N-glycan structures of Wisteria floribunda agglutinin-positive Mac2 binding protein in the serum of patients with liver fibrosis†.

The extent of liver fibrosis predicts prognosis and is important for determining treatment strategies for chronic hepatitis. During the fibrosis progression, serum levels of Mac2 binding protein (M2BP) increase and the N-glycan structure changes to enable binding to Wisteria floribunda agglutinin (WFA) lectin. As a novel diagnostic marker, glycosylation isomer of M2BP (M2BPGi) has been developed. However, its glycan structures recognized by WFA are unclear. In this study, we analyzed site-specific N-glycan structures of serum M2BP using Glyco-RIDGE (Glycan heterogeneity-based Relational IDentification of Glycopeptide signals on Elution profile) method. We evaluated five sample types: (1) M2BP immunoprecipitated from normal healthy sera (NHS-IP(+)), (2) M2BP immunoprecipitated from sera of patients with liver cirrhosis (stage 4; F4-IP(+)), (3) M2BP captured with WFA from serum of patients with liver cirrhosis (stage 4; F4-WFA(+)), (4) recombinant M2BP produced by HEK293 cells (rM2BP) and (5) WFA-captured rM2BP (rM2BP-WFA(+)). In NHS-IP(+) M2BP, bi-antennary N-glycan was the main structure, and LacNAc extended to its branches. In F4-IP(+) M2BP, many branched structures, including tri-antennary and tetra-antennary N-glycans, were found. F4-WFA(+) showed a remarkable increase in branched structures relative to the quantity before enrichment. In recombinant M2BP, both no sialylated-LacdiNAc and -branched LacNAc structures were emerged. The LacdiNAc structure was not found in serum M2BP. Glycosidase-assisted HISCL assays suggest that reactivity with WFA of both serum and recombinant M2BP depends on unsialylated and branched LacNAc and in part of recombinant depends on LacdiNAc. On M2BPGi, the highly branched LacNAc, probably dense cluster of LacNAc, would be recognized by WFA.

Study of glycosylation of prostate-specific antigen secreted by cancer tissue-originated spheroids reveals new candidates for prostate cancer detection.

Prostate-specific antigen (PSA) is the most frequently used biomarker for the screening of prostate cancer. Understanding the structure of cancer-specific glycans can help us improve PSA assay. In the present study, we analysed the glycans of PSA obtained from culture medium containing cancer tissue-originated spheroids (CTOS) which have similar characteristics as that of the parent tumour to explore the new candidates for cancer-related glycoforms of PSA. The glycan profile of PSA from CTOS was determined by comparing with PSA from normal seminal plasma and cancer cell lines (LNCaP and 22Rv1) using lectin chromatography and mass spectrometry. PSA from CTOS was mostly sialylated and the content of Wisteria floribunda agglutinin reactive glycan (LacdiNAc) was similar to that of PSA derived from seminal plasma and 22Rv1. Conversely, concanavalin A (Con A)-unbound PSA was definitely detected from the three cancer origins but was almost negligible in seminal PSA. Two novel types of PSA were elucidated in the Con A-unbound fraction: one is a high molecular weight PSA with highly branched N-glycans, and the other is a low molecular weight PSA without N-glycans. Furthermore, the existence of Lewis X antigen group on PSA was indicated. These PSAs will be candidates for new cancer-related markers.

Quantitative changes in perineuronal nets in development and posttraumatic condition.

Perineuronal net (PNN) is a highly structured portion of the CNS extracellular matrix (ECM) regulating synaptic plasticity and a range of pathologic conditions including posttraumatic regeneration and epilepsy. Here we studied Wisteria floribunda agglutinin-stained histological sections to quantify the PNN size and enrichment of chondroitin sulfates in mouse brain and spinal cord. Somatosensory cortex sections were examined during the period of PNN establishment at postnatal days 14, 21 and 28. The single cell PNN size and the chondroitin sulfate intensity were quantified for all cortex layers and specifically for the cortical layer IV which has the highest density of PNN-positive neurons. We demonstrate that the chondroitin sulfate proteoglycan staining intensity is increased between P14 and P28 while the PNN size remains unchanged. We then addressed posttraumatic changes of the PNN expression in laminae 6 and 7 of cervical spinal cord following hemisection injury. We demonstrate increase of the chondroitin sulfate content at 1.6-1.8 mm rostrally from the injury site and increase of the density of PNN-bearing cells at 0.4-1.2 mm caudally from the injury site. We further demonstrate decrease of the single cell PNN area at 0.2 mm caudally from the injury site suggesting that the PNN ECM takes part in the posttraumatic tissue rearrangement in the spinal cord. Our results demonstrate new insights on the PNN structure dynamics in the developing and posttraumatic CNS.

Meta-analysis of the diagnostic value of Wisteria floribunda agglutinin-sialylated mucin1 and the prognostic role of mucin1 in human cholangiocarcinoma.

OBJECTIVE: Serum carbohydrate antigen 19-9 (CA19-9) is a widely used tumour marker for cholangiocarcinoma (CCA). However, it is not a necessarily good CCA marker in terms of diagnostic accuracy. The purpose of this study is to evaluate the diagnostic value of Wisteria floribundaagglutinin-sialylated Mucin1 (WFA-MUC1) and the prognostic role of Mucin1 (MUC1) in human CCA. DESIGN: Meta-analysis. DATA SOURCES: Studies published in PubMed, Web of Science, The Cochrane Library and the China National Knowledge Infrastructure up to 11 October 2017. ELIGIBILITY CRITERIA: We included reports assessing the diagnostic capacity of WFA-MUC1 and the prognostic role of MUC1 in CCA. The receiver operating characteristic curve (ROC) of WFA-MUC1 and/or CA19-9 was described, and the HRs including 95% CI and the corresponding p value for MUC1 can be extracted. DATA EXTRACTION AND SYNTHESIS: Two independent researchers extracted data and assessed risk of bias. The diagnostic sensitivity and specificity data of WFA-MUC1 were extracted and analysed as bivariate data. Pooled HRs and its 95% CI for MUC1 were calculated with a random-effects meta-analysis model on overall survival of resectable CCA. RESULTS: Sixteen reports were included in this study. The pooled sensitivity and specificity of WFA-MUC1 were 0.76 (95% CI 0.71 to 0.81) and 0.72 (95% CI 0.59 to 0.83) in serum, 0.85 (95% CI 0.81 to 0.89) and 0.72 (95% CI 0.64 to 0.80) in bile and 0.72 (95% CI 0.50 to 0.87) and 0.85 (95% CI 0.70 to 0.93) in tissue, respectively. The summary ROC (SROC) were 0.77 (95% CI 0.73 to 0.81) in serum, 0.88 (95% CI 0.85 to 0.90) in bile and 0.86 (95% CI 0.83 to 0.89) in tissue, respectively. Furthermore, the pooled sensitivity and specificity and the SROC of CA19-9 in serum were 0.67 (95% CI 0.61 to 0.72), 0.86 (95% CI 0.75 to 0.93) and 0.75 (95% CI 0.71 to 0.79), respectively. The pooled HRs for MUC1 was 2.20 (95% CI 1.57 to 3.01) in CCA and 4.17 (95% CI 1.71 to 10.17) in mass-forming intrahepatic CCA. CONCLUSIONS: Compared with CA19-9, WFA-MUC1 was shown to possess stronger diagnostic capability. MUC1 could serve as a prognosis factor for poor outcomes of CCA, particularly, mass-forming intrahepatic CCA.

Wisteria floribunda agglutinin-sialylated mucin core polypeptide 1 is a sensitive biomarker for biliary tract carcinoma and intrahepatic cholangiocarcinoma: a multicenter study.

BACKGROUND: Wisteria floribunda agglutinin (WFA)-sialylated mucin core polypeptide 1 (MUC1) was investigated as a new glycoprotein marker for cholangiocarcinoma (CC) using glycoproteomics technologies. In this multicenter study, WFA-sialylated MUC1 levels in serum and bile samples were measured to determine their diagnostic capability in biliary tract carcinoma (BTC) and intrahepatic (Ih) CC. METHODS: The study included 244 patients with BTC, 59 patients with IhCC, 287 patients with benign biliary tract diseases, and 44 control subjects. RESULTS: Serum WFA-sialylated MUC1 levels were significantly higher in patients with either BTC or IhCC than in control subjects and those with benign biliary tract diseases. Patients with IhCC showed higher WFA-sialylated MUC1 levels than patients with tumors at other sites. No significant differences in WFA-sialylated MUC1 levels were found with regard to cancer stage or tissue type. Receiver operating characteristic curve analysis showed that WFA-sialylated MUC1 was superior to carbohydrate antigen 19-9 (CA19-9) and carcinoembryonic antigen (CEA) for the diagnosis of benign biliary tract diseases, BTC, and IhCC, as well as for stage I and II carcinomas. Significantly higher levels of biliary WFA-sialylated MUC1 were observed in BTC/IhCC than in benign biliary tract diseases. The diagnostic capability of biliary WFA-sialylated MUC1 was also superior to that of CA19-9, and diagnostic sensitivity was higher than that of biliary cytology for BTC/IhCC. CONCLUSIONS: WFA-sialylated MUC1 is a useful novel biomarker for BTC/IhCC. In the future, this measurement should be applied in the clinical setting.

Verification of WFA-Sialylated MUC1 as a Sensitive Biliary Biomarker for Human Biliary Tract Cancer.

BACKGROUND: The diagnostic accuracy of biliary cytology is limited. A novel sandwich enzyme-linked immunosorbent assay that combined Wisteria floribunda agglutinin (WFA) and anti-sialylated mucin 1 (MUC1) monoclonal antibody to target bile samples was recently developed. This study was designed to verify the diagnostic accuracy of WFA-sialylated MUC1 as a sensitive biliary biomarker for human biliary tract cancer. METHODS: Bile samples from 27 patients with benign disease and 174 patients with biliary tract cancer were analyzed. A receiver-operated characteristic curve analysis for biliary WFA-sialylated MUC1 and serum CA19-9 levels was performed to determine the cutoff value for the prediction of the presence of biliary tract cancer. RESULTS: Biliary WFA-sialylated MUC1 levels were significantly higher in the biliary tract cancer group compared with the benign group (P < 0.001). The cutoff value of WFA-sialylated MUC1 for discriminating biliary tract cancer was 10.5. The sensitivity of WFA-sialylated MUC1 in discriminating biliary tract cancer was much higher (82.2 %) than that of cytology (23.6 %) when this cutoff value was used. The cutoff value of serum CA19-9 for discriminating biliary tract cancer was 38 IU/L in the same cohort. All patients with biliary WFA-sialylated MUC1 and serum CA19-9 above the cutoff values had biliary tract cancer, and no patient with benign disease was categorized in this group. CONCLUSIONS: Biliary WFA-sialylated MUC1 is a useful biomarker for the differentiation of biliary tract cancer. The sensitivity of WFA-sialylated MUC1 was clearly higher than that of biliary cytology. Further data collection is necessary to validate the clinical usefulness of this biomarker.

Pel is a cationic exopolysaccharide that cross-links extracellular DNA in the Pseudomonas aeruginosa biofilm matrix.

Biofilm formation is a complex, ordered process. In the opportunistic pathogen Pseudomonas aeruginosa, Psl and Pel exopolysaccharides and extracellular DNA (eDNA) serve as structural components of the biofilm matrix. Despite intensive study, Pel's chemical structure and spatial localization within mature biofilms remain unknown. Using specialized carbohydrate chemical analyses, we unexpectedly found that Pel is a positively charged exopolysaccharide composed of partially acetylated 1→4 glycosidic linkages of N-acetylgalactosamine and N-acetylglucosamine. Guided by the knowledge of Pel's sugar composition, we developed a tool for the direct visualization of Pel in biofilms by combining Pel-specific Wisteria floribunda lectin staining with confocal microscopy. The results indicate that Pel cross-links eDNA in the biofilm stalk via ionic interactions. Our data demonstrate that the cationic charge of Pel is distinct from that of other known P. aeruginosa exopolysaccharides and is instrumental in its ability to interact with other key biofilm matrix components.

Novel glycobiomarker for ovarian cancer that detects clear cell carcinoma.

Epithelial ovarian cancer (EOC) is often asymptomatic and thus diagnosed at advanced stages with a poor prognosis. False-negative results for the conventional marker CA125 frequently occur in cases of clear cell carcinoma (CCC), a type of EOC; therefore, it is necessary to develop biomarkers with greater sensitivity. We previously reported a strategy to discover glycobiomarker candidates by combined lectin microarray and IGOT-LC/MS analysis. We have now optimized this strategy for discovering EOC biomarkers. Glycopeptides possessing cancerous glycans were enriched from the ascites fluids and culture supernatants of cancer cell lines with a fucose-binding lectin, AAL. IGOT-LC/MS analysis of CCC samples yielded 144 candidate glycoproteins. We selected WFA by lectin microarray as the optimal lectin to distinguish EOC from gastric and colon cancer. The candidates were narrowed by Western analysis of the WFA-bound fraction of ascites fluids. One of the final candidates, WFA-reactive ceruloplasmin, produced higher signals in the ascites fluids of EOC patients, including CCC, in comparison with the benign samples, while CA125 levels were comparable in the sandwich ELISA. Thus, our glycoproteomic strategy featuring efficient enrichment of glycans with disease-related alterations is applicable to various diseases.

Application of a glycoproteomics-based biomarker development method: alteration in glycan structure on colony stimulating factor 1 receptor as a possible glycobiomarker candidate for evaluation of liver cirrhosis.

The importance of diagnosis and therapies for liver cirrhosis (LC) is indisputable. Thus, a reliable method for monitoring the progression of liver fibrosis and resultant LC is urgently needed. Previously, using a lectin-assisted glycoproteomic method, we identified 26 serum glycoproteins as promising glycobiomarker candidates for monitoring the progression of liver diseases. In this study, we identified colony stimulating factor 1 receptor (CSF1R) as a promising LC marker candidate and then established Wisteria floribunda agglutinin (WFA)-reactive CSF1R (WFA(+)-CSF1R) as a novel possible glycobiomarker candidate by utilizing a glycoproteomics-based strategy. The serum level of WFA(+)-CSF1R in patients with hepatitis C virus (HCV)-infected liver disease was measured by an antibody-lectin sandwich ELISA. In a proof-of-concept experiment of the strategy preceding to future clinical studies, LC patients showed a high serum WFA(+)-CSF1R level in selected samples (P = 1.3 × 10(-17)). This result suggests WFA(+)-CSF1R is a possible biomarker candidate for evaluation of LC. Our results verified feasibility of this strategy for glycobiomarker development.

G2/M cell cycle arrest by an N-acetyl-D-glucosamine specific lectin from Psathyrella asperospora.

A new N-acetyl-D-glucosamine (GlcNAc) specific lectin was identified and purified from the fruiting body of the Australian indigenous mushroom Psathyrella asperospora. The functional lectin, named PAL, showed hemagglutination activity against neuraminidase treated rabbit and human blood types A, B and O, and exhibited high binding specificity towards GlcNAc, as well as mucin and fetuin, but not against asialofetuin. PAL purified to homogeneity by a combination of ammonium sulfate precipitation, chitin affinity chromatography and size exclusion chromatography, was monomeric with a molecular mass of 41.8 kDa, was stable at temperatures up to 55 °C and between pH 6-10, and did not require divalent cations for optimal activity. De novo sequencing of PAL using LC-MS/MS, identified 10 tryptic peptides that revealed substantial sequence similarity to the GlcNAc recognizing lectins from Psathyrella velutina (PVL) and Agrocybe aegerita (AAL-II) in both the carbohydrate binding and calcium binding sites. Significantly, PAL was also found to exert a potent anti-proliferative effect on HT29 cells (IC50 0.48 µM) that was approximately 3-fold greater than that observed on VERO cells; a difference found to be due to the differential expression of cell surface GlcNAc on HT29 and VERO cells. Further characterization of this activity using propidium iodine staining revealed that PAL induced cell cycle arrest at G2/M phase in a manner dependent on its ability to bind GlcNAc.

Terminal N-acetylgalactosamine-specific leguminous lectin from Wisteria japonica as a probe for human lung squamous cell carcinoma.

Millettia japonica was recently reclassified into the genus Wisteria japonica based on chloroplast and nuclear DNA sequences. Because the seed of Wisteria floribunda expresses leguminous lectins with unique N-acetylgalactosamine-binding specificity, we purified lectin from Wisteria japonica seeds using ion exchange and gel filtration chromatography. Glycan microarray analysis demonstrated that unlike Wisteria floribunda and Wisteria brachybotrys lectins, which bind to both terminal N-acetylgalactosamine and galactose residues, Wisteria japonica lectin (WJA) specifically bound to both α- and ß-linked terminal N-acetylgalactosamine, but not galactose residues on oligosaccharides and glycoproteins. Further, frontal affinity chromatography using more than 100 2-aminopyridine-labeled and p-nitrophenyl-derivatized oligosaccharides demonstrated that the ligands with the highest affinity for Wisteria japonica lectin were GalNAcß1-3GlcNAc and GalNAcß1-4GlcNAc, with K(a) values of 9.5 × 10(4) and 1.4 × 10(5) M(-1), respectively. In addition, when binding was assessed in a variety of cell lines, Wisteria japonica lectin bound specifically to EBC-1 and HEK293 cells while other Wisteria lectins bound equally to all of the cell lines tested. Wisteria japonica lectin binding to EBC-1 and HEK293 cells was dramatically decreased in the presence of N-acetylgalactosamine, but not galactose, mannose, or N-acetylglucosamine, and was completely abrogated by ß-hexosaminidase-digestion of these cells. These results clearly demonstrate that Wisteria japonica lectin binds to terminal N-acetylgalactosamine but not galactose. In addition, histochemical analysis of human squamous cell carcinoma tissue sections demonstrated that Wisteria japonica lectin specifically bound to differentiated cancer tissues but not normal tissue. This novel binding characteristic of Wisteria japonica lectin has the potential to become a powerful tool for clinical applications.

Glycoproteomics-based cancer marker discovery adopting dual enrichment with Wisteria floribunda agglutinin for high specific glyco-diagnosis of cholangiocarcinoma.

Cholangiocarcinoma (CC) is a lethal malignancy because it exhibits asymptomatic growth infiltrating the surrounding structures and therefore is usually detected at an advanced stage. The mainstay of treatment for CC is complete resection with negative surgical margins. Therefore, its diagnosis at a relatively early stage is demanded for performing relevant surgical resection. Since the definitive CC diagnosis depends on invasive methods such as biliary cytology and biopsy, a noninvasive assay with high diagnostic accuracy is keenly required. We therefore developed a CC marker with high specificity by the Wisteria floribunda agglutinin (WFA)-assisted glycoproteomics approach. WFA-positive glycoproteins were enriched by the direct dissection of the WFA-stained CC tissue region and following WFA-agarose column chromatography. Subsequent analysis by mass spectrometry identified 71 proteins as candidate markers. Screening of these candidates by gene expression profiling and immunohistochemistry resulted in the selection of L1 cell adhesion molecule (L1CAM) as the most specific CC marker. We confirmed the importance of WFA-positivity for L1CAM using both bile and serum of CC and benign bile duct disease patients. Specifically, WFA-positive L1CAM was enriched from serum by the WFA-assisted affinity capturing, with which CC was efficiently distinguished from benign. In the primary verification study using bile from CC patients (n=29) and that of benign bile duct disease (n=29), WFA-positive L1CAM distinguished CC with high specificity (sensitivity=0.66, specificity=0.93, overall accuracy=0.79, area under the receiver operating curve [AUC]=0.82). The combined use of the WFA-positive L1CAM assay with the high sensitive assay detecting WFA-positive sialylated mucin 1 sufficiently improved the diagnostic accuracy of CC (overall accuracy=0.84, AUC=0.93). This combination will possibly be a precise procedure for CC diagnosis compared with conventional diagnostic techniques. BIOLOGICAL SIGNIFICANCE: In this study, we constructed the system for verification of the candidate molecules that exhibit disease specific glyco-alterations and discovered a useful CC marker by the glycoproteomics-assisted strategy for biomarker discovery. Based on the strategy, we previously found that WFA is the best probe to detect CC-specific glycosylation and WFA-positive sialyl MUC1 as a possible biomarker candidate. While the diagnostic specificity of WFA-positive sialyl MUC1 was not superb, we proposed a new biomarker candidate WFA-positive L1CAM with high specificity in bile and serum to complement the previous one. We proved that the novel combination assay of WFA-L1CAM and WFA-sialyl MUC1 selected based on our strategy has the possibility to become a reliable serological test. This study represents application of our strategy, which can be extrapolated to discovery of marker candidates for other diseases.

Experience-dependent development of perineuronal nets and chondroitin sulfate proteoglycan receptors in mouse visual cortex.

Perineuronal nets (PNNs) are extracellular matrix structures consisting of chondroitin sulfate proteoglycans (CSPGs), hyaluronan, link proteins and tenascin-R (Tn-R). They enwrap a subset of GABAergic inhibitory interneurons in the cerebral cortex and restrict experience-dependent cortical plasticity. While the expression profile of PNN components has been widely studied in many areas of the central nervous system of various animal species, it remains unclear how these components are expressed during the postnatal development of mouse primary visual cortex (V1). In the present study, we characterized the developmental time course of the formation of PNNs in the mouse primary visual cortex, using the specific antibodies against the two PNN component proteins aggrecan and tenascin-R, or the lectin Wisteria floribunda agglutinin (WFA) that directly binds to glycosaminoglycan chains of chondroitin sulfate proteoglycans (CSPGs). We found that the fluorescence staining signals of both the WFA staining and the antibody against aggrecan rapidly increased in cortical neurons across layers 2-6 during postnatal days (PD) 10-28 and reached a plateau around PD42, suggesting a full construction of PNNs by the end of the critical period. Co-staining with antibodies to Ca(2+) binding protein parvalbumin (PV) demonstrated that the majority of PNN-surrounding cortical neurons are immunoreactive to PV. Similar expression profile of another PNN component tenascin-R was observed in the development of V1. Dark rearing of mice from birth significantly reduced the density of PNN-surrounding neurons. In addition, the expression of two recently identified CSPG receptors - Nogo receptor (NgR) and leukocyte common antigen-related phosphatase (LAR), showed significant increases from PD14 to PD70 in layer 2-6 of cortical PV-positive interneurons in normal reared mice, but decreased significantly in dark-reared ones. Taken together, these results suggest that PNNs form preferentially in cortical PV-positive interneurons in an experience-dependent manner, and reach full maturation around the end of the critical period of V1 development.

Human gastric TFF2 peptide contains an N-linked fucosylated N,N'-diacetyllactosediamine (LacdiNAc) oligosaccharide.

In the human stomach, the peptide trefoil factor family 2 (TFF2) is secreted together with the mucin MUC6 by mucous neck cells (MNCs) and antral gland cells. TFF2 is strongly associated with the gastric mucus and promotes gastric restitution. Here, TFF2 was purified from the human corpus and antrum, respectively, by size-exclusion chromatography, and the N-linked glycan structure at N-15 of the mature peptide was determined. As a hallmark, the unusual monofucosylated N,N'-diacetylhexosediamine (tentatively assigned as GalNAcß1 â†’ 4GlcNAc, LacdiNAc) modification was detected as the terminal structure of a bi-antennary complex type N-glycan exhibiting also core fucosylation. Replicate analyses did not show microheterogeneities in the fraction of peptide-N-glycosidase F cleaved and permethylated N-glycans when analyzed by matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS). On the glycopeptide level, a minor glycan microheterogeneity was evident in liquid chromatography-electrospray ionization (ESI)-MS, demonstrating the presence of underfucosylated species. The tryptic TFF2 N-glycopeptide p34-39 (LSPHNR N-glycosylated with Fuc3Hex3HexNAc6) was identified by both ESI-tandem mass spectrometry and MALDI-post-source decay analysis. Lectin analyses with the Wisteria floribunda agglutinin indicated the potential presence of LacdiNAc terminating glycans and revealed minor differences between TFF2 from fundic units, i.e. MNCs, and antral units, i.e. antral gland cells. Strikingly, on the level of the primary structure, there was no indication that the formation of the proposed LacdiNAc structure is cis-controlled by a peptidic determinant related to the published sequences.

Identification, cloning, and characterization of two N-acetylgalactosamine-binding lectins from the albumen gland of Helix pomatia.

Helix pomatia agglutinin (HPA), the lectin from the albumen gland of the Roman snail, has been used in histochemical studies relating glycosylation changes to the metastatic potential of solid tumors. To facilitate the use of HPA in a clinical (diagnostic) setting, detailed analysis of the lectin, including cloning and recombinant production of HPA, is required. A combination of isoelectric focusing, amino acid sequence analysis, and cloning revealed two polypeptides in native HPA preparations (HPAI and HPAII), both consistent with GalNAc-binding lectins of the H-type family. Pairwise sequence alignment showed that HPAI and HPAII share 54% sequence identity whereas molecular modeling using SWISS-MODEL suggests they are likely to adopt similar tertiary structure. The inherent heterogeneity of native HPA highlighted the need for production of functional recombinant protein; this was addressed by preparing His-thioredoxin-tagged fusion products in Escherichia coli Rosetta-gami B (DE3) cells. The recombinant lectins agglutinated human blood group A erythrocytes whereas their oligosaccharide specificity, evaluated using glycan microarrays, showed that they predominantly bind glycans with terminal α-GalNAc residues. Surface plasmon resonance with immobilized GalNAc-BSA confirmed that recombinant HPAI and HPAII bind strongly with this ligand (K(d) = 0.60 nm and 2.00 nm, respectively) with a somewhat higher affinity to native HPA (K(d) = 7.67 nm). Recombinant HPAII also bound the breast cancer cells of breast cancer tissue specimens in a manner similar to native lectin. The recombinant HPA described here shows important potential for future studies of cancer cell glycosylation and as a reagent for cancer prognostication.

Wisteria floribunda agglutinin-positive mucin 1 is a sensitive biliary marker for human cholangiocarcinoma.

UNLABELLED: Cholangiocarcinoma (CC) is an aggressive malignant tumor for which useful markers are not presently available for early and precise diagnosis. The aim of this study was therefore to identify a high-performance diagnostic marker with a special focus on glyco-alteration of glycoproteins. In the course of study, we found that Wisteria floribunda agglutinin (WFA) is the best probe to differentiate intrahepatic cholangiocarcinoma (ICC) lesions from normal bile duct epithelia (BDE) (P < 0.0001). The subsequent histochemical study confirmed ICC-specific WFA staining on 165 tissue specimens. On the other hand, the WFA staining was shown to be closely associated with that of MY.1E12 established previously against sialylated mucin 1 (MUC1) by double-staining experiments. Moreover, glyco-alteration of MUC1 could be verified by western blotting of WFA-captured bile samples from patients with CC patients. Thus, we attempted to construct an enzyme-linked immunosorbent assay system for more convenient CC diagnosis, where WFA-coated plates, the specific monoclonal antibody MY.1E12, and the bile specimens from CC including ICC (n = 30) and benign diseases (n = 38) were combined. As a result, CC was clearly distinguished from benign diseases with statistical scores (sensitivity = 90.0%, specificity = 76.3%, and area under the curve = 0.85). As a particular note, the obtained sensitivity is the highest score among those having been so far reported. CONCLUSION: Our approach focusing significant glyco-alteration of a particular glycoprotein yielded a novel diagnostic system for CC with satisfactory clinical scores.

Vimentin and desmin possess GlcNAc-binding lectin-like properties on cell surfaces.

Vimentin and desmin are intermediate filament proteins found in various mesenchymal and skeletal muscle cells, respectively. These proteins play an important role in the stabilization of the cytoplasmic architecture. Here, we found, using artificial biomimicking glycopolymers, that vimentin and desmin possess N-acetylglucosamine (GlcNAc)-binding lectin-like properties on the cell surfaces of various vimentin- and desmin-expressing cells such as cardiomyocytes and vascular smooth muscle cells. The rod II domain of these proteins was demonstrated to be localized to the cell surface and to directly bind to the artificial biomimicking GlcNAc-bearing polymer, by confocal laser microscopy and surface plasmon resonance analysis. These glycopolymers strongly interact with lectins and are useful tools for the analysis of lectin-carbohydrate interactions, since glycopolymers binding to lectins can induce the clustering of lectins due to multivalent glycoside ligand binding. Moreover, immunocytochemistry and pull-down assay with His-tagged vimentin-rod II domain protein showed that the vimentin-rod II domain interacts with O-GlcNAc proteins. These results suggest that O-GlcNAc proteins might be one candidate for physiological GlcNAc-bearing ligands with which vimentin and desmin interact. These findings demonstrate a novel function of vimentin and desmin that does not involve stabilization of the cytoplasmic architecture by which these proteins interact with physiological GlcNAc-bearing ligands such as O-GlcNAc proteins on the cell surface through their GlcNAc-binding lectin-like properties.

Purification and characterization of an N-acetylglucosamine specific lectin from marine bivalve Macoma birmanica.

A calcium independent lectin of molecular mass 47kDa was isolated from the foot muscle of marine bivalve Macoma birmanica by ammonium sulphate precipitation followed by affinity chromatography on immobilized GlcNAc column and designated as M. birmanica agglutinin (MBA). The lectin agglutinated rabbit erythrocytes strongly compared to human erythrocytes over a wide pH range from 5 to 9 and up to 50 degrees C. MBA is a glycoprotein and consists of 7.63% sugar. Among the tested sugars for analysis of carbohydrate recognition properties, Me-betaGlcNAc was the most potent inhibitor followed by Me-alphaMan. Enzyme linked solid phase assay revealed that MBA interacted well with complex type N-linked glycans and moderately to high mannose type N-linked glycans. Fluorescence study of MBA indicated that tryptophan was present in a non-hydrophobic region and its binding to GlcNAc was neither quenched nor altered lambda(max) position. The denaturation of MBA induced by urea was a reversible process and urea could not significantly change the Trp environment. MBA interacted with both Gram-positive and Gram-negative bacteria by recognizing their surface exposed GlcNAc containing antigens.

Development of an all-in-one technology for glycan profiling targeting formalin-embedded tissue sections.

An ultra-sensitive method for glycan analysis targeting small tissue sections (1.5mm in diameter) is described as an application of a recently-established lectin microarray technology. The developed system achieved a high level of detection of a tissue section consisting of approximately 500 cells for differential profiling, where both N- and O-glycans attached to a pool of glycoproteins are subjected to multiplex analysis with 43 lectins. By using an optimized protocol for differential glycan analysis, sections of adenocarcinoma (n=28) and normal epithelia (n=12) of the colon were analyzed in an all-in-one manner. As a result, Wisteria floribunda agglutinin (WFA) was found to clearly differentiate cancerous from normal epithelia with P<0.0001. The obtained results correlated well with the subsequent histochemical study using biotinylated WFA. Thus, the developed technology proved to be valid for expanding the lectin microarray applications to tissue-based glycomics, and hence, should accelerate a discovery phase of glycan-related biomarkers.

Presence of beta-linked GalNAc residues on N-glycans of human thyroglobulin.

Hepatic asialoglycoprotein receptor, which may mediate the clearance of circulating thyroglobulin, is known to have a high affinity for GalNAc. Recently, the receptor has been reported to be present also in the thyroid, implicating interaction with thyroglobulin. Here, mammalian thyroglobulins were analyzed for GalNAc termini by Western blotting with GalNAc-recognizing lectins labeled with peroxidase or (125)I. Wistaria floribunda lectin was found to bind human thyroglobulin and, to some extent, bovine, but not porcine thyroglobulin. After desialylation, the lectin bound all of the thyroglobulins tested. The binding was inhibited by competitive inhibitor GalNAc. Peptide N-glycanase treatment of human desialylated thyroglobulin resulted in the complete loss of reactivity with W. floribunda lectin, indicating that the binding sites are exclusively on N-glycans. The binding sites on human desialylated thyroglobulin were partly sensitive to beta-galactosidase, and the remainder was essentially sensitive to beta-N-acetylhexosaminidase. On the other hand, the binding sites of bovine and porcine desialylated thyroglobulins were totally sensitive to beta-galactosidase. Thus the lectin binds beta-Gal termini, as well as beta-GalNAc. GalNAc-specific Dolichos biflorus lectin also bound human thyroglobulin weakly. In contrast to W. floribunda lectin, desialylation diminished binding, suggesting that these two lectins recognize different GalNAc-terminated structures. Again, the binding was inhibited by GalNAc and by treatment with peptide N-glycanase. These results strongly indicate the presence of distinct GalNAc termini of N-glycans on human thyroglobulin.