Aleuria Aurantia Lectin Research Articles

A lectin-coupled, multiple reaction monitoring based quantitative analysis of human plasma glycoproteins by mass spectrometry

Aberrant protein glycosylation may be closely associated with cancer pathology. To measure the abundance of protein glycoforms with a specific glycan structure in plasma samples, we developed a lectin-coupled multiple reaction monitoring (MRM)-based mass spectrometric method. It was confirmed that the method could provide reproducible results with precision sufficient to distinguish differences in the abundance of protein glycoforms between individuals. Plasma samples prepared from hepatocellular carcinoma (HCC) patients without immuno-depletion of highly abundant plasma proteins were fractionated by use of fucose-specific aleuria aurantia lectin (AAL) immobilized on magnetic beads by use of a biotin-streptavidin conjugate. The lectin-captured fractions were digested by trypsin and profiled by tandem mass spectrometry. From the proteomic profiling data, target glycoproteins were selected and analyzed quantitatively by MRM-based analysis. The reproducibility of MRM-based quantification of the selected target proteins was reliable, with precision (CV; ≤14% for batch-to-batch replicates and ≤19% for replicates over three days) sufficient to distinguish differences in the abundance of AAL-captured glycoforms between individual plasma samples. This lectin-coupled, MRM-based method, measuring only lectin-captured glycoforms of a target protein rather than total target protein, is a tool for monitoring differences between individuals by measuring the abundance of aberrant glycoforms of a target protein related to a disease. This method may be further applied to rapid verification of biomarker candidates involved in aberrant protein glycosylation in human plasma.

A multiplex lectin-channel monitoring method for human serum glycoproteins by quantitative mass spectrometry

A mass profiling method and multiple reaction monitoring (MRM)-based quantitative approach were used to analyze multiple lectin-captured fractions of human serum using different lectins such as aleuria aurantia lectin (AAL), phytohemagglutinin-L(4) (L-PHA), concanavalin A (Con A), and Datura stramonium agglutinin (DSA) to quantitatively monitor protein glycosylation diversity. Each fraction, prepared by multiple lectin-fractionation and tryptic digestion, was analyzed by 1-D LC-MS/MS. Semi-quantitative profiling showed that the list of glycoproteins identified from each lectin-captured fraction is significantly different according to the used lectin. Thus, it was confirmed that the multiplex lectin-channel monitoring (LCM) using multiple lectins is useful for investigating protein glycosylation diversity in a proteome sample. Based on the semi-quantitative mass profiling, target proteins showing lectin-specificity among each lectin-captured fraction were selected and analyzed by the MRM-based method in triplicate using each lectin-captured fraction (average CV 7.9%). The MRM-based analysis for each lectin-captured fraction was similar to those obtained by the profiling experiments. The abundance of each target protein measured varied dramatically, based on the lectin-specificity. The multiplex LCM approach using MRM-based analyses is useful for quantitatively monitoring target protein glycoforms selectively fractionated by multiple lectins. Thus through multiplex LCM rather than single, we could inquire minutely into protein glycosylation states.

Development of an antibody-lectin enzyme immunoassay for fucosylated α-fetoprotein

BackgroundFucosylation is one of the most important types of glycosylations related to cancer. Our previous studies of the enzymatic basis and structural studies of α-fetoprotein (AFP) samples from liver cancer patients indicated that core-fucosylation by α1,6-fucosyltransferase (FUT8) resulted in the production of fucosylated AFP, and in fact fucosylated AFP allowed differential diagnosis in some types of liver cancer from liver cirrhosis. This served as a predictive biomarker for the development of liver cancer 3 to 18months before it could be detected using imaging techniques. Fucosylated AFP is currently measured by means of a liquid-phase binding assay (LBA) or by an electrokinetic analyte transport assay (EATA). However, these methods require special instrumentation that is currently available only in major medical laboratories. To overcome this problem, we attempted to develop an enzyme immunoassay (EIA) based on the sandwich technique with specific antibody and lectin. ResultsDilute solutions of highly fucosylated AFP in human sera were assayed using a microtiter plate coated with a periodate-oxidized anti-AFP antibody, a fucose-specific biotinylated Aleuria aurantia lectin (AAL), a peroxidase-conjugated streptoavidin, and a chemiluminescent detection system. The technique was able to measure highly fucosylated AFP diluted to 5 to 80ng/ml in human sera using the developed antibody-lectin EIA in combination with the enrichment of AFP. ConclusionA simple method using an antibody-lectin EIA for quantifying fucosylated AFP that does not require special instrumentation was developed. General significanceThe method can be generally applied to the quantitative measurement of various fucosylated glycoproteins using specific antibodies. This article is part of a Special Issue entitled Glycoproteomics.

Elucidating the selectivity of recombinant forms of Aleuria aurantia lectin using weak affinity chromatography

Aberrant glycosylation is connected to several pathological conditions and lectins are useful tools to characterize glycosylated biomarkers. The Aleuria aurantia lectin (AAL) is of special interest since it interacts with all types of fucosylated saccharides. AAL has been expressed in Escherichia coli as a fully functional recombinant protein. Engineered variants of AAL have been developed with the aim of creating monovalent lectins with more homogenous binding characteristics. Four different forms of AAL were studied in the present work: native AAL purified from A. aurantia mushrooms, recombinant AAL dimer, recombinant AAL monomer and recombinant AAL site 2 (S2-AAL). The affinities of these AAL forms toward a number of saccharides were determined with weak affinity chromatography (WAC). Disaccharides with fucose linked α1–3 to GlcNAc interacted with higher affinity compared to fucose linked α1–6 or α1–4 and the obtained dissociation constants (Kd) were in the range of 10μM for all AAL forms. Tetra- and pentasaccharides with fucose in α1–2, α1–3 or α1–4 had Kd values ranging from 0.1 to 7mM while a large α1–6 fucosylated oligosaccharide had a Kd of about 20μM. The recombinant multivalent AAL forms and native AAL exhibited similar affinities toward all saccharides, but S2-AAL had a lower affinity especially regarding a sialic acid containing fucosylated saccharide. It was demonstrated that WAC is a valuable technique in determining the detailed binding profile of the lectins. Specific advantages with WAC include a low consumption of non-labeled saccharides, possibility to analyze mixtures and a simple procedure using standard HPLC equipment.

Characterization and Scope of S-layer Protein O-Glycosylation in Tannerella forsythia

Cell surface glycosylation is an important element in defining the life of pathogenic bacteria. Tannerella forsythia is a Gram-negative, anaerobic periodontal pathogen inhabiting the subgingival plaque biofilms. It is completely covered by a two-dimensional crystalline surface layer (S-layer) composed of two glycoproteins. Although the S-layer has previously been shown to delay the bacterium's recognition by the innate immune system, we characterize here the S-layer protein O-glycosylation as a potential virulence factor. The T. forsythia S-layer glycan was elucidated by a combination of electrospray ionization-tandem mass spectrometry and nuclear magnetic resonance spectroscopy as an oligosaccharide with the structure 4-Me-β-ManpNAcCONH(2)-(1→3)-[Pse5Am7Gc-(2→4)-]-β-ManpNAcA-(1→4)-[4-Me-α-Galp-(1→2)-]-α-Fucp-(1→4)-[-α-Xylp-(1→3)-]-β-GlcpA-(1→3)-[-β-Digp-(1→2)-]-α-Galp, which is O-glycosidically linked to distinct serine and threonine residues within the three-amino acid motif (D)(S/T)(A/I/L/M/T/V) on either S-layer protein. This S-layer glycan obviously impacts the life style of T. forsythia because increased biofilm formation of an UDP-N-acetylmannosaminuronic acid dehydrogenase mutant can be correlated with the presence of truncated S-layer glycans. We found that several other proteins of T. forsythia are modified with that specific oligosaccharide. Proteomics identified two of them as being among previously classified antigenic outer membrane proteins that are up-regulated under biofilm conditions, in addition to two predicted antigenic lipoproteins. Theoretical analysis of the S-layer O-glycosylation of T. forsythia indicates the involvement of a 6.8-kb gene locus that is conserved among different bacteria from the Bacteroidetes phylum. Together, these findings reveal the presence of a protein O-glycosylation system in T. forsythia that is essential for creating a rich glycoproteome pinpointing a possible relevance for the virulence of this bacterium.

Oral microparticulate vaccine for melanoma using M-cell targeting

Cancer vaccines are limited in their use, because of their inability to mount a robust anti-tumor immune response. Thus, targeting M-cells in the small intestine, which are responsible for entry of many pathogens, will be an attractive way to elicit a strong immune response toward particulate antigens. Therefore, in the present investigation, we demonstrated that efficient oral vaccination against melanoma antigens could be accomplished by incorporating the antigens in an albumin-based microparticle with a ligand AAL (Aleuria aurantia lectin) targeted specifically to M-cells. The oral microparticulate vaccine effectively protected the mice from subcutaneous challenge with tumor cells in prophylactic settings. The animals were vaccinated with antigen microparticles having a size range of around 1–1.25 µm where one prime and four booster doses were administered every 14 days over 10 weeks of duration, followed by challenge with live tumor cells, which showed complete tumor protection after oral vaccination. With the inclusion of ligand in the microparticles, we observed significantly higher IgG titers (1565 μg/mL) as compared to the microparticle formulations without AAL (872 μg/mL). This data suggests that ligand loaded microparticles may have the potential to target antigens to M-cells for an efficient oral vaccination.

Application of partial-filling capillary electrophoresis using lectins and glycosidases for the characterization of oligosaccharides in a therapeutic antibody

Oligosaccharides in therapeutic recombinant antibodies play important roles in regulation of various biological functions. To monitor the glycosylation profiles of antibody pharmaceuticals in the manufacturing process, a highly sensitive and specific method is required. We extended partial-filling techniques using lectins and exoglycosidases in capillary electrophoresis for the characterization of 8-aminopylene-1,3,6-trisulfonic acid labeled N-linked oligosaccharides derived from the therapeutic antibody rituximab. In the lectin-filling method, Galb1–4GlcNAc-specific Erythrina cristagali agglutinin, a1, 6-linked Fuc-specific Aleuria aurantia lectin and Neu5Aca2–3Gal-specific Maackia amurensis lectin were used. The oligosaccharides migrated through the lectin plug during separation; the changes in separation profiles were observed according to the interaction with the lectins. The glycosidase-filling method allowed rapid digestion as suggested by the electropherograms. Partial-filling CE methods can avoid tedious hands-on procedures such as overnight incubation and optimization reaction condition with lectins and exoglycosidases. Combination of these partial-filling capillary electrophoresis methods makes the characterization of oligosaccharide profiles of therapeutic antibodies easier and faster.

Abstract A27: Comprehensive analysis of glycans on α1-acid glycoprotein from various cancer patients by MALDI-TOF-MS for assigning a prognostic predictor

Abstract α1-Acid glycoprotein (AGP) is well-known as an acute-phase protein, a major plasma glycoprotein with highly glycosylated, branched N-linked glycans and one of the markers for inflammation. In our previous study (Cancer 101:2825–2836, 2004), with the aid of crossed affinoimmunoelectrophoresis (CAIE), glycoforms of plasma AGP from various cancer patients could be easily determined based on the degrees of branching and extent of fucosylation characterized by reactivities with Con A lectin and Aleuria aurantia lectin, respectively. Results clearly indicated that patients with advanced malignancies who had AGP glycoforms that contained highly fucosylated tri- and tetraantennary glycans for long periods after surgery were likely to have a poor prognosis. However, patients who had AGP glycoforms with no such changes were expected to have a good prognosis irrespective of their clinical stages. In this study, we purified AGP from plasma samples of the same patients and then analysed detailed structures of their glycans involving a new tumor marker using a high throughput glycosylation kit (BlotGlyco, Sumitomo Bakelite, Tokyo, Japan) combined with MALDI-TOF-MS analysis. More than two hundreds of glycan structures were assigned and relative abundance of each glycan was calculated. Follow-up studies of the relative abundance of glycans indicated that the abundance of fucosylated tri- and tetraantennary glycans in patients with a poor prognosis was quite high, and most of which were estimated to form sialyl-LeX structures (NeuAcα2,3Galβ1,4[Fucα1,3]GlcNAcβ) with the non-reducing end sialyl residues. Whereas, in those with a good prognosis, such an abundance was found to be as low as in healthy controls. These results indicated that no distinct difference was observed from our previous CAIE data and that more precise glycan structures instead of glycoforms could be easily assigned by the present method, providing an aberrant marker for predicting the fate of patients after surgery. Citation Information: Cancer Prev Res 2011;4(10 Suppl):A27.

α1‐3/4 fucosylation at Asn 241 of β‐haptoglobin is a novel marker for colon cancer: A combinatorial approach for development of glycan biomarkers

Aberrant glycosylation has been observed in many types of cancer, but the mechanism of glycosylation change is still poorly understood. To elucidate relationships between glycosylation and colon cancer progression, we analyzed glycosylation status of β-haptoglobin (β-Hp) obtained from 46 cancer patients, 14 inflammatory bowel disease patients and 38 normal subjects. Aleuria aurantia lectin reactivity with cancer β-Hp was much higher than in the other two study groups. These results were confirmed by lectin blotting and microarray assay using other lectins directed to fucosyl residues. Levels of such glycans were correlated with stage of colon cancer progression. Reactivity with fucosylated glycans was eliminated by treatment with α1-3/4 fucosidase but not α1-6 fucosidase, indicating that enhanced lectin reactivity with the fucose moiety of colon cancer β-Hp is due to Fucα1-3/4GlcNAc. Moreover, site-specific glycan occupancy was determined by sequential LC/MS analysis. Mass spectrometric analysis showed that fucosylation of β-Hp was higher in colon cancer patients than in other subjects. In particular, fucosylation at Asn 241 of β-Hp in sera of colon cancer patients was clearly higher than in the other groups, and the ratio of fucosylated glycopeptides containing Asn 241 decreased greatly after treatment with α1-3/4 fucosidase. In conclusion, the level of α1-3/4 fucosyl epitope at Asn 241 of β-Hp is potentially useful as a novel marker for colon cancer.

Development of recombinant Aleuria aurantia lectins with altered binding specificities to fucosylated glycans

Changes in glycosylation have long been associated with disease. While there are many methods to detect changes in glycosylation, plant derived lectins are often used to determine changes on specific proteins or molecules of interest. One change in glycosylation that has been observed by us and by others is a disease or antigen associated increase in fucosylation on N-linked glycans. To measure this change, the fucose binding Aleuria aurantia lectin (AAL) is often utilized in plate and solution based assays. AAL is a mushroom derived lectin that contains five fucose binding sites that preferentially bind fucose linked (α-1,3, α-1,2, α-,4, and α-1,6) to N-acetyllactosamine related structures. Recently, several reports by us and by others have indicated that specific fucose linkages found on certain serum biomarker glycoprotein’s are more associated with disease than others. Taking a site-directed mutagenesis approach, we have created a set of recombinant AAL proteins that display altered binding affinities to different analytes containing various fucose linkages.

Use of fluorescent lectin binding to distinguish Teladorsagia circumcincta and Haemonchus contortus eggs, third-stage larvae and adult worms

Lectin binding to carbohydrates on parasite surfaces has been investigated as a method of distinguishing adult worms, eggs and sheathed and exsheathed L3 of Teladorsagia circumcincta and Haemonchus contortus, economically important abomasal parasites in temperate climates. Both species were maintained as pure laboratory cultures of field isolates from New Zealand. Each of the four life cycle stages could be distinguished by the binding of at least one lectin: adult worms by Sambucus nigra agglutinin (SNA); eggs by peanut agglutinin (PNA), ConcavalinA and Lens culinaris agglutinin (LCA); exsheathed L3 by Griffonia simplicifolia-I lectin (GSL-I) and Lotus tetragonolobus lectin (LTL) and sheathed L3 by Aleuria aurantia lectin (AAL). The whole surface of both adult T. circumcincta and H. contortus strongly bound lectins specific for N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), mannose and fucose, but the two species could be distinguished by SNA binding only to T. circumcincta. Eggs could be distinguished by the binding of mannose-specific PNA to H. contortus and GalNAc-specific LCA and PSA to T. circumcincta eggs. GalNAc, GlcNAc and mannose lectins bound to the cuticle and over the excretory pores of a large proportion of sheathed L3 of both species, but only the H. contortus surface had exposed fucose or sialic acid complexes. The distinguishing lectin for sheathed L3 was AAL, which did not bind to T. circumcincta, but bound weakly to the head region of all fresh H. contortus and to 50-90% after 3months storage. The cuticle of exsheathed L3 was unresponsive to all 19 lectins, and any binding was restricted to the head and tail regions. L3 exsheathed after 2-4months storage could be distinguished by the binding of GSL-I and LTL to H. contortus but not to T. circumcincta. Lectin binding could be a useful adjunct in identifying L3, but lacked the consistency to be definitive, whereas it could be further developed as a practical method of distinguishing parasitic nematodes at other stages in the life cycle, particularly the eggs.

Partial-filling affinity capillary electrophoresis of glycoprotein oligosaccharides derivatized with 8-aminopyrene-1,3,6-trisulfonic acid

Partial-filling affinity capillary electrophoresis has been applied to the simultaneous analysis of interactions between glycoprotein oligosaccharides and certain plant lectins. A lectin solution and a mixture of glycoprotein-derived oligosaccharides labeled with 8-aminopyrene-1,3,6-trisulfonic acid were introduced to a neutrally coated capillary in this order, and separated by application of a negative voltage. Interaction of a lectin with each oligosaccharide in the mixture was observed as the specific retardation or dissipation of peaks, in addition to the size/charge separation of oligosaccharides by zone electrophoresis in the remainder (≈90%) of the capillary. The strength of the interaction with lectin was controlled by introducing an appropriate volume of lectin solution. Application of various specificities of lectins indicated characteristic migration profiles of the oligosaccharides. Moreover, sequential injection of four lectins ( Maachia amurensis mitogen, Sambucus sieboldiana agglutinin, Erythrina cristagalli agglutinin, Aleuria aurantia lectin) induced complete dissipation of complex-type oligosaccharides and enabled specific determination of the presence of high-mannose oligosaccharides without the interference or alteration of the electropherogram in porcine thyroglobulin. This method was also applied to determine the binding constants of ovalbumin-derived oligosaccharides to wheat germ agglutinin.

A comparative evaluation of Golgi protein-73, fucosylated hemopexin, α-fetoprotein, and PIVKA-II in the serum of patients with chronic hepatitis, cirrhosis, and hepatocellular carcinoma

Golgi protein-73 (GP73) and fucosylated proteins have been proposed as potential serum markers for liver disease and/or hepatocellular carcinoma (HCC). The purpose of this study was to compare the sensitivity and specificity of serum GP73 and fucosylated hemopexin (Fuc-HPX) with α-fetoprotein (AFP) and with protein induced by the absence of vitamin K or antagonist-II (PIVKA-II) for diagnosing chronic hepatitis, cirrhosis, and HCC. The concentration of GP73 in human sera was determined using an enzyme-linked immunosorbent assay employing mouse monoclonal and rabbit polyclonal GP73 antibodies. Fuc-HPX was detected using a lectin chemiluminescence-linked immunosorbent assay using a mouse monoclonal anti-hemopexin antibody and Aleuria aurantia lectin. A total of 229 serum samples from patients with chronic hepatitis, cirrhosis, and HCC, as well as from normal individuals were evaluated using these four markers. GP73 and Fuc-HPX showed significantly higher values in samples from patients with cirrhosis and HCC than in samples from patients with hepatitis and from normal individuals. The areas under the curves (AUCs) for GP73, Fuc-HPX, AFP, and PIVKA-II were 0.90, 0.77, 0.74, and 0.88, respectively, for liver cirrhosis and HCC samples vs. hepatitis and normal samples. The AUCs of GP73, Fuc-HPX, AFP, and PIVKA-II were 0.78, 0.72, 0.81, and 0.90, respectively, for HCC samples vs. all other samples. PIVKA-II showed superior sensitivity and specificity for HCC compared with the other three markers. GP73 may be useful for detecting cirrhosis as a risk factor for HCC. Fuc-HPX showed inferior sensitivity and specificity compared to the other markers.

Alterations of glycan branching and differential expression of sialic acid on alpha fetoprotein among hepatitis patients

The level of serum glycoproteins and their glycosylation pattern change in liver diseases including hepatocellular carcinoma (HCC). Some of them, especially alpha fetoprotein (AFP), serve as useful biomarkers for HCC. The present investigation showed high level of AFP in hepatitis B cirrhosis (HBV-LC) and hepatitis C cirrhosis (HCV-LC) patients. However, increase of AFP level was not significantly high in chronic hepatitis B (HBV-CH) as determined by ELISA using monoclonal anti-human AFP (mAb-AFP). The differential expression of sialic acid linkage was observed in HBV-CH and HCV-LC by ELISA; the former bound strongly with Sambucus nigra agglutinin (SNA), which has exclusive binding specificity for NeuAcα2-6-, whereas HCV-LC reacted preferably with Maackia amurensis agglutinin (MAA) which recognizes NeuAcα2-3-. There was significantly high glycan branching in HBV-LC and HCV-LC in comparison to controls as illustrated by concanavalin A. This was further confirmed by Phaseolus vulgaris erythroagglutinin (E-PHA) and Datura stramonium agglutinin (DSA). Enhanced fucosylation of AFP was observed in HBV-LC, HCV-LC and HCC patients by ELISA using fucose binding Aleuria aurantia lectin; however, maximum binding was found in HCC. Fucosylation with α1-6 linkage was further confirmed by Lens culinaris agglutinin (LCA). From the above results it is concluded that the changes in concentration of AFP, differential expression of sialic acid, increase of glycan branching and fucosylation have a prognostic value of hepatitis and it could be possible that lectin-based assay with AFP can aid in diagnosis of hepatitis diseases besides clinical examination and routine laboratory investigation.

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

BackgroundWe 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 FindingsPatients 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/SignificanceThis 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.

A lectin affinity workflow targeting glycosite-specific, cancer-related carbohydrate structures in trypsin-digested human plasma

Glycans are cell-type-specific, posttranslational protein modifications that are modulated during developmental and disease processes. As such, glycoproteins are attractive biomarker candidates. Here, we describe a mass spectrometry-based workflow that incorporates lectin affinity chromatography to enrich for proteins that carry specific glycan structures. As increases in sialylation and fucosylation are prominent among cancer-associated modifications, we focused on Sambucus nigra agglutinin (SNA) and Aleuria aurantia lectin (AAL), lectins which bind sialic acid- and fucose-containing structures, respectively. Fucosylated and sialylated glycopeptides from human lactoferrin served as positive controls, and high-mannose structures from yeast invertase served as negative controls. The standards were spiked into Multiple Affinity Removal System (MARS) 14-depleted, trypsin-digested human plasma from healthy donors. Samples were loaded onto lectin columns, separated by HPLC into flow-through and bound fractions, and treated with peptide: N-glycosidase F to remove N-linked glycans. The deglycosylated peptide fractions were interrogated by ESI HPLC-MS/MS. We identified a total of 122 human plasma glycoproteins containing 247 unique glycosites. Importantly, several of the observed glycoproteins (e.g., cadherin 5 and neutrophil gelatinase-associated lipocalin) typically circulate in plasma at low nanogram per milliliter levels. Together, these results provide mass spectrometry-based evidence of the utility of incorporating lectin-separation platforms into cancer biomarker discovery pipelines.

Identification of fucosylated glycoconjugates in Xenopus laevis testis by lectin histochemistry

Glycoconjugates play roles in many physiological and pathological processes. Previous works have shown important functions mediated by glycans in spermatogenesis, and the carbohydrate composition of testis has been studied by several approaches, including lectin-histochemical methods. However, the testis of Xenopus laevis, an animal model extensively employed in biochemical, cell and developmental research, has not yet been analysed. The aim of this work was to carry out a histochemical study of the fucose (Fuc)-containing glycoconjugates of Xenopus testis by means of lectins, combined with deglycosylation pretreatments. Four Fuc-binding lectins were used: orange peel (Aleuria aurantia) lectin (AAL), gorse seed (Ulex europaeus) agglutinin-I (UEA-I), fresh water eel (Anguilla anguilla) agglutinin (AAA), and asparagus pea (Lotus tetragonolobus) agglutinin (LTA), each recognizing different forms of fucosylated glycans. Labelling with UEA-I, which preferably binds Fucalpha(1,2) containing oligosaccharides, did not show any appreciable staining. LTA, specific for Fucalpha(1,3), and AAA, which binds Fucalpha(1,2), labelled spermatocytes and spermatids, but no labelling was seen when the histochemical procedure was carried out after either beta-elimination (which removes O-linked oligosaccharides) or incubation with PNGase F (which removes N-linked oligosaccharides), suggesting that fucosylated glycans are of both N- and O-linked types. AAL, which has its highest affinity to Fucalpha(1,6), but also recognizes Fucalpha(1,2) and Fucalpha(1,3), labelled the whole testis, and the staining remained when the histochemical method was performed after either beta-elimination or incubation with PNGase F. Labelling with AAL could be explained by the fact that this lectin could be binding to diverse fucosylated glycans in N- and O-glycans, and even in glycolipids. The importance of these glycans is discussed.

Clinical application of a lectin-antibody ELISA to measure fucosylated haptoglobin in sera of patients with pancreatic cancer

Recent advanced techniques in glycobiology have produced a number of tumor marker candidates. As a result from the glycomic approach, we found that fucosylated haptoglobin in sera was a possible tumor marker for pancreatic cancer (PC). Although Aleuria aurantia lectin (AAL) blotting can detect fucosylated haptoglobin, it is difficult to quantify fucosylated haptoglobin precisely. To overcome this problem, we developed a fucosylated haptoglobin detection kit as a sandwich enzyme-linked immune sorbent assay (ELISA) using AAL and the Fab portion of anti-haptoglobin antibody. In the present study, we investigated the clinical application of this lectin-antibody ELISA kit to measure fucosylated haptoglobin in PC. We measured fucosylated haptoglobin in patients with PC with a lectin-antibody ELISA kit. The fucosylated haptoglobin measured with this assay was compared with lectin blotting data, and the discrepancy was analyzed by immunoprecipitation methods. The concentration of fucosylated haptoglobin was investigated with respect to the clinical stage of PC. We also measured fucosylated haptoglobin, using 397 cases of several types of cancers including PC, benign diseases, and normal controls. The sensitivity and specificity for the differential diagnosis of PC from normal controls was 50% and 91%, respectively. The results from lectin-antibody ELISA were significantly correlated with data from previous AAL blotting studies. Positive rates of fucosylated haptoglobin with this method in patients with PC were significantly higher in cases of stage IV compared with other clinical stages. Fucosylated haptoglobin was increased in several types of cancers, in which fucosylated haptoglobin was reported to increase. While certain cases showed a discrepancy in fucosylated haptoglobin concentrations between the lectin-antibody ELISA and conventional lectin blotting, this novel type of lectin-antibody ELISA might be useful for a tumor marker for PC.

Зміна гепарин- і лектин-зв’язувальної активності фібронектину при проліферативних захворюваннях крові

Fibronectin concentration, its heparin-binding activity and interaction with Lens Culinaris Agglutinin (LCA), Aleuria Aurantia Lectin (AAL) and Laburnum Anagyroides Agglutinin (LABA) in patients with erythremia and subleukemic myelosis were investigated. Reliable decreasing of concentration and heparin-binding activity of plasma fibronectin as well as a connection between its functional activity and lectin-affinity were detected. Increasing of the level of core fucose in N-glycans of fibronectin in proliferative blood diseases was revealed. Presence of fucose in O-glycans of fibronectin and dependence of fucosylation level of O-glycans in the glycoprotein on the type of pathological processes were proved. Keywords: fibronectin, heparin, lectin, fucose, erythremia, subleukemic myelosis.

N-glycosylation status of β-haptoglobin in sera of patients with prostate cancer vs. benign prostate diseases

N-glycosylation status of purified beta-haptoglobin separated from sera of patients with prostate cancer was studied in comparison to that of sera from patients with benign prostate diseases, or normal subjects. Two different approaches, as summarized below, one based on binding of lectins and antibodies to beta-haptoglobin, the other on mass spectrometry of released N-linked glycans from beta-haptoglobin, were performed. Some of the results were useful for distinction of prostate cancer vs. benign prostate diseases. i) Binding of Phaseolus vulgaris-L lectin (PHA-L), defining the GlcNAcbeta6Manalpha6Man side chain present in tri- or tetra-antennary N-linked glycans, to beta-haptoglobin was higher for cases of prostate cancer and high-grade prostate intraepithelial neoplasia than for benign diseases. Binding of Aleuria aurantia lectin (AAL) defining Fucalpha3-, alpha4-, or alpha6-GlcNAc, or monoclonal antibody directed to sialyl-Le(x), to beta-haptoglobin was also higher for some of the cancer cases than for benign diseases. Many other lectins and antibodies showed no binding to beta-haptoglobin, or showed no significant difference between cancer vs. benign diseases. ii) Mass spectrometric analysis of N-linked glycans of beta-haptoglobin released by Peptide N-glycosidase-F showed enhanced expression of monosialyl tri-antennary structures in prostate cancer cases. Thus, binding of PHA-L to affinity-purified beta-haptoglobin from sera of patients could lead to development of useful tools for differential diagnosis of prostate cancer vs. benign prostate diseases.