Enhanced expression of α1-acid glycoprotein and fucosylation in hepatitis B patients provides an insight into pathogenesis

Altered glycosylation and concentration of α1-acid glycoprotein has been known to be related to the pathogenesis of the hepatic diseases. The present study investigated enhanced fucosylation of AGP in the sera of chronic hepatitis B (HBV-CH) and hepatitis B cirrhosis (HBV-LC) patients by high performance anion exchange chromatography and by ELISA using fucose binding Aleuria aurantia lectin. The concentration of AGP determined by ELISA using monoclonal anti-human AGP (mAb-AGP) showed high level of AGP in HBV-CH and HBV-LC patients. This was further judged by association constant (K _A) measured by surface plasmon resonance analysis. There was no apparent linkage variation of sialic acid among different patient groups when tested with two sialic acid binding lectins viz., Maackia amurensis agglutinin (MAA, NeuAc α2-3-) and Sambucus nigra agglutinin (SNA, NeuAc α2-6-) respectively. There was no change of oligosaccharide branching in HBV-CH in comparison to controls whereas a slight change was observed in HBV-LC using ConA. The above results suggest that the changes in concentration of AGP and fucosylation have a prognostic value of hepatitis diseases and it could be possible to use AGP as diagnostic marker besides clinical examination and routine laboratory investigation.     

Altered glycosylation,expression of serum haptoglobin and alpha-1-antitrypsin in chronic hepatitis C,hepatitis C induced liver cirrhosis and hepatocellular carcinoma patients

Liver cirrhosis with hepatitis C viral infection (HCV-LC) causes high risk to develop hepatocellular carcinoma (HCC). Besides diagnosis of liver cirrhosis by biochemical test, imaging techniques, assessment of structural liver damage by biopsy shows several disadvantages. Our aim was to monitor the changes in the expression level of serum proteins and their glycosylation pattern among chronic hepatitis C (HCV-CH), HCV-LC and HCC patients with respect to controls. 2D gel electrophoresis of HCV-CH, HCV-LC and HCC patients’ sera showed several protein spots, which were identified by LC-MS. The change in the expression of two prominent protein spots, haptoglobin (Hp) and alpha 1-antitrypsin (AAT) was evaluated by western blot and ELISA. The changes in glycosylation pattern of these serum proteins were assayed using different lectins. Increased level of Hp and AAT was observed in HCV-LC and HCC patients’ group whereas those were found to be present less in HCV-CH patient groups with respect to control as determined by ELISA using monoclonal antibodies. Decreased level of sialylation in both Hp and AAT was observed in HCV-LC and HCV-CH patients’ group whereas increased level of sialylation was observed in HCC patient groups by ELISA using Sambucus nigra agglutinin. On the other hand increased level of fucosylation in two serum glycoproteins was observed in HCV-LC and HCC patients’ group using Lens culinarris agglutinin. High glycan branching was found in HCV-LC and HCC patient groups in Hp but not in HCV-CH as determined by Datura stramonium agglutinin. However, there was no such change observed in glycan branching in AAT of HCV-CH and HCV-LC patients’ groups, to the contrary high glycan branching was observed in HCC patients’ group. Increased level of exposed galactose in both serum proteins was observed in both HCC patients’ group as determined by Ricinus communis agglutinin. The present glycoproteomics study could predict the progression of HCV-CH, HCV-LC and HCC without the need of liver biopsy.     

Glycans as receptors for influenza pathogenesis

Influenza A viruses, members of the Orthomyxoviridae family, are responsible for annual seasonal influenza epidemics and occasional global pandemics. The binding of viral coat glycoprotein hemagglutinin (HA) to sialylated glycan receptors on host epithelial cells is the critical initial step in the infection and transmission of these viruses. Scientists believe that a switch in the binding specificity of HA from Neu5Acα2-3Gal linked (α2-3) to Neu5Acα2-6Gal linked (α2-6) glycans is essential for the crossover of the viruses from avian to human hosts. However, studies have shown that the classification of glycan binding preference of HA based on sialic acid linkage alone is insufficient to establish a correlation between receptor specificity of HA and the efficient transmission of influenza A viruses. A recent study reported extensive diversity in the structure and composition of α2-6 glycans (which goes beyond the sialic acid linkage) in human upper respiratory epithelia and identified different glycan structural topologies. Biochemical examination of the multivalent HA binding to these diverse sialylated glycan structures also demonstrated that high affinity binding of HA to α2-6 glycans with a characteristic umbrella-like structural topology is critical for efficient human adaptation and human-human transmission of influenza A viruses. This review summarizes studies which suggest a new paradigm for understanding the role of the structure of sialylated glycan receptors in influenza virus pathogenesis.     

Fractionation and characterization of boar seminal plasma spermadhesin PSP-II glycoforms reveal the presence of uncommon N-acetylgalactosamine-containing N-linked oligosaccharides

Lectin mapping, carbohydrate analysis and electrospray mass spectrometry of boar seminal plasma PSP-II glycoforms show that its single N-glycosylation site displays a repertoire of carbohydrate structures consisting of the basic pentasaccharide core Manα 1–6[Manα 1–3]Manβ1-4GlcNAcβ1-4GlcNAc with a fucosyl residue α1-6-linked to the innermost N-acetylglucosamine residue. Other glycoforms display fucosylated hybrid-type or monoantennary complex-type chains, some of which contain α2-6-linked sialic acid. N-acetylgalactosamine, possibly in Galβ1-3GalNAc sequence, is present in most of the PSP-II glycoforms. Abbreviations: PSP-I and PSP-II, porcine seminal plasma proteins I and II; PNGaseF, peptide-N4-(N-acetyl-β-D-glucosaminyl) asparagine amidase (EC 3.5.1.52) from Flavobacterium meningosepticum; ConA, Cannavalia ensiformis (jack bean) agglutinin; GNA, Galanthus nivalis (snowdrop) agglutin; SNA, Sambucus nigra (elderberry) agglutinin; MAA, Maackia amurensis (maakia) agglutinin; PNA, Arachis hypogaea (peanut) agglutinin; DSA, Datura stramonium (jimson weed) agglutinin; AAA, Aleuria aurantia agglutinin This revised version was published online in November 2006 with corrections to the Cover Date.     

Carbohydrate-based measurements on alpha-fetoprotein in the early diagnosis of hepatocellular carcinoma

Serum alpha-fetoprotein (AFP) is a useful marker for the diagnosis of hepatocellular carcinoma (HCC), although this protein also increases moderately in benign liver diseases. The serum concentration of AFP in HCC at the time of initial diagnosis is now lower than before because of advancements in techniques for imaging the liver. The AFP concentration alone cannot distinguish between HCC and benign liver diseases, especially when it is less than 1000 ng ml^–1. These circumstances lead to the need to discriminate between these diseases. This has been achieved by determining the carbohydrate structures of AFP by its reactivity withLens culinaris agglutinin (LCA). The percentage of LCA-reactive species of AFP is significantly higher in HCC than in benign liver diseases. The fucosylation of the sugar chain at the innermostN-acetylglucosamine is the molecular basis of this variation. Therefore, the term fucosylation index has been introduced to express the percentage of LCA-reactive species of AFP. This index is useful for the diagnosis of HCC even if the carcinoma is at an early stage. Furthermore, it can predict the development of HCC in the follow-up of chronic liver diseases. Thus, the qualitative and quantitative measurements of carbohydrate in AFP provide us with very valuable information for the differential diagnosis of various liver diseases.     

Relative sialylation and fucosylation of synovial and plasma fibronectins in relation to the progression and activity of rheumatoid arthritis

The expressions of terminal sugars in synovial and plasma fibronectins were studied in relation to rheumatoid arthritis (RA) progression defined according to the early, established and late radiological changes in the patients’ hands. The relative amounts of sialic acid and fucose were analyzed by lectin-ELISA using appropriate sialic acid-linked α2-3 (Maackia amurensis) and α2-6 (Sambucus nigra) lectins as well as fucose-linked α1-6 (Aleuria aurantia), α1-2 (Ulex europaeus), and α1-3 (Tetragonolobus purpureus). In the early RA group, the synovial fibronectin reactivities were the lowest with the all lectins used. In the established and late groups, relative sialylation and fucosylation significantly increased. However, sialylation negligibly decreased, whereas fucosylation remained at nearly the same level in the late group. Moreover, the expression of α1-6-linked fucose was found to be related to disease activity. In contrast, plasma fibronectin reactivity with lectins showed different dynamic alterations. In the early RA group, the reactivity of fibronectin with the lectins used was similar to that of healthy individuals, whereas it increased significantly in the established RA group compared with the early and normal plasma groups. In the late RA group it decreased to a level similar to that of the normal group. The lower expressions of terminal sugars in synovial fibronectin were mainly associated with the early degenerative processes of RA. In conclusion, such alterations may be applicable as a stage-specific marker for diagnosis and therapy of RA patients. The higher expression of terminal sugars in fibronectin could be associated with repair and adaptation processes in longstanding disease.     

Profiling terminal N-acetyllactosamines of glycans on mammalian cells by an immuno-enzymatic assay

Profiling of carbohydrate structures on cell membranes has been difficult to perform because of the complexity and the variations of such structures on cell surface glycans. This study presents a novel method for rapid profiling of cell surface glycans for terminal N-acetyllactosamines (Galβ1-(3)4GlcNAc-R) that are uncapped, capped with sialic acid as SA-Galβ1-(3)4GlcNAc-R, or with α1,3galactosyls as the α-gal epitope- Galα1-3Galβ1-(3)4GlcNAc-R. This method includes two enzymatic reactions: (1) Terminal sialic acid is removed by neuraminidase, and (2) α-gal epitopes are synthesized on the exposed N-acetyllactosamines by α1,3galactosyltransferase. Existing and de novo synthesized α-gal epitopes on cells are quantified by a modification of radioimmunoassay designated as “ELISA inhibition assay,” which measures binding of the monoclonal anti-Gal antibody M86 to α-gal epitopes. This binding is proportional to the number of cell surface α-gal epitopes. The amount of free M86 antibody molecules remaining in the solution is determined by ELISA using synthetic α-gal epitopes linked to albumin as solid phase antigen. The number of α-gal epitopes on cells is estimated by comparing binding curves of M86 incubated with the assayed cells, at various concentrations of the cells, with the binding of M86 to rabbit red cells expressing 2 × 10⁶ α-gal epitopes/cell. We could demonstrate large variations in the number of sialic acid capped N-acetyllactosamines, α-gal epitopes and uncapped N-acetyllactosamines on different mammalian red blood cells, and on nucleated cells originating from a given tissue in various species. This method may be useful for rapid identification of changes in glycosylation patterns in cells subjected to various treatments, or in various states of differentiation.     

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 (α-1,3) fucosylation. Increases in core (α-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.     

Glycosylation ofα 1 glycoprotein in septic shock: Changes in degree of branching and in expression of sialyl Lewisx groups

The occurrence of differences in acute-phase response, with respect to concentration and glycosylation of ₁-acid glycoprotein (AGP) was studied in the sera of patients surviving or not from septic shock. Crossed affino-immunoelectrophoresis was used with concanavalin A andAleuria aurantia lectin for the detection of the degree of branching and fucosylation, respectively, and the monoclonal CSLEX-1 for the detection of sialyl Lewis^x (SLeX) groups on AGP. Septic shock apparently induced an acute-phase response as indicated by the increased serum levels and changed glycosylation of AGP. In the survivor group a transient increase in diantennary glycan content was accompanied by a gradually increasing fucosylation and SLeX expression, comparable to those observed in the early phase of an acute-inflammatory response. Remarkably, in the non-survivor group a modest increase in diantennary glycan content was accompanied by a strong elevation of the fucosylation of AGP and the expression of SLeX groups on AGP, typical for the late phase of an acute-phase response. Our results suggest that these changes in glycosylation of AGP can have a prognostic value for the outcome of septic shock.Abbreviations AAL Aleuria aurantia lectin - AGP ₁-acid glycoprotein - CAIE crossed affinoimmunoelectrophoresis - Con A Concanavalin A - HSPC human serum protein calibrator - IL-1 interleukin 1 - IL-6 interleukin 6 - LIF leukaemia inhibitory factor - LPS lipopolysaccharide - SLeX sialyl Lewis^x - TNF tumour necrosis factor     

Phosphorylation of elongation factor-2 kinase differentially regulates the enzyme's stability under stress conditions

Chronic infection with hepatitis B virus (HBV) is associated with the majority of cases of hepatocellular carcinoma (HCC) in China. Despite this, there is no effective method for the early detection of HBV-induced liver cancer. Aberrant fucosylation is known to occur during the development of HCC. We, therefore, developed a method of applying matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to analyze the relationship between aberrant fucosylation, tumor genesis and progression of HBV-associated HCC, and to establish proteomic profiling of serum for early diagnosis of HCC. The MALDI-TOF MS was based on Lens culinaris agglutinin (LCA) lectin magnetic beads and their affinity for separation. The method was applied initially to a 'training' cohort of 111 serum samples obtained from subjects in China with no liver disease (n=26), chronic hepatitis B without cirrhosis (n=21), HBV-infected cirrhosis (n=32), or HBV-infected HCC (n=32). In contrast to previous findings, the results of our profiling analysis demonstrated defucosylation on some of the glycoproteins involved in HCC. HCC was then diagnostically classified in a 'blind test' cohort (n=96). In this group we demonstrated that, HCC could be distinguished from all serum samples, HBV-associated chronic liver disease, and HBV-associated cirrhosis with a sensitivity/specificity of 70%/70%, 78%/74%, and 81%/82%, respectively. When combined with serum alpha-fetoprotein detection (AFP>20 ng/mL), the sensitivity/specificity improved to 78%/88%, 85%/88%, and 89%/91%, respectively. In conclusion, serum glycoprotein fucosylation abnormalities have diverse forms in patients with HCC. MALDI-TOF MS profiling of aberrant serum fucosylated glycoproteins distinguished HCC from controls with high accuracy.     

Production of sialyltrisaccharides using β-galactosidase andtrans-sialidase in one pot

Sialyltrisaccharides based on β-galactosyldisaccharides were synthesized using β-galactosidase andtrans-sialidase in one pot. Using β-galactosidase fromBacillus circulans andtrans-sialidase fromTrypanosoma cruzi simultaneously, 6 mM sialyltrisaccharides composed of about 95% NeuAcα(2,3)Galβ(1,4)GlcNAc and 5% NeuAcα(2,3)Galβ(1,6)GlcNAc were produced from a reaction mixture containing 25 mM 0-nitrophenyl-β-D-galactopyranoside, 100 mM N-acety lglucosamine and 10 mM p-nitrophenyl-α-D-N-acetylneuraminic acid. One beauty of this reaction was that a secondary hydrolysis of the disaccharide intermediate occurring between the activated galactopyranoside and N-acetylglucosamine was prevented. Using β-galactosidase fromEscherichia coli and the sametrans-sialidase, 15 mM sialyltrisaccharides composed of about 90% NeuAcα(2,3)Galβ(1,6)GlcNAc and 10% NeuAcα(2,3)Galβ (1,4)GlcNAc were produced from a reaction mixture containing 400 mM galactose, 800 mM N-acetylglucosamine and 20 mMp-nitrophenyl-α-D-N-acetylneuraminic acid. In this study, the reverse-galactosylation reaction between galactose and N-acetylglucosamine was dominant since the disaccharide intermediate mainly resulted in the sialylated product.     

Sialic acid-binding lectins: submolecular specificity and interaction with sialoglycoproteins and tumour cells

We examined the specificity of limulin,Limax flavus agglutinin (LFA) andSambucus nigra agglutinin I (SNA I) at the submolecular level of sialic acid, and characterized their interactions with a panel of structurally distinct sialoglycoproteins. In haemagglutination inhibition assays NeuAc--glycosides were stronger inhibitors for limulin and LFA than nativeN-acetylneuraminic acid (NeuAc). TheN-acetyl of NeuAc was crucial for binding to both lectins. N-thioacetylated NeuAc lost affinity for LFA, but still bound to limulin. Thus, distinct intermolecular interactions are involved in binding of sialic acid to the lectins. The glyceryl side chain was required for interaction with LFA, but not with limulin. SNA I specifically bound NeuAc2 6Gal1 4Glc, but not monomeric sialic acids. Limulin and LFA strongly interacted with O-chain glycoproteins, whereas SNA I preferred N-chain proteins that carry NeuAc2 6 residues. The lectins were compared with those fromCepaea hortensis andTachypleus tridentatus (TTA) and to wheat-germ agglutinin, and were then used to probe tumour cell lines for cell surface sialylation. With the exception of TTA, all lectins interacted with the tumour cells. Limulin distinguished between the low (Eb) and highly (ESb) metastatic mouse lymphoma lines by selectively agglutinating sialidase-treated ESb cells.Abbreviations BSM bovine submaxillary mucin - CHA I Cepaea hortensis agglutinin I - LFA Limax flavus agglutinin - NeuAc N-acetylneuraminic acid - OSM ovine submaxillary mucin - SNA I Sambucus nigra agglutinin I - THP Tamm-Horsfall protein - TTA Tachypleus tridentatus agglutinin     

Immunohistochemical expression of pi class glutathione S-transferase and alpha-fetoprotein in hepatocellular carcinoma and chronic liver disease

OBJECTIVE: To determine whether tumor marker pi glutathione transferase (GST-pi) is expressed in hepatocellular carcinoma (HCC) and other chronic liver diseases and to compare its expression with that of alpha-fetoprotein (AFP). STUDY DESIGN: Samples used were formalin-fixed, paraffin-embedded liver tissues: normal (n = 3), chronic hepatitis B (n = 15), cirrhosis (n = 15) and HCC (n = 30). The expression of AFP and GST-pi was detected by using immunohistochemistry with the peroxidase-antiperoxidase method. AFP immunoreactivity was based on the cytoplasm of the hepatocytes, while GST-pi immunoreactivity was based on the nuclei of hepatocytes. RESULTS: In normal liver tissues, AFP was not expressed. However, there was strong staining of GST-pi in bile duct epithelium cells and weak staining in hepatocytes. Our results showed higher AFP immunoreactivity in cases of HCC (36.7%) as compared to cirrhosis (6.7%) and hepatitis B (0%), whereas GST-pi immunoreactivity was lower in cases of HCC (53.3%) as compared to cases of cirrhosis (100.0%) and hepatitis B (93.3%). Percent sensitivity of AFP determination for HCC was 36.7% as compared to 53.3% for GST-pi, thus making GST-pi a more sensitive marker for detection of HCC. This study showed a significant relationship between the intensity and percentage of cells stained in hepatitis B, cirrhosis and HCC for GST-pi immunoreactivity (P < .001, .001 and .05, respectively) but not for AFP (P > .05). Statistical analysis showed that there was no significant relationship between expression of AFP and GST-pi in cirrhosis and HCC cases. Hepatitis B virus infection in HCC cases showed a positive rate of 46.7%, with AFP staining positively in 42.9% of tissues and GST-pi staining positively in 57.1% of tissues. CONCLUSION: AFP is a diagnostic but rather insensitive tissue marker for HCC. However, the absence of AFP in benign chronic liver disease makes this marker useful in differentiating between HCC and other chronic liver diseases, whereas GST-pi can be used as a diagnostic marker for HCC as well as in detecting other chronic liver diseases.     

The substrate specificity of the enzyme Endo-α-N-acetyl-D-galactosaminidase from Diplococcus pneumonia

The substrate specificity of the enzyme endo-α-N-acetyl-D-galactosaminidase from Diplococcus pneumonia was re-examined using bovine submaxillary mucin and remodelled antifreeze glycoprotein as substrates. Incubation with desialylated bovine submaxillary mucin, which contains six O-linked core types, indicated that the disaccharide Galβ1-3GalNAc, which is present in very small amount, was the only glycan released, while the disaccharide GlcNAcβ1-3GalNAc, which is the major structure present, and other disaccharides, were not released. To test whether the core disaccharide Galβ1-3GalNAc with sialic acid linked α2-3 to the Gal or linked α2-6 to the GalNAc was released, the enzyme was incubated with remodelled antifreeze glycoprotein containing (1) [3H]NeuAcα2-3Galβ1-3GalNAc and (2) Galβ1-3[[14C]NeuAcα2-6]GalNAc as substrates. No NeuAc-containing trisaccharide was released. These results serve to clarify the doubts of many researchers regarding the activity of this enzyme on some newly-described core types and on sialylated substrates. This revised version was published online in November 2006 with corrections to the Cover Date.     

Proteomic analysis of serum associated fucosylated glycoproteins in the development of primary hepatocellular carcinoma

Changes in N-linked glycosylation are known to occur during the development of cancer. For example, increased branching of oligosaccharides has been associated with metastasis and has been correlated to tumor progression in human cancers of the breast, colon and melanomas. Increases in core fucosylation have also been associated with the development of hepatocellular carcinoma (HCC). Chronic infection with the hepatitis B virus is associated with more than 55% of all cases of hepatocellular carcinoma. We show here that increased levels of core fucosylation can be observed via glycan analysis of total serum and are associated with the development of HCC. In a blinded study, the serum glycoproteins derived from people diagnosed with HBV induced liver cancer were found to possess a dramatically higher level of fucosylation. This change occurs on both immunoglobulin molecules and on other serum glycoproteins. Targeted glycoproteomic analysis was used to identify those glycoproteins that are hyperfucosylated in cancer. In total, 19 proteins were found to be hyperfucosylated in cancer. The potential of these proteins as biomarkers of cancer is discussed.     

Thomsen-Friedenreich-related carbohydrate antigens in normal adult human tissues: a systematic and comparative study

A broad variety of normal human tissues were examined for the expression of Thomsen-Friedenreich (TF)-related histo-blood group antigens. TF (Galβ1-3GalNAcα1-R), Tn (TF precursor, GalNAcα1-R), sialosyl-Tn (NeuAcα2-6GalNAcα1-R), considered to be useful in cancer diagnosis and immunotherapy, and sialosyl-TF, the cryptic form of TF. These antigens or, more correctly, glycotopcs, were determined by immunohistochemistry with at least two monoclonal antibodies (mAbs) each (except sialosyl-TF) as well as by lectin histochemistry. For a better dissection of sialosyl-TF and TF glycotopes, tissue sections were pretreated with galactose oxidase or the galactose oxidase-Schiff sequence. Staining with mAbs appeared to be more restricted than with the lectins used. Distribution patterns among normal epithelia were different for all four antigens. These antigens were also detected in some non-epithelial tissues. They can be classified in the following sequence according to the frequency of their occurrence in normal tissues: sialosyl-TF> >sialosyl-Tn>Tn>TF. Most of the positively staining sites for TF, Tn, and sialosyl-Tn are located in immunologically privileged areas. The complex results obtained with anti-TF mAbs (after treatment of the tissue sections with sialidase fromVibrio cholerae) and the lectins amaranthin and jacalin revealed a differential distribution of the subtypes of sialosyl-TF [NeuAcα2-3Galβ1-3GalNAcα1-R and Galβ1-3 (NeuAcα2-6)GalNAcα1-R] in normal human tissues. From our data it can be inferred that TF, Tn, and sialosyl-Tn are promising targets for a cancer vaccine.     

Galectins as tools for glycan mapping in histology: comparison of their binding profiles to the bovine zona pellucida by confocal laser scanning microscopy

Gene divergence has given rise to the galectin family of mammalian lectins. Since selective binding to distinct β-galactosides underlies the known bioactivities of galectins, they could find application in cyto- and histochemistry. The pertinent question on the characteristics of their individual reactivity profiles therefore needs to be answered. Toward this end, comparative studies of a panel of galectins in defined systems are required. We here characterise the staining profiles of seven human lectins as well as five natural derivatives originating from proteolytic truncation and serine phosphorylation and one engineered variant. As test system, bovine germinal vesicle oocytes with their glycoprotein envelope (zona pellucida), which presents bi- to tetraantennary complex-type N-glycans with N-acetyllactosamine repeats and core fucosylation, were processed. Technically, confocal laser scanning microscopy was used, first with plant lectins to map the sialylation status. Hereby, α2,3/6-sialylation was detected in the superficial filamentous meshwork of the zona pellucida, while sialic acid-free glycan chains were found to characterise the main inner part of the compact layer of the zona pellucida. Galectin staining was specific and non-uniform. Significant differences in reactivity were detected for the superficial filamentous meshwork and the compact layer of the zona pellucida between galectins-1 to -4 versus galectins-8 and -9. The typical staining profiles intimate a spatially organised display of N-glycans in the different layers of the zona pellucida, underscoring the potential of galectins as cyto- and histochemical tools. Our results encourage further comparative analysis and research to trace the underlying structural and/or topological properties.     

Sequence analysis and receptor specificity of the hemagglutinin of a recent influenza H2N2 virus isolated from chicken in North America

Influenza viruses bind host cells following an interaction between the viral hemagglutinin (HA) protein and host cell sialylated glycoproteins and glycolipids. Differences in binding affinities of the HAs for different types of sialic acid linkages (α2-3 vs. α2-6) contribute to determining the host range of an influenza virus. The ability of an avian influenza virus HA to bind the human form of the receptor may be one requirement for an avian virus to propagate in the human population. In this paper, we describe the characterization of the HA from an H2N2 virus isolated from a Pennsylvania chicken farm in 2004. Sequence analysis revealed that this HA is a member of the Eurasian clade, and receptor binding studies show that it maintains its specificity for the avian influenza virus α2-3 linked sialic acid receptor.     

Construction of an in vitro trans-sialylation system: surface display of Corynebacterium diphtheriae sialidase on Saccharomyces cerevisiae

Sialidases can be used to transfer sialic acids from sialoglycans to asialoglycoconjugates via the trans-glycosylation reaction mechanism. Some pathogenic bacteria decorate their surfaces with sialic acids which were often scavenged from host sialoglycoconjugates using their surface-localized enzymes. In this study, we constructed an in vitro trans-sialylation system by reconstructing the exogenous sialoglycoconjugate synthesis system of pathogens on the surfaces of yeast cells. The nanH gene encoding an extracellular sialidase of Corynebacterium diphtheriae was cloned into the yeast surface display vector pYD1 based on the Aga1p–Aga2p platform to immobilize the enzyme on the surface of the yeast Saccharomyces cerevisiae. The surface-displayed recombinant NanH protein was expressed as a fully active sialidase and also transferred sialic acids from pNP-α-sialoside, a sialic acid donor substrate, to human-type asialo-N-glycans. Moreover, this system was capable of attaching sialic acids to the glycans of asialofetuin via α(2,3)- or α(2,6)-linkage. The cell surface-expressed C. diphtheriae sialidase showed its potential as a useful whole cell biocatalyst for the transfer of sialic acid as well as the hydrolysis of N-glycans containing α(2,3)- and α(2,6)-linked sialic acids for glycoprotein remodeling.     

AFP-L3蛋白芯片的建立与应用

目的:建立一种可同时检测血清中甲胎蛋白(AFP)及甲胎蛋白异质体(AFP-L3)的蛋白芯片方法,为AFP-L3检测提供经济、便捷、省时、有效的新途径。方法:将鼠源AFP单克隆抗体和小扁豆凝集素点样固定在醛基玻片上,制备出AFP抗体和小扁豆凝集素蛋白芯片。利用抗原抗体特异性结合以及盐藻糖与小扁豆凝集素特异性结合的原理,用蛋白芯片方法检测血清样本中的AFP和AFP-L3。结果:肝癌血清39份,其中37份检测到AFP;26份肝癌血清中同时检测到AFP和AFP-L3,2份肝癌血清均未检测到AFP和AFP-L3。肝细胞癌组血清样本中AFP和AFP-L3水平明显高于健康对照组(P0.001),健康对照组与空白对照组统计学上无差异(P0.05)。结论:本研究成功地建立AFP和AFP-L3同时检测的蛋白芯片方法,与ELISA法和凝集素微量离心柱法相比,是一种切实可行,经济、便捷、省时、有效的方法。