown to play a role in generating ATIII-binding HS anticoagulants whereas 3-OST2, 3-OST3, 3-OST4 and 3OST-6 have been shown to play a vital role in generating gD-binding HS chains that permit the entry of herpes simplex virus type 1 into cells. 3-OST5 has been found to generate both ATIII- and gD-binding HS motifs. Previous studies have examined the substrate specificities of all the 3-OST isoforms using HS polysaccharides. However, very few studies have examined the contribution of the epimer configuration of neighboring uronic acid residues next to the target site to 3-OST action. In this study, we utilized a well-defined synthetic oligosaccharide library to examine the substrate specificity of 3-OST3a and compared it to 3-OST1. We found that both 3-OST1 and 3-OST3a preferentially sulfate the 6-O-sulfated, N-sulfoglucosamine when an adjacent iduronyl residue is located to its reducing side. On the other hand, 2-O-sulfation of this uronyl residue can inhibit the action of 3-OST3a on the target residue. The results reveal novel substrate sites for the enzyme actions of 3-OST3a. It is also evident that both these enzymes have promiscuous and overlapping actions that are differentially regulated by iduronyl 2-O-sulfation.
Structural data on mammalian proteins are often difficult to obtain by conventional NMR approaches because of an inability to produce samples with uniform isotope labeling in bacterial expression hosts. Proteins with sparse isotope labels can be produced in eukaryotic hosts by using isotope-labeled forms of specific amino acids, but structural analysis then requires information from experiments other than nuclear Overhauser effects. One source of alternate structural information is distance-dependent perturbation of spin relaxation times by nitroxide spin-labeled analogs of natural protein ligands. Here, we introduce spin-labeled analogs of sugar nucleotide donors for sialyltransferases, specifically, CMP-TEMPO (CMP-4-O-[2,2,6,6-tetramethylpiperidine-1-oxyl]) and CMP-4carboxyTEMPO (CMP-4-O-[4-carboxy-2,2,6,6-tetramethylpiperidinine-1-oxyl]). An ability to identify resonances from active site residues and produce distance constraints is illustrated on a 15N phenylalanine-labeled version of the structurally uncharacterized, α-2,6-linked sialyltransferase, ST6Gal I.
Mannostatin A is a potent inhibitor of the mannose trimming enzyme Golgi α-mannosidase II (GMII), which acts late in the N-glycan processing pathway. Inhibition of this enzyme provides a route to blocking the transformation–associated changes in cancer cell surface oligosaccharide structures. In this paper, we report on the synthesis of new Mannostatin derivatives and analyze their binding in the active site of Drosophila GMII by X-ray crystallography. The results indicate that the interaction with the backbone carbonyl of Arg876 is crucial to the high potency of the inhibitor, an effect enhanced by the hydrophobic interaction between the thiomethyl group and an aromatic pocket vicinal to the cleavage site. The various structures indicate that differences in the hydration of protein-ligand complexes are also important determinant of plasticity as well as selectivity of inhibitor binding.
The modular synthesis of heparan sulfate fragments is greatly facilitated by employing an anomeric aminopentyl linker protected by a benzyloxycarbonyl group modified by a perfluorodecyl tag, which made it possible to purify highly polar intermediates by fluorous solid phase extraction. This tagging methodology made it also possible to perform repeated glycosylations to drive reactions to completion.
Infection by Opisthorchis viverrini (liver fluke) is a major public health problem in southeastern Asia, resulting in hepatobiliary disease and cholangiocarcinoma. Fluke surface glycoconjugates are prominently presented to the host, thereby constituting a crucial immunological interface that can determine the parasite’s success in establishing infection. Therefore, N- and O-linked glycoprotein glycan profiles of the infective metacercarial stage and of the mature adult were investigated by nanospray ionization-linear ion trap mass spectrometry (NSI-MSn). Glycan immunogenicity was investigated by immunobloting with serum from infected humans. Metacercariae and adult parasites exhibit similar glycan diversity, although the prevalence of individual glycans and glycan classes varies by stage. The N-glycans of the metacercaria are mostly high mannose and monofucosylated, truncated-type oligosaccharides (62.7%), with the remainder processed to complex and hybrid type glycans (37.3%). The N-linked glycan profile of the adult is also dominated by high mannose and monofucosylated, truncated-type oligosaccharides (80.0%), with a smaller contribution from complex and hybrid type glycans (20.0%). At both stages, complex and hybrid type glycans are detected as mono-, bi-, tri-, or tetra-antennary structures. In metacercariae and adults, O-linked glycans are detected as mono- to pentasaccharides. The mucin type core 1 structure, Galβ1-3GalNAc, predominates in both stages but is less prevalent in the adult than in the metacercaria. Immunogenic recognition of liver fluke glycoproteins is reduced after deglycosylation but infected human serum was unable to recognize glycans released from peptides. Therefore, the most potent liver fluke antigenic epitopes are mixed determinants, comprised of glycan and polypeptide elements.
Liver fluke; Opisthorchis viverrini glycoproteins; N-linked glycans; O-linked glycans; Antigenic epitopes