Highly Sensitive Glycan Profiling of Hepatitis B Viral Particles and a Simple Method for Dane Particle Enrichment.

Hepatitis B virus (HBV) is a double-stranded DNA virus composed of three types of viral particles. The virions are called Dane particles and the others are noninfectious subviral particles (SVPs). In blood, SVPs are detected in abundance, about 1000-10000 fold higher than Dane particles.

Engineering of recombinant Wisteria floribunda agglutinin specifically binding to GalNAcβ1,4GlcNAc (LacdiNAc).

Wisteria floribunda agglutinin (WFA) is a useful probe for distinguishing glycan structural alterations in diseases such as intrahepatic bile duct carcinoma and hepatic fibrosis; however, the gene encoding WFA has not been identified. Here, we identified the gene encoding WFA, and recombinant WFA (rWFA) was expressed in Escherichia coli and purified. The natural complementary DNA sequence obtained from wisteria seeds contained an open reading frame of 861 nucleotides encoding a WFA precursor, which included a hydrophobic signal peptide at the N-terminus, a propeptide at the C-terminus and a single cysteine (Cys) residue for dimer formation.

Hepatic stellate cells secreting WFA -M2BP: Its role in biological interactions with Kupffer cells.

Background And Aim: Hepatic stellate cells (HSCs) play a central role in hepatic fibrosis and are regulated by Kupffer cells (KCs). Wisteria floribunda agglutinin-positive Mac-2 binding protein (WFA -M2BP) was recently identified as a serum marker for hepatic fibrosis. Although WFA -M2BP was identified as a ligand of Mac-2, the function of WFA -M2BP in hepatic fibrosis remains unclear.

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.

Alteration of matrix metalloproteinase-3 O-glycan structure as a biomarker for disease activity of rheumatoid arthritis.

Background: Nearly all secreted proteins are glycosylated, and serum glycoproteins that exhibit disease-associated glycosylation changes have potential to be biomarkers. In rheumatoid arthritis (RA), C-reactive protein (CRP), and matrix metalloproteinase-3 (MMP-3) are widely used as serologic biomarkers, but they lack sufficient specificity or precision. We performed comparative glycosylation profiling of MMP-3 using a recently developed antibody-overlay lectin microarray technology that allows semicomprehensive and quantitative analysis of specific protein glycosylation to develop an RA-specific disease activity biomarker.

Large-scale mutational analysis in the EXT1 and EXT2 genes for Japanese patients with multiple osteochondromas.

Background: Multiple osteochondroma (MO) is an autosomal dominant skeletal disorder characterized by the formation of multiple osteochondromas, and exostosin-1 (EXT1) and exostosin-2 (EXT2) are major causative genes in MO. In this study, we evaluated the genetic backgrounds and mutational patterns in Japanese families with MO.

Results: We evaluated 112 patients in 71 families with MO.

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.

Development of M2BPGi: a novel fibrosis serum glyco-biomarker for chronic hepatitis/cirrhosis diagnostics.

Many proteins in the living body are glycoproteins, which present glycans linked on their surface. Glycan structures reflect the degree of cell differentiation or canceration and are cell specific. These characteristics are advantageous in the development of various disease biomarkers.

Large-Scale Identification of N-Glycan Glycoproteins Carrying Lewis x and Site-Specific N-Glycan Alterations in Fut9 Knockout Mice.

The Lewis x (Le(x)) structure (Galβ1-4(Fucα1-3)GlcNAc-R) is a carbohydrate epitope comprising the stage-specific embryonic antigen-1 (SSEA-1) and CD15, and it is synthesized by α1,3-fucosyltransferase 9 (Fut9). Fut9 is expressed specifically in the stomach, kidney, brain, and in leukocytes, suggesting a specific function in these tissues. In this study, the N-linked glycan mass spectrometry profile of wild-type mouse kidney glycoproteins revealed the presence of abundant terminal fucoses, which were lost following knockout of the Fut9 gene; the terminal fucose was therefore concluded to be Le(x).

Lectin Microarray-Based Sero-Biomarker Verification Targeting Aberrant O-Linked Glycosylation on Mucin 1.

Glycoform of mucin 1 (MUC1) in cancerous cells changes markedly with cell differentiation, and thus, qualitative detection and verification of the MUC1 glycosylation changes have potential diagnostic value. We have developed an ultrasensitive method to detect the changes in cholangiocarcinoma (CC), which produces MUC1, and applied it in the diagnostics development. The focused glycan analysis using 43-lectin-immobilized microarray could obtain the glycan profiles of sialylated MUC1 in 5 μL of sera.

Large-scale identification of secretome glycoproteins recognized by Wisteria floribunda agglutinin: A glycoproteomic approach to biomarker discovery.

One of the promising approaches to the development of cancer diagnostic systems is quantification of a specific protein carrying cancerous glycans. Potential utility of Wisteria floribunda agglutinin (WFA) for such assays has been suggested for several cancer types. To develop such diagnostic systems, identification of WFA-recognized glycoproteins is essential.

Clinicopathological characteristics and diagnostic performance of Wisteria floribunda agglutinin positive Mac-2-binding protein as a preoperative serum marker of liver fibrosis in hepatocellular carcinoma.

Background: Wisteria floribunda agglutinin positive Mac-2-binding protein (WFA(+)-M2BP) is a novel serum marker of liver fibrosis identified in glycoproteomic biomarker screening studies, and its clinicopathological characteristics have yet to be elucidated sufficiently for clinical utilization.

Methods: We retrospectively analyzed the clinicopathology data and serum WFA(+)-M2BP levels in 376 hepatocellular carcinoma patients undergoing liver surgery. WFA(+)-M2BP was quantified in frozen serum samples collected at the time of surgery using the FastLec-Hepa method.

LecT-Hepa facilitates estimating treatment outcome during interferon therapy in chronic hepatitis C patients.

Background: A combination treatment of interferon and ribavirin is the standard and the commonly used treatment for chronic hepatitis C (CHC). Developing noninvasive tests like serum indicators that can predict treatment outcome at an early stage of therapy is beneficial for individualized treatment and management of CHC. A glyco-indicator based on the glyco-alteration of serum α1-acid glycoprotein, LecT-Hepa, was discovered by glycomics technologies as a robust indicator of liver fibrosis.

Glycoproteomics approach for identifying Glycobiomarker candidate molecules for tissue type classification of non-small cell lung carcinoma.

Histopathological classification of lung cancer has important implications in the application of clinical practice guidelines and the prediction of patient prognosis. Thus, we focused on discovering glycobiomarker candidates to classify the types of lung cancer tissue. First, we performed lectin microarray analysis of lung cancer tissue specimens and cell lines and identified Aleuria aurantia lectin (AAL), Hippeastrum hybrid lectin (HHL), and Concanavalia ensiformis agglutinin (ConA) as lectin probes specific to non-small cell lung carcinoma (NSCLC).

IgA nephropathy caused by unusual polymerization of IgA1 with aberrant N-glycosylation in a patient with monoclonal immunoglobulin deposition disease.

Immunoglobulin A nephropathy (IgAN) is a form of chronic glomerulonephritis characterized by the deposition of IgA immune complexes in the glomerular region. The cause of IgAN is unknown, but multiple mechanisms have been suggested. We previously reported a rare case of mesangioproliferative glomerulonephritis in a patient with monoclonal immunoglobulin deposition disease associated with monoclonal IgA1.

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.

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.

Multilectin-assisted fractionation for improved single-dot tissue glycome profiling in clinical glycoproteomics.

To survey the glycome shift in cancer, single-dot tissue glycome profiling was improved by incorporating a lectin-assisted fractionation. The differential analysis of tissue specimens from colorectal cancer patients (n = 45) revealed that unfucosylated/α2,6-sialylated glycoproteins significantly increased in patients with poor prognoses. The detailed annotation will be an indispensable supplement for cancer-related glyco-biomarker discovery.

Reconstruction of a robust glycodiagnostic agent supported by multiple lectin-assisted glycan profiling.

Purpose: Wisteria floribunda agglutinin positive human Mac-2-binding protein (WFA(+)-hM2BP) was recently validated as a liver fibrosis glycobiomarker with a fully automated lectin-antibody sandwich immunoassay. In this study, we supplied recombinant WFA(+)-hM2BP as the standard glycoprotein and the overlaid antibody to enhance the robustness of WFA(+)-hM2BP quantification.

Experimental Design: The optimum conditions for producing recombinant WFA(+)-hM2BP were selected by cell glycome analysis based on a lectin microarray.

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

Unlabelled: 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.

A serum "sweet-doughnut" protein facilitates fibrosis evaluation and therapy assessment in patients with viral hepatitis.

Although liver fibrosis reflects disease severity in chronic hepatitis patients, there has been no simple and accurate system to evaluate the therapeutic effect based on fibrosis. We developed a glycan-based immunoassay, FastLec-Hepa, to fill this unmet need. FastLec-Hepa automatically detects unique fibrosis-related glyco-alteration in serum hyperglycosylated Mac-2 binding protein within 20 min.

Co-translational function of Cosmc, core 1 synthase specific molecular chaperone, revealed by a cell-free translation system.

The core 1 structure of the mucin type O-glycan is synthesized by core 1 β1,3-galactosyltransferase (C1GalT). Core 1 synthase specific molecular chaperone (Cosmc), a molecular chaperone specific for C1GalT, is essential for the expression of functional C1GalT in mammalian cells. In this study, we have established a procedure for detecting the chaperone activity of Cosmc by using a wheat germ cell-free translation system.

Multilectin assay for detecting fibrosis-specific glyco-alteration by means of lectin microarray.

Background: Despite the progress made in understanding glyco-alterations of specific glycoproteins such as α1-acid glycoprotein (AGP) associated with liver fibrosis, there has been no useful diagnostic assay with a lectin recognizing the fibrosis-specific alteration and an antibody against the core protein. We therefore developed a compatible multiple lectin-antibody sandwich immunoassay on the basis of the results obtained by the lectin microarray analysis for monitoring fibrosis.

Methods: AGP-enriched fractions derived from 0.

Beta3GnT2 (B3GNT2), a major polylactosamine synthase: analysis of B3GNT2-deficient mice.

The polylactosamine structure is a fundamental structure of carbohydrate chains and carries a lot of biofunctional carbohydrate epitopes. To investigate the biological function of polylactosamine chains, here we generated and analyzed knockout mice lacking the gene B3gnt2, which encodes a major polylactosamine synthase. In beta1,3-N-acetylglucosaminyltransferase (B3gnt2) B3gnt2-deficient (B3gnt2-/-) mice, the number of polylactosamine structures was markedly lower than in wild-type mice.