Linkage Specific Fucosylation of Alpha-1-Antitrypsin in Liver Cirrhosis and Cancer Patients: Implications for a Biomarker of Hepatocellular Carcinoma

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).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.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

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

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.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.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.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 Levels of Galactose-Deficient Anti-Gal Immunoglobulin G in the Sera of Hepatitis C Virus-Infected Individuals with Fibrosis and Cirrhosis▿

Hepatitis B and C viruses are major causative agents of liver fibrosis, cirrhosis, and liver cancer. Using comparative glycoproteomics, we identified a glycoprotein that is altered both in amount and in glycosylation as a function of liver fibrosis and cirrhosis. Specifically, this altered glycoprotein is an immunoglobulin G (IgG) molecule reactive to the heterophilic alpha-Gal epitope [Galα-1-3Galβ1-(3)4GlcNAc-R]. While similar changes in glycosylation have been observed in several autoimmune diseases, the specific immunoglobulins and their antigen recognition profiles were not determined. Thus, we provide the first report identifying the specific antigenic recognition profile of an immunoglobulin molecule containing altered glycosylation as a function of liver disease. This change in glycosylation allowed increased reactivity with several fucose binding lectins and permitted the development of a plate-based assay to measure this change. Increased lectin reactivity was observed in 100% of the more than 200 individuals with stage III or greater fibrosis and appeared to be correlated with the degree of fibrosis. The reason for the alteration in the glycosylation of anti-Gal IgG is currently unclear but may be related to the natural history of the disease and may be useful in the noninvasive detection of fibrosis and cirrhosis

Peripheral markers of LPS exposure in patients with liver fibrosis correlate with the level of LRAGG.

<p>A) There is a statistically significant increase in the level of LPS Binding Protein (LBP) detected in the sera of patients with mild liver fibrosis (P = 0.0002) and also as a function of liver cirrhosis (P = 0.0013). B) A similar increase is seen in soluble CD14 (sCD14) with the progression of liver fibrosis to liver cirrhosis (P = 0.0020). C) There is a direct correlation between LBP and LRAGG (r_S = 0.4168, P<0.0001) and between sCD14 and LRAGG (r_S = 0.5287, P<0.0001) (D). r_S denotes the Spearman’s correlation coefficient. For panels A & B, samples size is Normal, n = 20; Stage 1–2, n = 21; and Stage 5–6 (Cirrhosis), n = 39.</p

Anti-gal specific antibodies have poor complement mediated killing ability and are unable to induce phagocytosis of opsonized target cells.

<p>A) Results from a hemoglobin release assay using serum from control patients and patients with cirrhosis. Compared to normal serum, serum from cirrhosis patients has an over 60% decrease in the capacity to induce complement mediated killing of target rRBCs. For panel A, sample size is: Normal, n = 20 and Cirrhotics, n = 20. As a control, complement alone was used to indicate the level of the alternative pathway (complement alone). In addition, if normal samples were heat inactivated or not treated with serum or complement, no lysis was observed. B) Results from a bactericidal assay using human serum show the growth pattern of bacteria alone (–), bacteria incubated with functional complement (+), bacteria with functional complement and normal human serum (NHS), or bacteria with functional complement and serum from a pool of 20 cirrhosis patients (Cirr). Error bars are indicated. C) Bacteria incubated with serum from a pool of 20 cirrhosis patients, in the presence of functional complement, show a significantly increased survival rate compared to those exposed to normal human serum (P = 0.013). Data normalized to those that did not receive serum addition. D) Results from an opsonization/phagocytosis assay. Bottom panel show target cells opsonized with serum from cirrhosis patients are not phagocytosed by monocytes, while top panel shows target cells opsonized with purchased normal serum are phagocytosed. Black peak represents monocytes alone, blue peak represents monocytes incubated with non-opsonized target cells, and gray filled peak represents opsonized target cells incubated with monocytes.</p