Protein glycosylation plays an important role in the pathogenesis and progression of various liver diseases. However, little is known about the precise alterations in protein glycosylation or the potential correlation between glycan-related genes expression and glycan profiles in liver fibrosis. The aim of the study was to investigate potential associations between glycan-related genes expression and glycan profiles to evaluate liver fibrosis in a mouse model. Analyses of glycan-related genes expression and glycan profiles were performed using oligonucleotide microarrays and lectin microarrays, respectively. Real-time PCR and Western blot were used to confirm any altered glycan-related genes expression levels and protein levels. Moreover, altered glycan patterns on the surface of hepatocytes were verified by lectin histochemistry. These results revealed that the mRNA levels of 10 glycan-related genes were significantly altered in fibrotic liver. Furthermore, we observed an increase in multivalent sialic acid, poly-LacNAc, sialyl-T-antigen, Fucoseα-1,3/6GlcNAc, and GalNAcα1–3Gal in fibrotic liver specimens, whereas GlcNAc oligomers was decreased in fibrotic liver. Our findings indicated that the synthetic pathway of “Tn antigen → T antigen (core-1) → sialyl-T antigen” was activated for O-glycan during the process of liver fibrosis.Keywords: gene expression microarray; glycan structure; glycan-related genes; hepatic fibrosis; lectin histochemistry; lectin microarray; tumorigenesis;

Recent studies have elucidated that expression of certain glycoproteins in human saliva is increased or decreased according to age; meanwhile, human saliva may inhibit viral infection and prevent viral transmission. However, little is known about the age- and sex-associated differences in the glycopatterns of human salivary glycoproteins and their significant roles against influenza A virus (IVA). Here, we investigate the glycopatterns of human salivary glycoproteins with 180 healthy saliva samples divided into six age/sex groups using lectin microarrays and fabricate saliva microarrays to validate the terminal carbohydrate moieties of glycoproteins in individual saliva samples. Furthermore, we assess the inhibiting and neutralizing activity of saliva against two strains of influenza A (H9N2) virus. We find that seven lectins (e.g., MAL-II and SNA) show significant age differences in both females and males, and seven lectins (e.g., WFA and STL) show significant sex differences in children, adults and elderly people. Interestingly, we observe that elderly individuals have strongest resistance to IVA partly by presenting more terminal α2–3/6-linked sialic acid residues in their saliva, which bind with the influenza viral hemagglutinations. We conclude that age- and sex-associated differences in the glycopatterns of human salivary glycoproteins may provide pivotal information to help understand some age related diseases and physiological phenomena.Keywords: age; glycopattern; influenza A virus; lectin microarrays; saliva; saliva microarrays; sex;

Diagnosis of bladder cancer, one of the most common types of human cancer, at an early (nonmuscle-invasive) stage is the best way to reduce the mortality rate. Tumor malignancy in general is closely associated with alterations of glycan expression. Glycosylation status, particularly global glycomes, in bladder cancer has not been well studied. We integrated lectin microarray and mass spectrometry (MS) methods to quantitatively analyze and compare glycan expression in four bladder cancer cell lines (KK47, YTS1, J82, T24) and one normal bladder mucosa cell line (HCV29). Glycopattern alterations were analyzed using lectin microarray analysis and confirmed by lectin staining and lectin blotting. Associations of glycopatterns with diverging stages were evaluated by lectin histochemistry on tissue microarrays. N-Glycans were derivatized by amidation of sialylated glycans with acetohydrazide and reductive amination with the stable isotope tags [12C6]- and [13C6]-aniline, and were quantitatively analyzed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). N-Glycan biosynthesis-associated proteins were quantitatively analyzed by a stable isotope labeling by amino acids in cell culture (SILAC) proteomics method, which revealed significant differences in expression of 13 glycosyltransferases and 4 glycosidases. Our findings indicate that sialyl Lewis X (sLex), terminal GalNAc and Gal, and high mannose-type N-glycans were more highly expressed in bladder cancer cells and tissues than in normal cells. Bladder cancer cells showed high expression of core-fucosylated N-glycans but low expression of terminally fucosylated N-glycans. Each of these glycome changes may be directly related to bladder cancer progression.Keywords: bladder cancer; lectin microarray analysis; mass spectrometry; N-glycans; quantitative glycomics; SILAC method;

•The distribution and evolution of N-glycosylated sites in the HA and NA of H9N2 virus were explored.•One strain of the H9N2 subtypes was proliferated human embryonic lung fibroblast cells.•The viral N-glycan profiles were analyzed by a glycomic method that combined the lectin microarray and MALDI-TOF/TOF-MS.N-glycosylation can affect the host specificity, virulence and infectivity of influenza A viruses (IAVs). In this study, the distribution and evolution of N-glycosylation sites in the hemagglutinin (HA) and neuraminidase (NA) of H9N2 virus were explored using phylogenetic analysis. Then, one strain of the H9N2 subtypes was proliferated in the embryonated chicken eggs (ECE) and human embryonic lung fibroblast cells (MRC-5) system. The proliferated viral N-glycan profiles were analyzed by a glycomic method that combined the lectin microarray and MALDI-TOF/TOF-MS. As a result, HA and NA of H9N2 viruses prossess six and five highly conserved N-glycosylation sites, respectively. Sixteen lectins (e.g., MAL-II, SNA and UEA-I) had increased expression levels of the glycan structures in the MRC-5 compared with the ECE system; however, 6 lectins (e.g., PHA-E, PSA and DSA) had contrasting results. Eleven glycans from the ECE system and 13 glycans from the MRC-5 system were identified. Our results showed that the Fucα-1,6GlcNAc(core fucose) structure was increased, and pentaantennary N-glycans were only observed in the ECE system. The SAα2-3/6 Gal structures were highly expressed and Fucα1-2Galβ1-4GlcNAc structures were only observed in the MRC-5 system. We conclude that the existing SAα2-3/6 Gal sialoglycans make the offspring of the H9N2 virus prefer entially attach to each other, which decreases the virulence. Alterations in the glycosylation sites for the evolution and role of IAVs have been widely described; however, little is known about the exact glycan structures for the same influenza strain from different hosts. Our findings may provide a novel way for further discussing the molecular mechanism of the viral transmission and virulence associated with viral glycosylation in avian and human hosts as well as vital information for designing a vaccine against influenza and other human viruses.

•RNApc (RNA polymerase complex) segments in this research covered overall subtypes.•Tracing the cladistic distribution, co-evolutionary relationship could be inferred.•RNApc segments showed a much closer correlation than others.The RNA polymerase complex (RNApc) in influenza A viruses (IVs) is composed of the PB2, PB1 and PA subunits, which are encoded by the three longest genome segments (Seg1-3) and are responsible for the replication of vRNAs and transcription of viral mRNAs. However, the co-evolutionary relationships of the three segments from the known 126 subtypes IVs are unclear. In this study, we performed a detailed analysis based on a total number of 121,191 nucleotide sequences. Three segment sequences were aligned before the repeated, incomplete and mixed sequences were removed for homologous and phylogenetic analyses. Subsequently, the estimated substitution rates and TMRCAs (Times for Most Recent Common Ancestor) were calculated by 175 representative IVs. Tracing the cladistic distribution of three segments from these IVs, co-evolutionary patterns and trajectories could be inferred. The further correlation analysis of six internal protein coding segments reflect the RNApc segments have the closer correlation than others during continuous reassortments. This global approach facilitates the establishment of a fast antiviral strategy and monitoring of viral variation.

Hepatitis B is a potentially life-threatening liver infection caused by the hepatitis B virus (HBV), which can lead to chronic liver disease and put people at high risk of death from cirrhosis of the liver and liver cancer. However, little is known about the correlation of salivary N-linked glycans related to HBV-infected liver diseases. Here we investigated N-linked glycome in saliva from 200 subjects (50 healthy volunteers (HV), 40 HBV-infected patients (HB), 50 cirrhosis patients (HC), and 60 hepatocellular carcinoma patients (HCC) using MALDI-TOF/TOF-MS. Representative MS spectra of N-glycans with signal-to-noise ratios >6 were annotated using the GlycoWorkbench program. A total of 40, 47, 29, and 33 N-glycan peaks were identified and annotated from HV, HB, HC, and HCC groups, respectively. There were 15 N-glycan peaks (e.g., m/z 1647.587, 1688.613 and 2101.755) were present in all groups. Three N-glycan peaks (m/z 2596.925, 2756.962, and 2921.031) were unique in HV group, 2 N-glycan peaks (m/z 1898.676 and 1971.692) were unique in HB group, 5 N-glycan peaks (m/z 1954.677, 2507.914, 2580.930, 2637.952, and 3092.120) were unique in HC group, and 3 N-glycan peaks (m/z 2240.830, 2507.914, and 3931.338) were unique in HCC group. The proportion of fucosylated N-glycans was apparently increased in the HCC group (84.8%) than in any other group (73.1% ± 0.01), however, the proportion of sialylated N-glycans was decreased in HCC group (12.1%) than in any other group (17.23% ± 0.003). Our data provide pivotal information to distinguish between HBV-associated hepatitis, cirrhosis and HCC, and facilitate the discovery of biomarkers for HCC during its early stages based on precise alterations of N-linked glycans in saliva.

In our recent study, we found that the expression levels of total xylose-binding proteins (XBPs) were up-regulated significantly in activated hepatic stellate cells (HSCs); however, the denomination, distribution, and function of the XBPs were uncharted. Herein, 70 XBPs from activated HSCs and 64 XBPs from quiescent HSCs were isolated, identified and annotated. A total of 30 XBPs were up-regulated (all fold change ≥ 1.5, p ≤ 0.05) and 14 XBPs were down-regulated (all fold change ≤ 0.67, p ≤ 0.05) in the activated HSCs. The XBPs were localized at the cytoplasm and cytoplasmic membrane in HSCs and cirrhotic liver tissues by cy/histochemistry. The XBPs (i.e. PDIA6 and CFL2) responsible for the regulation of protein binding were up-regulated and those responsible for the regulation of catalytic activity (i.e. TUBB and MX1) were up-regulated in the activated HSCs. 2 candidates (i.e. PDIA6 and APOA1) were then selected for further verification in the sera of patients with HBV-induced chronic hepatitis/cirrhosis using western blotting and serum microarrays. PDIA6 showed a higher discrimination (Area Under Curves, AUCs = 0.8985, p < 0.0001) relative to APOA1 (AUCs = 0.8738, p < 0.0001) in the sera of patients as biomarker candidate. In conclusion, the precision alteration of the XBPs associated with pathological changes in HSCs during liver fibrosis/cirrhosis may provide pivotal information needed to discover potential glycan-binding protein-related biomarkers for diagnosis of liver fibrosis/cirrhosis and for development of new anti-fibrotic strategies.

The incidence of hepatocellular carcinoma (HCC) is closely correlated with hepatitis B virus (HBV)-induced liver cirrhosis. Structural changes in the glycans of serum and tissue proteins are reliable indicators of liver damage. However, little is known about the alteration of liver glycopatterns during cirrhosis and tumor progression induced by HBV infection. This study compared the differential expression of liver glycopatterns in 7 sets of normal pericarcinomatous tissues (PCTs), cirrhotic, and tumor tissues from patients with liver cirrhosis and HCC induced by HBV using lectin microarrays. Fluorescence-based lectin histochemistry and lectin blotting were further utilized to validate and assess the expression and distribution of certain glycans in 9 sets of corresponding liver tissue sections. Eight lectins (e.g., Jacalin and AAL) revealed significant difference in cirrhotic tissues versus PCTs. Eleven lectins (e.g., EEL and SJA) showed significant alteration during cirrhotic and tumor progression. The expression of Galα1-3(Fucα1-2)Gal (EEL) and fucosyltransferase 1 was mainly increasing in the cytoplasm of hepatocytes during PCTs-cirrhotic-tumor tissues progression, while the expression of T antigen (ACA and PNA) was decreased sharply in cytoplasm of tumor hepatocytes. Understanding the precision alteration of liver glycopatterns related to the development of hepatitis, cirrhosis, and tumor induced by HBV infection may help elucidate the molecular mechanisms underlying the progression of chronic liver diseases and develop new antineoplastic therapeutic strategies.

Although aberrant glycosylation of human glycoproteins is related to liver fibrosis that results from chronic damage to the liver in conjunction with the activation of hepatic stellate cells (HSCs), little is known about the precision alteration of protein glycosylation referred to the activation of HSCs by transforming growth factor-β1 (TGF-β1). The human HSCs, LX-2 were activated by TGF-β1. The lectin microarrays were used to probe the alteration of protein glycosylation in the activated HSCs compared with the quiescent HSCs. Lectin histochemistry was used to further validate the lectin binding profiles and assess the distribution of glycosidic residues in cells. As a result, 14 lectins (e. g. AAL, PHA-E, ECA and ConA) showed increased signal while 7 lectins (e. g. UEA-I and GNA) showed decreased signal in the activated LX-2 compared with the quiescent LX-2. Meanwhile, AAL, PHA-E and ECA staining showed moderate binding to the cytoplasma membrane in the quiescent LX-2, and the binding intensified in the same regions of the activated LX-2. In conclusion, the precision alteration of protein glycosylation related to the activation of the HSCs may provide useful information to find new molecular mechanism of HSC activation and antifibrotic therapeutic strategies.Highlights► We predict glycosylation site in proteins which are related to the activation of HSCs. ► The alteration of protein glycosylation in the activated LX-2 is detected and validated. ► Tn and T antigen increase significantly in the activated LX-2. ► Fucose residues with various linkages distribute in different regions of activated LX-2. ► Biantennary N-glycans binder PHA-E showed increased signal on the cell membrane of the activated LX-2.

The discovery, isolation, and subsequent identification of cell membrane glycoproteins involved in the structure and function of the cell surface are becoming more and more important. Here, concanavalin A–magnetic particle conjugates were employed to isolate the special membrane glycoproteins from living HepG-2 cells. The isolated glycoproteins were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and liquid chromatography–tandem mass spectrometry as well as annotated. A total of 37 membrane glycoproteins were identified, and 25 of them were ascertained to locate in the extracellular region.

•A gold nanoparticle-based microarray for simultaneously detecting seven swine viruses.•All targets were labeled during gold nanoparticle-based multiplex asymmetric PCR.•Single stranded GNP-labeled amplicons were directly used for hybridization.•The assay is appropriate for the routine detection of multiple infections in pigs.A gold nanoparticle (GNP) based oligonucleotide microarray assay (GNMA) that combined GNP-based multiplex asymmetric PCR with silver enhancement detection, was developed for simultaneous detection of seven important swine viruses in intensive swine production. Multiplex PCR was first performed to enable the target fragments of seven viruses containing a universal sequence, which were labeled simultaneously with GNPs by multiplex asymmetric PCR in the presence of excess GNP-conjugated primer. Target labeled products were captured by virus-specific oligonucleotide probes immobilized on microarrays, followed by silver staining for signal enhancement. Black image of microarray spots was easily detected by the naked eye or a simple flatbed scanner and quantified. The results for purified plasmid constructs indicated that the assay was highly specific for detecting the seven viruses in single or multiple infections, and as few as 6–80 copies/μl of specific viral target fragments were detected successfully. Fifty-seven archived samples were tested by this assay, and the results were 100% consistent with previous results based on real-time PCR and those obtained by conventional PCR/RT-PCR and sequencing. The assay is appropriate for the routine diagnosis of viral infections in pigs due to its simplicity, low-cost, high specificity and sensitivity.