Recombinant protein production and functional analysis of a M60-like-2 metallopeptidase enzyme from the carcinogenic liver fluke Opisthorchis viverrini.

Mucin plays a crucial role in safeguarding mucosal tissues by obstructing the translocation of microorganisms. Mucosal tissue-dwelling parasites must devise a strategy to surmount this mucin barrier in order to establish colonization. In a recent discovery, it was observed that the liver fluke Opisthorchis viverrini secretes two mucinases, namely Ov-M60-like-1 and Ov-M60-like-2. Ov-M60-like-1 was previously characterized. Here, we study the Ov-M60-like-2 by utilizing the wheat germ expression system to produce recombinant proteins and conducted a functional analysis of its enzymatic activity on bovine submaxillary mucin (BSM). Subsequently, we delved deeper into understanding the role of this enzyme in host-parasite interactions by evaluating its mucinase activity on mucins from the bile duct of O. viverrini-infected hamsters. Through successful production of recombinant proteins using the wheat germ expression system, we observed that this enzyme displayed mucinase activity over a wide pH range (pH 2 to pH 10) against BSM. Our investigations revealed it ability to digest mucin from the bile duct. These findings suggest that Ov-M60-like-2 possess a mucinase activity, together with Ov-M60-like-1, enabling the liver fluke to successful colonization of the host's bile duct.

Altered O-linked glycosylation in benign and malignant meningiomas.

Background: Changes in protein glycosylation have been reported in various diseases, including cancer; however, the consequences of altered glycosylation in meningiomas remains undefined. We established two benign meningioma cell lines-SUT-MG12 and SUT-MG14, WHO grade I-and demonstrated the glycan and glycosyltransferase profiles of the mucin-type O-linked glycosylation in the primary benign meningioma cells compared with two malignant meningioma cell lines-HKBMM and IOMM-Lee, WHO grade III. Changes in O-linked glycosylation profiles in malignant meningiomas were proposed. Methods: Primary culture technique, morphological analysis, and immunocytochemistry were used to establish and characterize two benign meningioma cell lines. The glycan profiles of the primary benign and malignant meningiomas cell lines were then analyzed using lectin cytochemistry. The gene expression of O-linked glycosyltransferases, mucins, sialyltransferases, and fucosyltransferases were analyzed in benign and malignant meningioma using the GEO database (GEO series GSE16581) and quantitative-PCR (qPCR). Results: Lectin cytochemistry revealed that the terminal galactose (Gal) and N-acetyl galactosamine (GalNAc) were highly expressed in primary benign meningioma cells (WHO grade I) compared to malignant meningioma cell lines (WHO grade III). The expression profile of mucin types O-glycosyltransferases in meningiomas were observed through the GEO database and gene expression experiment in meningioma cell lines. In the GEO database, C1GALT1-specific chaperone (COSMC) and mucin 1 (MUC1) were significantly increased in malignant meningiomas (Grade II and III) compared with benign meningiomas (Grade I). Meanwhile, in the cell lines, Core 2 ß1,6-N-acetylglucosaminyltransferase-2 (C2GNT2) was highly expressed in malignant meningiomas. We then investigated the complex mucin-type O-glycans structures by determination of sialyltransferases and fucosyltransferases. We found ST3 ß-galactoside α-2,3-sialyltransferase 4 (ST3GAL4) was significantly decreased in the GEO database, while ST3GAL1, ST3GAL3, α1,3 fucosyltransferases 1 and 8 (FUT1 and FUT8) were highly expressed in malignant meningioma cell lines-(HKBMM)-compared to primary benign meningioma cells-(SUT-MG12 and SUT-MG14). Conclusion: Our findings are the first to demonstrate the potential glycosylation changes in the O-linked glycans of malignant meningiomas compared with benign meningiomas, which may play an essential role in the progression, tumorigenesis, and malignancy of meningiomas.

Piperlongumine and bortezomib synergically inhibit cholangiocarcinoma via ER stress-induced cell death.

Cholangiocarcinoma (CCA) is a lethal malignancy of the cholangiocytes lining the biliary tree. Only 25% of affected patients are eligible for resection due to late-stage diagnosis. Systemic chemotherapy is recommended for those inoperable patients; however, an inadequate response to such treatment remains a significant obstacle. Piperlongumine (PL) is a biologically active alkaloid that selectively kills various cancer cells through the induction of reactive oxygen species (ROS). The role of PL has been shown through its inhibiting the ubiquitin-proteasome system. The mechanism of PL-induced CCA cell death was investigated by inhibiting the UPS and testing the therapeutic potential of combining PL and the proteasome inhibitor bortezomib. A single treatment with PL or BTZ suppressed CCA cell growth. Combined treatment with PL with BTZ produced a synergistic interaction, evidenced by (1) a combination index of < 1 and (2) induction of cell cycle arrest and down-regulation of cell cycle markers. PL induced the accumulation of poly-ubiquitinated proteins in CCA cells but did not affect proteasome activity. PL, in combination with BTZ, amplified the accumulation of poly-ubiquitinated proteins in CCA cells, leading to an endoplasmic reticulum (ER) stress response through the induction of X-box binding protein mRNA splicing. Moreover, PL-combined BTZ promoted the activation of a proapoptotic unfolded protein response via the ATF4-CHOP axis. PL induced CCA cell death via increased accumulation of the poly-ubiquitinated proteins. PL also enhanced the anti-cancer activity of BTZ via ER stress-induced CCA cell death. Thus, the combination of PL and BTZ has potential as an alternative therapeutic option for CCA.

Inhibition of Ceramide Glycosylation Enhances Cisplatin Sensitivity in Cholangiocarcinoma by Limiting the Activation of the ERK Signaling Pathway.

Cholangiocarcinoma (CCA) is an aggressive tumor of the biliary epithelium with poor survival that shows limited response to conventional chemotherapy. Increased expression of glucosylceramide synthase (GCS) contributes to drug resistance and the progression of various cancers; the expression profiles of GCS (UGCG) and the genes for glucocerebrosidases 1, 2, and 3 (GBA1, GBA2, and GBA3) were therefore studied in CCA. The biological functions of GCS for cell proliferation and cisplatin sensitivity in CCA were explored. GCS expression was higher in CCA tumor tissues than that of GBA1, GBA2, and GBA3. Verification of GCS expression in 29 paired frozen CCA tissues showed that 8 of 29 cases (27.6%) had high GCS expression. The expression of GCS and GBA2 was induced in CCA cell lines following low-dose cisplatin treatment. Suppression of GCS by either palmitoylamino-3-morpholino-1-propanol (PPMP), GCS knockdown or a combination of the two resulted in reduced cell proliferation. These treatments enhanced the effect of cisplatin-induced CCA cell death, increased the expression of apoptotic proteins and reduced phosphorylation of ERK upon cisplatin treatment. Taken together, inhibition of the GCS increased cisplatin-induced CCA apoptosis via the inhibition of the ERK signaling pathway. Thus, targeting GCS might be a strategy for CCA treatment.

Down-Regulation of C1GALT1 Enhances the Progression of Cholangiocarcinoma through Activation of AKT/ERK Signaling Pathways.

Alteration of mucin-type O-glycosylation is implicated in tumor progression and metastasis of cholangiocarcinoma (CCA). Core 1 ß1-3 Galactosyltransferase (C1GALT1) is a primary enzyme that regulates the elongation of core 1-derived mucin-type O-glycans. Dysregulation of C1GALT1 has been documented in multiple cancers and is associated with aberrant core 1 O-glycosylation and cancer aggressiveness; however, the expression of C1GALT1 and its role in CCA progression remains unknown. Our study demonstrated that C1GALT1 was downregulated in CCA tissues at both the mRNA and protein levels. The biological function of C1GALT1 using siRNA demonstrated that suppression of C1GALT1 in the CCA cell lines (KKU-055 and KKU-100) increased CCA progression, evidenced by: (i) Induction of CCA cell proliferation and 5-fluorouracil resistance in a dose-dependent manner; (ii) up-regulation of growth-related genes, ABC transporter genes, and anti-apoptotic proteins; and (iii) an increase in the activation/phosphorylation of AKT and ERK in silencing C1GALT1 cells. We demonstrated that silencing C1GALT1 in CCA cell lines was associated with immature core 1 O-glycosylation, demonstrated by high expression of VVL-binding glycans and down-regulation of other main O-linked glycosyltransferases ß1,3-N-acetylglucosaminyltransferase 6 (B3GNT6) and ST6 N-Acetylgalactosaminide Alpha-2,6-Sialyltransferase 1 (ST6GALNAC1) in C1GALT1 knockdown. Our findings demonstrate that down-regulation of C1GALT1 in CCA increases the expression of immature core 1 O-glycan, enhancing CCA progression, including growth and 5-fluorouracil resistance via the activation of the AKT/ERK signaling pathway.

RNF43 overexpression attenuates the Wnt/ß-catenin signalling pathway to suppress tumour progression in cholangiocarcinoma.

RING finger protein 43 (RNF43) is a ubiquitin E3 ligase that negatively regulates Wnt/ß-catenin signalling. Mutation, inactivation and downregulation of RNF43 in cholangiocarcinoma (CCA) are associated with a less favourable prognosis. Since the functional role of RNF43 in CCA has not yet been demonstrated, the present study aimed to assess the effect of its overexpression in mediating CCA suppression via Wnt/ß-catenin signalling pathway inhibition. Accordingly, RNF43 was overexpressed, and various malignant phenotypic changes studied, including cell proliferation, cell migration, chemotherapeutic sensitivity and the expression of several Wnt/ß-catenin target genes. Overexpression of RNF43 in the CCA cell-line KKU-213B hindered activation of Wnt/ß-catenin signalling, evidenced by: i) Accumulation of ß-catenin in the cytoplasmic fraction and downregulation of several known Wnt target genes at the mRNA level [AXIN2, survivin (BIRC5), CCND1, MMP-7, c-MYC and ABCB1 (MDR1)]; ii) a reduction of cell proliferation; iii) a significant decrease in KKU-213B cell migration with RNF43 overexpression via upregulation of E-cadherin (CDH1); and iv) a reduction in N-cadherin (CDH2), MMP-2, MMP-7 and MMP-9. In addition, overexpression of RNF43 increased 5-fluorouracil sensitivity and downregulation of ABC transporter genes [including ABCB1 and ABCC1 (MRP1)]. The current results demonstrate a functional role for RNF43 in CCA by: i) Blocking ß-catenin nuclear translocation; and ii) the subsequent downregulation of Wnt/ß-catenin target genes (the latter being involved in the progression of CCA and chemotherapeutic drug susceptibility). Therefore, the present findings suggest that RNF43 could serve a tumour suppressive role in CCA.

High expression of tissue O-linked glycans is associated with a malignant phenotype of cholangiocarcinoma.

OBJECTIVE: This study aimed to investigate the expression of O-linked glycoprotein glycans in tissue of patients with cholangiocarcinoma compared with adjacent normal tissue. METHODS: Sixty patients with cholangiocarcinoma were included in the study. Permethylated O-linked glycans from intrahepatic cholangiocarcinoma tissue and adjacent normal tissue were analyzed using nano-spray ionization-linear ion trap mass spectrometry. Histochemistry of peanut agglutinin lectin was used for detection and localization of galactose (Gal) 1, N-acetyl-galactosamine (GalNAc) 1. RESULTS: O-linked glycans from patients with cholangiocarcinoma were composed of di- to hexa-saccharides with a terminal galactose and sialic acids (N-acetylneuraminic acid [NeuAc]). A total of eight O-linked glycan structures were detected. Gal1GalNAc1 and Gal2 N-acetyl-glucosamine 1 GalNAc1 expression was significantly higher in tissue from patients with cholangiocarcinoma compared with adjacent normal tissue, while NeuAc1Gal1GalNAc1 expression was significantly lower. High Gal1GalNAc1 expression was significantly associated with the late stage of cholangiocarcinoma (stages II-IV), lymphatic invasion, and vascular invasion. CONCLUSION: Our study shows expression of O-linked glycans in progression of cholangiocarcinoma and highlights the association of Gal1GalNAc1 with lymphatic and vascular invasion of cholangiocarcinoma.

Enhancement of piperlongumine chemosensitivity by silencing heme oxygenase-1 expression in cholangiocarcinoma cell lines.

Piperlongumine (PL) produces reactive oxygen species (ROS) and induces G2/M-phase arrest in cholangiocarcinoma (CCA) cells via the JNK/ERK pathway. A differential response to PL was observed among all CCA cell lines However, the underlying mechanisms have remained to be fully elucidated. The aim of the present study was to investigate the molecular mechanisms of PL-induced heme oxygenase-1 (HO-1) expression in CCA cell lines. The anti-proliferative action of PL in the CCA cell lines KKU-100 and KKU-213A was analyzed using sulforhodamine B assays. Reverse transcription-quantitative PCR and western blot analyses were used to examine mRNA and protein expression. HO-1 inhibition was achieved using the chemical inhibitor zinc protophoryn or specific small interfering RNA to HO-1. Intracellular ROS was detected using a 2,7-dichlorodihydrofluorescein diacetate fluorescence assay. High expression of phase-II detoxification enzymes, including NADPH quinone oxidoreductase-1, heme oxygenase-1, superoxide dismutases and aldo-keto reductase 1 subunits C-1 and 3, were detected in the KKU-100 cell line. Of the CCA cell lines tested, KKU-100 was the least sensitive to PL. Dose-dependent upregulation of HO-1 expression via PI3K/Akt activation was detected in PL-treated CCA cells. Inhibition of HO-1 eliminated the antioxidant defense mechanisms, leading to increased anti-cancer activity of PL in the CCA cell lines via an increase in intracellular ROS levels and apoptotic protein expression. These observations indicated that HO-1 inhibition had a chemosensitizing effect on CCA to PL.

Ganglioside GM2: a potential biomarker for cholangiocarcinoma.

OBJECTIVE: To investigate the expression of glycosphingolipids in serum and tissue from patients with cholangiocarcinoma compared with healthy controls. METHODS: Nanospray ionization-linear ion trap mass spectrometry (NSI-MSn) was used to demonstrate the comparative structural glycomics of glycosphingolipids in serum from patients with cholangiocarcinoma (n=15), compared with healthy controls (n = 15). GM2 expression in cholangiocarcinoma tissues (n = 60) was evaluated by immunohistochemistry. RESULTS: Eleven glycosphingolipids were detected by NSI-MSn: CMH (ceramide monohexose), Lac-Cer (galactose (Gal)ß1-4 glucose (Glc)ß1-1'-ceramide), Gb3 (Galα1-4Galß1-4Glcß1-1'-ceramide), Gb4/Lc4 (N-acetylgalactosamine (GalNAc)ß1-3Galα1-4Galß1-4Glcß1-1'-ceramide/Galß1-4 N-acetylglucosamine (GlcNAc)ß1-3Galß1-4Glcß1-1'-ceramide), GM3 (N-acetylneuraminic acid (NeuAc)2-3Galß1-4Glcß1-1'-ceramide), GM2 (GalNAcß1-4[NeuAc2-3]Galß1-4Glcß1-1'-ceramide), GM1 (Galß1-3GalNAcß1-4[NeuAc2-3]Galß1-4Glcß1-1'-ceramide), hFA (hydroxylated fatty acid)-CMH, hFA-Lac-Cer, hFA-Gb3, and hFA-GM3. Lac-Cer was the most abundant structure among the lactosides and globosides (normal, 24.40% ± 0.11%; tumor, 24.61% ± 2.10%), while GM3 predominated among the gangliosides (normal, 29.14% ± 1.31%; tumor, 30.53% ± 4.04%). The two glycosphingolipids that significantly differed between healthy controls and patients with cholangiocarcinoma were Gb3 and GM2. High expression of GM2 was associated with vascular invasion in tissue from patients with cholangiocarcinoma. CONCLUSIONS: Altered expression of glycosphingolipids in tissue and serum from patients with cholangiocarcinoma may contribute to tumor growth and progression. The ganglioside GM2, which significantly increased in the serum of patients with cholangiocarcinoma, represents a promising target as a biomarker for cholangiocarcinoma.

Increased expression of the high-mannose M6N2 and NeuAc3H3N3M3N2F tri-antennary N-glycans in cholangiocarcinoma.

Changes in protein glycosylation have been reported in various types of cancer, including cholangiocarcinoma (CCA). Nanospray ionization-linear ion trap mass spectrometry (NSI-MS n ) was used in the present study to determine the comparative structural glycomics of the N-linked glycans in the serum of patients with CCA compared with healthy controls. A total of 5 high-mannose and 4 complex N-linked glycans were detected. Mannose7-N-acetyl-glucosamine2 was the most abundant structure among the high-mannose types (control 12.12±2.54 vs. CCA 9.27±2.66%), whereas NeuAc2H2N2M3N2 predominated the complex types (control 61.17±2.55 vs. CCA 64.68±4.23%). The expression of 3 different N-glycans differed significantly between the CCA cases and controls. These included mannose6-N-acetyl-glucosamine2 (P=0.044), mannose9-N-acetyl-glucosamine2 (Ρ=0.030) and NeuAc3H3N3M3N2F (Ρ=0.002). These three glycan structures may therefore be associated with tumor progression in CCA and may be useful for its diagnosis.

Genetic polymorphisms in TLR1, TLR2, TLR4, and TLR10 of Helicobacter pylori-associated gastritis: a prospective cross-sectional study in Thailand.

The toll-like receptors (TLRs) mediate the recognition of Helicobacter pylori and initiate the innate immune response to infection. We hypothesized those genetic polymorphisms in the TLR1, TLR2, TLR4, and TLR10 influence bacterial infection, affecting susceptibility H. pylori to disease outcomes. Genomic DNA was extracted and genotypes of TLR1 (rs4833095), TLR2 (rs3804099 and rs3804100), TLR4 (rs10759932), and TLR10 (rs10004195) polymorphism were detected by the TagMan single-nucleotide epolymorphisms genotyping assay using the real-time PCR hybridization probe method. The TLR1 (rs4833095), C allele and the TLR10 (rs10004195), A allele frequency was significantly increased risk in the H. pylori infection group (odds ratio=1.76, 95% confidence interval=1.84-2.15, P=0.01 and odds ratio=1.81, 95% confidence interval=1.18-3.26, P=0.04, respectively). The TLR1 (rs4833095), C allele and TLR10 (rs10004195), A allele are susceptible TLRs polymorphisms in the Thai population. These findings suggest that TLR1 rs4833095 and TLR10 rs10004195 may play crucial roles in H. pylori susceptibility and gastric pathogenesis.

Differential Expression of O-glycoprotein Glycans in Cholangiocarcinoma Cell Lines.

Protein glycosylation is the most common posttranslational modification in mammalian cells. Aberrant protein glycosylation has been reported in various diseases, including cancer. We identified and quantified the glycan structures of O-linked glycoprotein from cholangiocarcinoma (CCA) cell lines from different histological types and compared their profiles by nanospray ionization-linear ion trap mass spectrometry (NSI-MSn). Five human CCA cell lines, K100, M055, M139, M213 and M214 were characterized. The results showed that the O-linked glycans of the CCA cell lines comprised tri- to hexa-saccharides with terminal galactose and sialic acids: NeuAc1Gal1GalNAc1, Gal2GlcNAc1GalNAc1, NeuAc2Gal1GalNAc1 NeuAc1Gal2GlcNAc1GalNAc1 and NeuAc2Gal2GlcNAc1GalNAc1 All five CCA cell lines showed a similar glycan pattern, but with differences in their quantities. NeuAc1Gal1GalNAc1 proved to be the most abundant structure in poorly differentiated adenocarcinoma (K100; 57.1%), moderately differentiated adenocarcinoma (M055; 42.6%) and squamous cell carcinoma (M139; 43.0%), while moderately to poorly differentiated adenocarcinoma (M214; 40.1%) and adenosquamous cell carcinoma (M213; 34.7%) appeared dominated by NeuAc2Gal1GalNAc1. These results demonstrate differential expression of the O-linked glycans in the different histological types of CCA. All five CCA cell lines have abundant terminal sialic acid (NeuAc) O-linked glycans, suggesting an important role for sialic acid in cancer cells. Our structural analyses of glycans may provide important information regarding physiology of disease-related glycoproteins in CCA.

Ring finger protein 43 expression is associated with genetic alteration status and poor prognosis among patients with intrahepatic cholangiocarcinoma.

Ring finger E3 ligases have roles in processes central to maintenance of genomic integrity and cellular homeostasis. Many ring finger E3 ligases are implicated in malignancy. Ring finger protein 43 (RNF43) is a ring finger E3 ligase that negatively regulates the Wnt/ß-catenin signaling pathway. RNF43 is frequently mutated in several types of malignancy, including intrahepatic cholangiocarcinoma (ICC). The significance of its expression in ICC has not, however, been reported. We determined RNF43 expression and identified RNF43 polymorphisms in ICC tissues. We also investigated the correlation between RNF43 expression and RNF43 mutation status, RNF43 polymorphisms, clinicopathological features, and prognosis of ICC patients. RNF43 reduced expression in ICC, and the reduction of RNF43 messenger RNA expression was significantly correlated with the presence of rs2257205 and RNF43 somatic mutations, confirming that all RNF43 somatic mutations in ICC are inactivating. Overall survival was worst in patients with down-regulation of RNF43. Univariate and multivariate analyses revealed that RNF43 expression was an independent prognostic factor. There was no statistically significant association between RNF43 messenger RNA and protein expression nor any clinicopathological features or RNF43 polymorphisms. The results imply that RNF43 is down-regulated in ICC and may play a crucial role during development of ICC.

Correlation between Patterns of Mdm2 SNIP 309 and Histopathological Severity of Helicobacter pylori Associated Gastritis in Thailand.

BACKGROUND: The commonly held view of the tumor suppressor p53 is as a regulator of cell proliferation, apoptosis and many other biological processes as well as external and internal stress responses. Mdm2 SNIP309 is a negative regulator of p 53. Therefore, this study aimed to determine the correlation between the patterns of Mdm2 SNIP 309 and the inflammation grading of Helicobacter pylori associated gastritis in a Thai population. MATERIALS AND METHODS: A cross-sectional study was carried out from November 2014 through June 2015. Biopsy specimens were obtained from infected patients and infection was proved by positive histology. The gastric mucosa specimens were sent to the Molecular Genetic Unit, Institute of Medicine, Suranaree University of Technology where they were tested by molecular methods to detect the patterns of Mdm2 SNIP 309 using the real-time PCR hybridization probe method. The results were analyzed and compared with the Updated Sydney classification. RESULTS: A total of 100 infected patients were interviewed and gastric mucosa specimens were collected. In this study the percentage of Mdm2 SNIP 309 T/T homozygous and Mdm2 SNIP309 G/T heterozygous was 78% and 19 % respectively whereas Mdm2 SNIP309 G/G homozygous was 3%. Mdm2 SNIP 309 T/T homozygous and Mdm2 SNIP309 G/T heterozygous correlated with mild to moderate inflammation (P<0.01) whereas Mdm2 SNIP309 G/G homozygous correlated with severe inflammation (P<0.01). CONCLUSIONS: Our study found the frequency of Mdm2 SNP309 G/G in our Thai population to be very low, and suggests that this can explain to some extent the low incidence of severe inflammation and gastric cancer changes in the Thai population. Mild to moderate inflammation are the most common pathologic gradings due to the unique genetic polymorphism of Mdm2 SNIP 309 in the Thai population.

A serotonin-induced N-glycan switch regulates platelet aggregation.

Serotonin (5-HT) is a multifunctional signaling molecule that plays different roles in a concentration-dependent manner. We demonstrated that elevated levels of plasma 5-HT accelerate platelet aggregation resulting in a hypercoagulable state in which the platelet surface becomes occupied by several glycoproteins. Here we study the novel hypothesis that an elevated level of plasma 5-HT results in modification of the content of N-glycans on the platelet surface and this abnormality is associated with platelet aggregation. Mass spectrometry of total surface glycoproteins on platelets isolated from wild-type mice infused for 24 hours with saline or 5-HT revealed that the content of glycoproteins on platelets from 5-HT-infused mice switched from predominantly N-acetyl-neuraminic acid (Neu5Ac) to N-glycolyl-neuraminic acid (Neu5Gc). Cytidine monophosphate-N-acetylneuraminate hydroxylase (CMAH) synthesizes Neu5Gc from Neu5Ac. Up-regulation of Neu5Gc content on the platelet surface resulted from an increase in the catalytic function, not expression, of CMAH in platelets of 5-HT-infused mice. The highest level of Neu5Gc was observed in platelets of 5-HT-infused, 5-HT transporter-knock out mice, suggesting that the surface delineated 5-HT receptor on platelets may promote CMAH catalytic activity. These new findings link elevated levels of plasma 5-HT to altered platelet N-glycan content, a previously unrecognized abnormality that may favor platelet aggregation.

Stage-specific expression and antigenicity of glycoprotein glycans isolated from the human liver fluke, Opisthorchis viverrini.

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 ionisation-linear ion trap mass spectrometry (NSI-MS(n)). Glycan immunogenicity was investigated by immunoblotting 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, Galß1-3GalNAc, 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 recognise glycans released from peptides. Therefore, the most potent liver fluke antigenic epitopes are mixed determinants, comprised of glycan and polypeptide elements.