Maki Sogabe

Strategy for glycoproteomics: identification of glyco-alteration using multiple glycan profiling tools.

Glycan alterations of proteins, a common feature of cancer cells, are associated with carcinogenesis, invasion and metastasis. Glycomics, the study of glycans and glycan-binding proteins in various biological systems, is an emerging field in the postgenome and postproteomics era. However, systematic and robust strategies for glycomics are still not fully established because the structural analysis of glycans, which comprise different patterns of branching, various possible linkage positions as well as monomer anomericity, is technically difficult. Here, we introduce a new strategy for glyco-alteration analysis of glycoproteins by using multiple glycan profiling tools. To understand glycan alterations of proteins by correlating the glycosyltransferase expression profile with the actual glycan structure, we systematically used three glycan profiling tools: (1) multiplex quantitative PCR (qPCR) array format for profiling the expression pattern of glycogenes, (2) lectin microarray as a multiplex glycan-lectin interaction analysis system for profiling either a pool of cell glycoproteins or a target glycoprotein, and (3) tandem mass spectrometry for identifying the glycan structure connected to a target glycoprotein. Using our system, we successfully identified glycan alterations on alpha-fetoprotein (AFP), including a novel LacdiNAc structure in addition to previously reported alterations such as alpha1,6 fucosylation.

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 0.5-μL sera of 125 patients with staging-determined fibrosis (26.4% F0-F1, 25.6% F2, 24% F3, and 23.2% F4) were subjected to systematic analysis by antibody-overlay lectin microarray. Data were analyzed to statistically relate to the degree of fibrosis progression. Additionally, we applied an optimal lectin signal set on the microarray to distinguish 45 patients with cirrhosis from 43 patients with chronic hepatitis. RESULTS Signal patterns of the 12 selected lectins reflected fibrosis-associated glyco-alteration of AGP. Among the 12 lectins, we found a specific lectin at each stage of fibrosis (i.e., significant fibrosis, severe fibrosis, and cirrhosis) (P < 0.0001). The test for the detection of cirrhosis showed that combinational use of 3 lectins (AOL, MAL, and DSA) on the array enhanced the diagnostic value for liver cirrhosis to 95% diagnostic sensitivity and 91% diagnostic specificity. CONCLUSIONS The multiple lectin-antibody sandwich immunoassay targeting AGP enables monitoring of disease progression in chronic hepatitis patients at risk of developing hepatocellular carcinoma.

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

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.

Glycoproteomic discovery of serological biomarker candidates for HCV/HBV infection-associated liver fibrosis and hepatocellular carcinoma.

We previously proposed a high-throughput strategy to discover serological biomarker candidates of cancer. This strategy focuses on a series of candidate glycoproteins that are specifically expressed in the original tissues (cells) of the target cancer and that carry glycan structures associated with carcinogenesis [Narimatsu, H., et al. FEBS J.2010, 277(1), 95-105]. Here, we examined the effectiveness of our strategy in identifying biomarkers to assess progression of liver fibrosis and for the early detection of hepatocellular carcinoma (HCC). On the basis of the results of lectin array analyses in culture media of hepatoma cell lines, we captured glycopeptides carrying AAL-ligands (fucosylated glycans) or DSA-ligands (branched glycans) from digests of culture media proteins and sera from HCC patients with a background of liver cirrhosis (LC). Glycoproteins were identified by the IGOT-LC-MS method. In all, 21 candidates were selected from 744 AAL-bound glycoproteins for further verification according to (i) their abundance in serum, (ii) their specific expression in liver, and (iii) the availability of antibodies to the glycoproteins. All selected candidates showed enhancement of AAL-reactivity in sera of HCC patients compared with that of healthy volunteers (HV). These results indicate that our glycoproteomic strategy is effective for identifying multiple glyco-biomarker candidates in a high-throughput manner.

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.

Automated Latex Photometric Immunoassay for Total Plasminogen Activator Inhibitor-1 in Plasma

Plasminogen activator inhibitor-1 (PAI-1) is a key regulator of the fibrinolytic system (1). The continuously high PAI-1 concentrations make fibrins resistant to dissolution by tissue-type plasminogen activator (tPA), which leads to multiorgan dysfunction and a bad prognosis in patients (2). Several methods have been developed for measuring the functional activity of PAI-1 in plasma samples based on the principle of adding a specified amount of tPA in excess of the PAI-1 and measuring residual tPA activity after a short incubation period (3). These methods, however, are neither completely specific nor accurate, and many other protease inhibitors in plasma inhibit tPA (4). Although several assays have also been described for PAI-1 antigen that are based on two-step enzyme immunoassays using monoclonal and/or polyclonal antibodies (5)(6), they have a narrow measurement range (0–40 μg/L) and are time-consuming (7). Because PAI-1 is a labile molecule, the utmost care is needed during blood collection and sample handling to ensure accurate measurement of PAI-1 and tPA. The half-life for the transformation from the active form of PAI-1 into the inactive latent form is ∼4 h in vitro and even shorter in vivo (8). It is therefore essential to correctly evaluate the amount of PAI-1 released from endothelial cells and adipose tissue (9) to make such methods clinically useful. We have developed a latex photometric immunoassay (LPIA) for total PAI-1 within a dynamic measurement range that can detect all forms of PAI-1 without the influence of conformational changes. Samples were obtained from 47 patients with myocardial infarction (29 men and 18 women) and 276 hospital employees, who volunteered for this study, as controls [100 men (age range, 23–63 years) and 168 women (age range, 21–57 years)]. After informed consent, blood samples were collected in a 1/10 volume of a solution containing 38 g/L sodium …

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.