Glycan Profiles Of Glycoprotein In Sera From Chronic Hepatitis B- And C-Infected Patients

Background: Chronic hepatitis B (CHB) and hepatitis C (CHC) are the most common infectious diseases in the world. Chronic hepatitis is caused by virus infection in liver and led to liver damage and inflammatory diseases. Most of acute infections caused by HB V and HCV would become chronic carriers after 6 month, and even developed into cirrhosis and hepatocellular carcinoma(HCC). Glycosylation is a common post-translational modification of many secreted proteins and it is estimated that more than 70% of human proteins are glycosylated. Glycosylation alterations in serum proteins are highly associated with the development of liver disease. Serum glycan profiles are an early indicator for liver hepatocytes damage, and can provide critical diagnostic markers and insights into disease progression and pathogenesis. It is of great significance for early diagnosis of liver disease to analyze sera glycoproteins glycan structures of chronic hepatitis patients with fibrosis grading for F0-F1 period, which is the early fibrosis. The purpose of this study is to reveal the whole serum glycan profiles in chronic viral hepatitis patients and virus-associated specific alterations of N-glycan detailed compositions, and assist in the diagnosis of CHB or CHC. Serum glycan profiles may improve more accurate serologic diagnosis in chronic hepatitis. And it provides pivotal information to treat viral chronic hepatitis based on precise alterations of glycopatterns in sera.Method: Serum samples were collected from 54 CHB,55 CHC patients and 45 healthy volunteers (HVs). We employed a method combining the lectin microarrays and lectin-mediated affinity capture glycoproteins to enrich the glycoproteins specifically. Then the glycans were further released from the target glycoproteins with PNGase F. Glycans were cleaned up and collected using sepharose 4B and filter membrane. The matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/TOF-MS) was employed to identify glycan profiles in sera from patients with CHB, CHC and HVs. Finally we systematically compared different or similar alterations of glycoprotein glycopatterns between HBV and HCV-infected chronic hepatitis patients sera.Results:1. We applied lectin microarrays to screen the differences of the CHC and CHB patients compared with the health volunteers. There were 15 lectins (HHL, GSL-II, EEL, LEL, GSL-?, DBA, LCA, PTL-?, SBA, VVA, NPA, PSA, UEA-I, ACA and PHE-E+L) showed alterations of glycopattems in the CHB patients compared with the HVs. Meanwhile, there were 7 lectins (SBA, LEL, MAL-I, VVA, WGA, DBA and PHA-E+L) only changed in the CHC patients that revealed alterations of the glycopatterns compared with the HVs. Among the alterations of glycans recognized by lectins, there were 5 lectins (LEL, SBA, MAL-I, VVA and PHA-E+L) showed similar or different alterations of he glycopatterns simultaneously in both CHC and CHB patients.2. Notably, bi-antennary, bisecting GlcNAc, tri-and tetra-antennary complex-type N-glycan recognized by PHA-E+L were up regulated in the CHC patients while down regulated in the CHB patients. It is reported that the N-acetylglucosaminyltransferase ?(GnT-?) participated in the synthesis of bisecting GlcNAcglycan, which was closely related to liver disease. According to the results of SDS-PAGE and lectin blotting, different glycoproteins exhibit in molecular weight at 250 kDa protein bands and 55 kDa protein band and showed the glycoproteins enriched by PHA-E+L magnetic particles have high specificity and repeatability.3. The results of MALDI-TOF/TOF-MS indicated that ? Sera samples from HVs, CHB and CHC showed 18,23 and 26 glycan peaks respectively. Compared with HVs, CHB patients exclusive have m/z 1850.666,1866.465,2037.750,2151.793 and 2304.835 glycan peaks. Otherwise the CHC patients share these glycan peaks and exclusive have m/z 1460.502,2069.740 and 2174.772.? The glycans enriched by PHA-E+L in serum of patients with chronic hepatitis C are the most abundant and bisecting GlcNAc accounted for the vast majority. And the terminal of glycans are mostly galactose and N-acetylgalactose.?In patients with chronic hepatitis B and chronic hepatitis C, the enrichment glycans are mostly bi-antennary and tri-antennary N-glycans, only one appearance of tri-antennary complex N-glycan.4. In conclusion, some significant glycan alterations than can be detected in the early chronic hepatitis (slight fibrosis). In the serum of patients with chronic hepatitis B mostly terminal Gal glycosylation decreased, end of GalNAc and GlcNAc glycosylation increased. In chronic hepatitis C patients, the sialylation, fucosylation glycosylation and branch structures of glycan increased significantly.