Identification of fucosylated Fetuin-A as a potential biomarker for cholangiocarcinoma.

PURPOSE: Cholangiocarcinoma (CCA) is a malignancy of the bile ducts. The purpose of this discovery study was to identify effective serum markers for surveillance of cholangiocarcinoma. EXPERIMENTAL DESIGN: Using a glycomic method, patients with CCA were determined to have increased levels of alpha-1,3 and alpha-1,6 linked fucosylated glycan. Proteomic analysis of the serum fucosylated proteome identified proteins such as alpha-2-macroglobulin, kininogen, hemopexin, fetuin-A, alpha-1 anti-trypsin, and ceruloplasmin as being hyperfucosylated in HCC. The levels of these glycoproteins in 109 patients with CCA, primary sclerosing cholangitis (PSC), and control patients were compared to the performance of CA-19-9, the current "gold standard" assay for cholangiocarcinoma. RESULTS: Fucosylated Fetuin-A (fc-Fetuin-A) had the best ability to differentiate CCA from PSC, with an AUROC of 0.812 or 0.8665 at differentiating CCA from those with PSC or other liver disease. CA-19-9 had poor ability to differentiate PSC from cholangiocarcinoma (AUROC of 0.625). CONCLUSION AND CLINICAL RELEVANCE: Using glycomic and proteomic methods we identified a set of proteins that contain altered glycan in the sera of those with CCA. One of these proteins, fucosylated Fetuin-A may have value in the surveillance of people at risk for the development of cholangiocarcinoma.

Intrinsic hepatocyte dedifferentiation is accompanied by upregulation of mesenchymal markers, protein sialylation and core alpha 1,6 linked fucosylation.

Alterations in N-linked glycosylation have long been associated with cancer but for the most part, the reasons why have remained poorly understood. Here we show that increased core fucosylation is associated with de-differentiation of primary hepatocytes and with the appearance of markers indicative of a transition of cells from an epithelial to a mesenchymal state. This increase in core fucosylation was associated with increased levels of two enzymes involved in α-1,6 linked fucosylation, GDP-mannose 4, 6-dehydratase (Gmds) and to a lesser extent fucosyltransferase 8 (Fut8). In addition, the activation of cancer-associated cellular signaling pathways in primary rat hepatocytes can increase core fucosylation and induce additional glycoform alterations on hepatocyte proteins. Specifically, we show that increased levels of protein sialylation and α-1,6-linked core fucosylation are observed following activation of the ß-catenin pathway. Activation of the Akt signaling pathway or induction of hypoxia also results in increased levels of fucosylation and sialylation. We believe that this knowledge will help in the better understanding of the genetic factors associated with altered glycosylation and may allow for the development of more clinically relevant biomarkers.

Identification of IgM as a contaminant in lectin-FLISA assays for HCC detection.

Liver disease, in the form of hepatocellular carcinoma (HCC) accounts for > 700,000 deaths worldwide. A major reason for this is late diagnosis of HCC. The currently used biomarker, serum alpha-fetoprotein (AFP) is elevated in 40-60% of those with HCC and other markers that can either compliment or replace AFP are desired. Our previous work has identified a number of proteins that contain altered glycans in HCC. Specifically, these altered glycans were increased levels of core and outer arm fucosylation. To determine the clinical usefulness of those identified glycoproteins, a plate based assay was developed that allowed for the detection of fucosylated glycoforms. While this method was applicable to a number of independent patient sets, it was unable to specifically detect fucosylated glycoforms in many patient samples. That is, some material was present in serum that led to non-specific signal in the lectin- fluorescence -linked immunosorbent assay (lectin-FLISA). To address this issue, a systematic process was undertaken to identify the material. This material was found to be increased levels of lectin reactive IgM. Removal of both IgG and IgM using a multi-step protein A/G incubation and filtration step removed the contaminating signal and allowed for the analysis of specific protein glycoforms. This assay was subsequently used on two sample sets, one that was shown previously to be unable to be tested via a lectin FLISA and in a larger independent sample set. The clinical usefulness of this assay in the early detection of HCC is discussed.

Matrix assisted laser desorption ionization imaging mass spectrometry workflow for spatial profiling analysis of N-linked glycan expression in tissues.

A new matrix assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS) method to spatially profile the location and distribution of multiple N-linked glycan species in tissues is described. Application of an endoglycosidase, peptide N-glycosidase F (PNGaseF), directly on tissues followed by incubation releases N-linked glycan species amenable to detection by MALDI-IMS. The method has been designed to simultaneously profile the multiple glycan species released from intracellular organelle and cell surface glycoproteins, while maintaining histopathology compatible preparation workflows. A recombinant PNGaseF enzyme was sprayed uniformly across mouse brain tissue slides, incubated for 2 h, then sprayed with 2,5-dihydroxybenzoic acid matrix for MALDI-IMS analysis. Using this basic approach, global snapshots of major cellular N-linked glycoforms were detected, including their tissue localization and distribution, structure, and relative abundance. Off-tissue extraction and modification of glycans from similarly processed tissues and further mass spectrometry or HPLC analysis was done to assign structural designations. MALDI-IMS has primarily been utilized to spatially profile proteins, lipids, drug, and small molecule metabolites in tissues, but it has not been previously applied to N-linked glycan analysis. The translatable MALDI-IMS glycan profiling workflow described herein can readily be applied to any tissue type of interest. From a clinical diagnostics perspective, the ability to differentially profile N-glycans and correlate their molecular expression to histopathological changes can offer new approaches to identifying novel disease related targets for biomarker and therapeutic applications.

Total serum glycan analysis is superior to lectin-FLISA for the early detection of hepatocellular carcinoma.

PURPOSE: Hepatocellular carcinoma (HCC) is a primary cancer of the liver that is predominantly the result of infection with a hepatotropic virus such as hepatitis B virus or hepatitis C virus. As liver cancer is often asymptomatic, the development of sensitive noninvasive biomarkers is needed for early detection and improved survival. EXPERIMENTAL DESIGN: We have previously identified alterations in the N-linked glycosylation of serum proteins with the development of HCC and identified many of the proteins that contained the altered glycosylation. In the current study, we compared the ability of the identified proteins to diagnose HCC with the total serum glycan analysis. RESULTS: Surprisingly, glycan analysis of total serum had the greatest ability to distinguish HCC from cirrhosis with an AUROC of 0.851, a sensitivity of 73% at a specificity of 88%. When total glycan sequencing was combined with alpha-fetoprotein (AFP), the sensitivity increased to 95% at a specificity of 90%. CONCLUSION AND CLINICAL RELEVANCE: Changes in glycosylation as detected in whole serum could be used to diagnose HCC with greater sensitivity and specificity than that observed through the analysis of specific protein glycoforms or protein levels. Such an assay could have value in the management of those at risk for the development of HCC.

Altered functionality of anti-bacterial antibodies in patients with chronic hepatitis C virus infection.

BACKGROUND: Using comparative glycoproteomics, we have previously identified a glycoprotein that is altered in both amount and glycosylation as a function of liver cirrhosis. The altered glycoprotein is an agalactosylated (G0) immunoglobulin G molecule (IgG) that recognizes the heterophilic alpha-gal epitope. Since the alpha gal epitope is found on gut enterobacteria, it has been hypothesized that anti-gal antibodies are generated as a result of increased bacterial exposure in patients with liver disease. METHODS: The N-linked glycosylation of anti-gal IgG molecules from patients with fibrosis and cirrhosis was determined and the effector function of anti-bacterial antibodies from over 100 patients examined. In addition, markers of microbial exposure were determined. RESULTS: Surprisingly, the subset of agalactosylated anti-gal antibodies described here, was impaired in their ability to mediate complement mediated lysis and inhibited the complement-mediated destruction of common gut bacteria. In an analysis of serum from more than 100 patients with liver disease, we have shown that those with increased levels of this modified anti-gal antibody had increased levels of markers of bacterial exposure. CONCLUSIONS: Anti-gal antibodies in patients with liver cirrhosis were reduced in their ability to mediate complement mediated lysis of target cells. As bacterial infection is a major complication in patients with cirrhosis and bacterial products such as LPS are thought to play a major role in the development and progression of liver fibrosis, this finding has many clinical implications in the etiology, prognosis and treatment of liver disease.

Increased bisecting N-acetylglucosamine and decreased branched chain glycans of N-linked glycoproteins in expressed prostatic secretions associated with prostate cancer progression.

PURPOSE: Using prostatic fluids rich in glycoproteins like prostate-specific antigen and prostatic acid phosphatase (PAP), the goal of this study was to identify the structural types and relative abundance of glycans associated with prostate cancer status for subsequent use in emerging MS-based glycopeptide analysis platforms. EXPERIMENTAL DESIGN: A series of pooled samples of expressed prostatic secretions (EPS) and exosomes reflecting different stages of prostate cancer disease were used for N-linked glycan profiling by three complementary methods, MALDI-TOF profiling, normal-phase HPLC separation, and triple quadropole MS analysis of PAP glycopeptides. RESULTS: Glycan profiling of N-linked glycans from different EPS fluids indicated a global decrease in larger branched tri- and tetra-antennary glycans. Differential exoglycosidase treatments indicated a substantial increase in bisecting N-acetylglucosamines correlated with disease severity. A triple quadrupole MS analysis of the N-linked glycopeptides sites from PAP in aggressive prostate cancer pools was done to cross-reference with the glycan profiling data. CONCLUSION AND CLINICAL RELEVANCE: Changes in glycosylation as detected in EPS fluids reflect the clinical status of prostate cancer. Defining these molecular signatures at the glycopeptide level in individual samples could improve current approaches of diagnosis and prognosis.

Increased levels of tetra-antennary N-linked glycan but not core fucosylation are associated with hepatocellular carcinoma tissue.

BACKGROUND: Alterations in glycosylation have long been associated with the development of cancer. In the case of primary hepatocellular carcinoma (HCC), one alteration that has often been associated is increased amounts of fucose attached to the N-glycans of serum proteins secreted by the liver. METHODS: In an effort to determine the origin of this increased fucosylation, we have conducted N-linked glycan analysis of HCC tissue, the surrounding nontumor tissue, and compared this to tissue from a nondiseased adult liver. RESULTS: Surprisingly, no difference in the level of fucosylation was observed from the three donor groups, suggesting that the increased levels of fucosylation observed in serum of those with HCC is not the result of increased synthesis of fucosylated proteins in the cancer tissue. On the other hand, increased levels of a tetra-antennary glycan were observed in the HCC tissue as compared with the surrounding tissue or to the nondiseased livers. CONCLUSIONS: This represents, to our knowledge, one of the first reports associating increased levels of branching with the development of HCC. IMPACT: The identification of increased levels of tetra-antennary glycan on liver tumor tissue, as opposed to adjacent or nondiseased tissue may lead to improved detection of HCC.

Novel changes in glycosylation of serum Apo-J in patients with hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide and the occurrence of HCC has more than doubled in the United States in the past decade. Early detection is considered key to reducing the mortality of HCC. METHODS: Using two-dimensional gel electrophoresis and high-performance liquid chromatography we have analyzed the glycosylation of Apo-J from healthy controls, patients with liver cirrhosis, or those with HCC. RESULTS: Apo-J in the serum from patients with HCC had decreased levels of (ß-1,4) triantennary N-linked glycan compared with the healthy controls or patients with liver cirrhosis. We analyzed this change in an independent cohort of 76 patients with HCC, 32 with cirrhosis, and 43 infected with hepatitis C virus using the Datura stramonium lectin (DSL), which binds to (ß-1,4) triantennary N-linked glycan. The level of DSL-reactive Apo-J allowed us to differentiate HCC from cirrhosis with an area under the receiver operating characteristic curve (AUROC) of 0.852. When Apo-J was combined with other serum biomarkers such as α-fetoprotein (AFP) and fucosylated kininogen by using a multivariate logistic regression model, the AUROC increased to 0.944, a value much greater than that observed with AFP alone (AUROC of 0.765). CONCLUSIONS: The glycosylation of Apo-J is a useful marker when used alone or in combination with outer makers for the early detection of HCC. IMPACT: The potential use of a combination of AFP, DSL-reactive Apo-J, and fucosylated kininogen as a biomarker of HCC would have great value in the management of patients with liver disease.

Linkage specific fucosylation of alpha-1-antitrypsin in liver cirrhosis and cancer patients: implications for a biomarker of hepatocellular carcinoma.

BACKGROUND: We previously reported increased levels of protein-linked fucosylation with the development of liver cancer and identified many of the proteins containing the altered glycan structures. One such protein is alpha-1-antitrypsin (A1AT). To advance these studies, we performed N-linked glycan analysis on the five major isoforms of A1AT and completed a comprehensive study of the glycosylation of A1AT found in healthy controls, patients with hepatitis C- (HCV) induced liver cirrhosis, and in patients infected with HCV with a diagnosis of hepatocellular carcinoma (HCC). METHODOLOGY/PRINCIPAL FINDINGS: Patients with liver cirrhosis and liver cancer had increased levels of triantennary glycan-containing outer arm (alpha-1,3) fucosylation. Increases in core (alpha-1,6) fucosylation were observed only on A1AT from patients with cancer. We performed a lectin fluorophore-linked immunosorbent assay using Aleuria Aurantia lectin (AAL), specific for core and outer arm fucosylation in over 400 patients with liver disease. AAL-reactive A1AT was able to detect HCC with a sensitivity of 70% and a specificity of 86%, which was greater than that observed with the current marker of HCC, alpha-fetoprotein. Glycosylation analysis of the false positives was performed; results indicated that these patients had increases in outer arm fucosylation but not in core fucosylation, suggesting that core fucosylation is cancer specific. CONCLUSIONS/SIGNIFICANCE: This report details the stepwise change in the glycosylation of A1AT with the progression from liver cirrhosis to cancer and identifies core fucosylation on A1AT as an HCC specific modification.

Glucosidase inhibition enhances presentation of de-N-glycosylated hepatitis B virus epitopes by major histocompatibility complex class I in vitro and in woodchucks.

UNLABELLED: In this report, the possibility of pharmacologically altering the hepatitis B virus (HBV) epitopes presented by major histocompatibility complex class I on infected cells is demonstrated. The HBV middle envelope glycoprotein (MHBs) maturation appears to require calnexin-mediated folding. This interaction is dependent on glucosidases in the endoplasmic reticulum. Prevention of HBV envelope protein maturation in cultured cells through use of glucosidase inhibitors, such as 6-O-butanoyl castanospermine and N-nonyl deoxynorjirimycin, resulted in MHBs degradation by proteasomes. The de-N-glycosylation associated with polypeptide degradation was predicted to result in conversion of asparagine residues into aspartic acid residues. This prediction was confirmed by showing that peptides corresponding to the N-glycosylation sequons of MHBs, but with aspartic acid replacing asparagine, (1) can prime human cytotoxic T lymphocytes that recognize HBV-producing cells and (2) that the presentation of these envelope motifs by major histocompatibility complex class I is enhanced by incubation with glucosidase inhibitors. Moreover, although peripheral blood mononuclear cells isolated from woodchucks chronically infected with woodchuck hepatitis virus and vaccinated with woodchuck hepatitis virus surface antigen could be induced to recognize the natural MHBs asparagine-containing peptides, only cells isolated from animals treated with glucosidase inhibitor recognized the aspartic acid-containing peptides. CONCLUSION: These data suggest that pharmacological intervention with glucosidase inhibitors can alter the MHBs epitopes presented. This editing of the amino acid sequence of the polypeptide results in a new epitope, or "editope", with possible medical significance.