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

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

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

Change in LRAGG in response to IFN Treatment in patients with liver fibrosis.

<p>(A) The change in the level of LRAGG in patients who responded to IFN and cleared their HCV and had low LRAGG at start of treatment. As this figure shows, there is no significant change in patients who started with low levels of LRAGG in response to IFN therapy. (B) In contrast, those patients who responded to IFN and cleared their HCV and started had greater than 5-fold increase over normal at the start of treatment show a significant decrease following treatment (P<0.05) (C). However, patients who do not respond to IFN treatment show a significant increase (P<0.05) following treatment when they began with low levels (C) and stayed high when they started with greater than 5-fold increase in LRAGG over normal (D).</p

Increased lectin reactive anti-gal IgG from patients with increasing levels of liver fibrosis.

<p>A) Compared to commercially purchased normal human sera, patients with limited liver fibrosis (stage 1–2) have a 3.5 (±1.4) fold increase in lectin reactive anti-gal IgG (LRAGG). More advanced (Stage 5–6) fibrosis patients have a 7.9 (±2.4) fold increase in LRAGG. The fold increase in LRAGG is statistically significant (P<0.001). When serum from more advanced fibrotic patients is used on plates coated with HSA alone and not HSA-alpha-gal, no signal is observed (HSA lane). B) The level of anti-gal IgA, IgM and IgG bound to target rabbit red blood cells as a function of liver fibrosis. There is a statistically significant increase in anti-gal IgA from commercially purchased normal human serum to serum from patients with advanced fibrosis (P = 0.0048); Anti-gal IgA also significantly increases from limited to advanced fibrosis (P = 0.0133). Anti-gal IgM significantly increases from control to limited fibrosis (P = 0.02) and from control to advanced fibrosis (P = 0.002); there is also a significant increase in anti-gal IgM from limited to advanced fibrosis (P = 0.0018). Anti-gal IgG is significantly elevated in advanced fibrosis compared to control (P = 0.0075) and also from limited to advanced fibrosis (P = 0.0217). 50 mM of BSA-alpha-gal can prevent binding of antibodies to RBCC. See text for more details. For panels A & B, samples size is Normal, n = 21; Stage 1–2, n = 22; and Cirrhosis, n = 39.</p

Sample population characteristics.

1<p>Samples were provided from St. Louis University Medical School or from the University of Michigan. See text for more details.</p>2<p>Disease diagnosis was determined by MRI or by liver biopsy.</p>3<p>For Etiology: HBV, hepatitis B virus; HCV, hepatitis C virus; crypto, cryptogenic liver disease; alcohol, alcohol induced liver disease; other, liver disease of unknown origin.</p>4<p>ALT, Alanine transaminase.</p>5<p>AST, aspartate aminotransferase.</p>6<p>Total Bilirubin levels.</p>7<p>MELD: Model for end stag liver disease. N/A, not available.</p>8<p>The percent of patients with each Child-Pugh score is given as a percentage in each group.</p