Pregnancy associated glycoprotein-1, -6, -7, and -17 are major products of bovine binucleate trophoblast giant cells at midpregnancy

Pregnancy associated glycoproteins (PAGs) are extensively glycosylated secretory proteins of ruminant trophoblast cells. In cattle placenta several PAG cDNAs are expressed, but the variety of correspondent proteins and their degree of glycosylation are not well characterized. Thus, we purified PAGs by using a protocol which included a lectin (Vicia villosa agglutinin) affinity chromatography. Due to their specific glycosylation pattern, PAGs derived from binucleate trophoblast giant cells were highly enriched by this protocol. PAGs were purified from cotyledons of 2 day 100 placentas and from a single placenta at day 155 and 180. In all samples three major bands (75; 66; 56 kDa) were detected by one-dimensional SDS-PAGE. Mass-spectrometric analysis identified the 75 kDa band as a mixture of PAG-7 and PAG-6, the 66 kDa band as PAG-1 and the 56 kDa band as PAG-17. N-terminal sequencing of the day 100 sample confirmed the mass spectrometric identifications. Enzymatic release of N-glycans with peptide-N-glycanase-F from PAGs reduced the molecular weight to approximately 37 kDa which corresponds to the theoretical molecular mass of PAGs. Limited peptide-N-glycanase-F treatment revealed that all four N-glycosylation sites are quantitatively occupied in PAG-1. Compared to PAG-1 the number of potential N-glycosylation sites is lower in PAG-17 (three sites) and higher in PAG-6 and -7 (five and six sites, respectively). This suggests that the number of attached N-glycans is the main determinant of molecular mass of bovine PAGs. The degree of glycosylation may be a major factor regulating the plasma half life of PAGs.     

A tetraantennary glycan with bisecting N-acetylglucosamine and the Sd(a) antigen is the predominant N-glycan on bovine pregnancy-associated glycoproteins

Pregnancy-associated glycoproteins (PAGs) are major secretory proteins of trophoblast cells in ruminants. Binucleate trophoblast giant cells (BNCs) store these proteins in secretory granules and release them into the maternal organism after fusion with maternal uterine epithelial cells. By matrix assisted laser desorption ionisation-mass spectrometry (MALDI-MS) analysis and linkage analysis, we show that by far, the most abundant N-glycan of PAGs in midpregnancy is a tetraantennary core-fucosylated structure with a bisecting N-acetylglucosamine (GlcNAc). All four antennae consist of the Sd(a)-antigen (NeuAcalpha2-3[GalNAcbeta1-4]Galbeta1-4GlcNAc-). Immunohistochemistry with the mono- clonal antibody CT1, which recognizes the Sd(a)-antigen, shows that BNC granules contain the Sd(a)-antigen from gestation day (gd) 32 until a few days before parturition. Lectin histochemistry with Maackia amurensis lectin (MAL), which binds to alpha2-3sialylated lactosamine, shows that BNC granules are MAL-positive prior to gd 32 and also at parturition. The observed tetraantennary glycan is a highly unusual structure, since during the synthesis of N-glycans, the insertion of a bisecting GlcNAc inhibits the activity of the GlcNAc-transferases that leads to tri- and tetraantennary glycans. The study defines the substantial changes of PAG N-glycosylation in the course of pregnancy. This promotes the hypothesis that PAGs may have different carbohydrate-mediated functions at different stages of pregnancy.     

The B4 lectin from Vicia villosa seeds interacts with N-acetylgalactosamine residues on erythrocytes with blood group Cad specificity

We have previously shown that the B4 lectin from Vicia villosa seeds interacts with N-acetylgalactosamine alpha-linked to serine or threonine in cell surface glycoproteins. In the present study, we show that the lectin also binds to Cad erythrocytes (0.44-2.78 X 10(6) sites/cell) with an association constant of 0.61-0.84 X 10(7)M-1. Variability in the number of B4 lectin binding sites in Cad erythrocytes from different individuals parallels reactivity of these erythrocytes with other N-acetylgalactosamine-binding lectins. Agglutination of Cad erythrocytes with B4 lectin is inhibited by urinary Tamm-Horsfall Sda-active glycoprotein. Since the Cad and Sda determinants share the terminal GalNAc beta 1.4----Gal sequence, our results indicate that Vicia villosa B4 lectin can also interact with terminal beta-linked N-acetylgalactosamine in closely-spaced oligosaccharide units of cell surface glycoproteins.     

Binucleate trophoblast giant cells in the water buffalo (Bubalus bubalis) placenta

The binucleate trophoblast giant cells (BNC) of the water buffalo, Bubalus bubalis, placenta were studied, with emphasis on the synthesis of BNC-specific proteins. Placentomal tissues of 27 water buffalos (2-10 months of pregnancy) were processed for light and electron microscopy. The frequency of BNCs was 20% of the trophoblastic cells in 2-3-month placentas and increased to 27% in the later stages. Ultrastructurally, binucleate cells displayed a prominent granular endoplasmic reticulum and Golgi apparatus, typical of cells involved with protein synthesis and exportation. The buffalo BNCs contained periodic acid-Schiff (PAS)-positive granules and reacted with antisera against bovine placental lactogen, prolactin-related protein-I, and pregnancy-associated glycoproteins. Lectin histochemistry with Dolichos biflorus agglutinin, Vicia villosa agglutinin, and Phaseolus vulgaris leucoagglutinin showed specific staining of BNCs. Different stages of BNC migration and fusion with uterine epithelial cells were observed. Trinucleate feto-maternal hybrid cells were the typical outcome of cell fusions. These cells underwent degeneration, with typical morphological features of apoptosis. The results revealed a strong homology between water buffalo and cattle BNCs concerning cell morphology, protein expression, glycosylation pattern, and characteristics of cell migration and fusion.     

Protein blotting: detection of proteins with colloidal gold, and of glycoproteins and lectins with biotin-conjugated and enzyme probes

Methods to detect "native" proteins immobilized on nitrocellulose membranes in spot tests or on blots prepared from polyacrylamide slab gels after electrophoretic separation are described. Gold sols were found to be useful as general stains for proteins: They are polychromatic, yield an indelible record, and are complementary to india ink as protein stains because these two stains have different sensitivities for a number of proteins tested. For detection of wheat germ lectin (WGL)-binding glycoproteins, avidin-peroxidase was an effective enzyme probe, because the glycoportion of the avidin moiety possesses binding affinity to WGL. Glycocomponents in human parotid saliva were detected with this probe and with the following biotin-conjugated lectins as intermediary probes: soybean lectin, Bandeiraea simplicifolia lectin, Lotus tetragonolobus lectin, and kidney bean lectin. Autoclaving blots prior to probing eliminated endogenous peroxidase activity. Concanavalin A and WGL were separated by isoelectric focusing and detected on blots with horseradish peroxidase and avidin-peroxidase, respectively. The versatility of the biotin/avidin system was used to detect other lectins on similar blots using biotin-conjugated glycoproteins as intermediary probes: Helix pomatia lectin and B. simplicifolia lectin were detected with biotinyl neoglycoproteins, and kidney bean lectin with biotin-conjugated components of parotid saliva.     

Stage-specific alterations in the apical membrane glycoproteins of endometrial epithelial cells related to implantation in rabbits

The rabbit endometrial epithelium undergoes differentiation prior to the time of blastocyst implantation, including loss of surface negativity and a change in glycocalyx morphology. Nonpregnant (estrous) and pseudopregnant rabbits were used to study specific alterations in proteins and saccharide composition of the luminal epithelial membrane and its glycocalyx related to the acquisition of receptivity to implantation. Pregnant animals were used to study further modification of the luminal surface by implanting blastocysts. The apical surface of luminal epithelial cells was solubilized by a 15-min intraluminal incubation of 1% Triton X-100 containing protease inhibitors. Proteins in extract solutions were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Three new polypeptides (24 kDa, 42 kDa and 58 kDa) were identified in uteri from receptive rabbits. Binding of succinyl Wheat Germ Agglutinin (sWGA) and Ricinus communis Agglutinin (RCA-I) lectins to the 24 kDa and 42 kDa components on Western blots of extracts separated by SDS-PAGE identified them as glycoproteins. Additionally, other polypeptides (26 kDa, 80-86 kDa and 145 kDa) showed changes in affinity for WGA, RCA-I or concanavalin A (Con A), depending on the hormonal state. Correlating with these findings was an increased binding of these lectins to intact nonciliated cells in uteri of receptive rabbits compared to estrous animals; ciliated cells bound Dolichos biflorus Agglutinin (DBA) specifically, regardless of the hormonal condition. Treatment of uteri from estrous animals, or Western blots of proteins from these animals, with neuraminidase prior to lectin exposure suggested the presence of glycoproteins having a sialic acid-D-galactose terminus in nonreceptive rabbits. Reduced binding of lectin to intact cells at implantation sites and to blots of proteins isolated from these sites, compared to nonimplantation sites, was noted. These results provide evidence for stage-specific alterations in protein and saccharide composition of the apical surface of endometrial epithelium prior to implantation, and indicate that implanting blastocysts further modify the luminal surface.     

LOCALIZATION AND IDENTIFICATION OF GALACTOSE/N-ACETYLGALACTOSAMINE AND SIALIC ACID-CONTAINING PROTEINS IN CHINESE HAMSTER METAPHASE CHROMOSOMES

Four lectins were used to recognize galactose/N-acetyl-galactosamine (Gal/GalNAc) and sialic acid residues in proteins of Chinese hamster metaphase chromosomes. In situ binding pattern of a fluorescein isothiocyanate-labelled (Gal/GalNAc)-specific lectin Sophora japonica agglutinin (SJA) showed that chromosomal SJA-binding proteins are primarily localized to the helically coiled substructure of chromatids. Numerous SJA-binding proteins were identified in Western blots of chromosomal proteins, their molecular weights ranging from 26 to 200kDa. Another Gal/GalNAc-specific lectin, peanut agglutinin (PNA), with a slightly different sugar binding specificity, did not bind to Chinese hamster metaphase chromosomes, and in Western blots only two chromosomal protein bands were faintly stained. The in situ labelling patterns of two sialic acid-specific lectins, Maackia amurensis (MAA) and Sambucus nigra (SNA) agglutinins, both showed that the helically coiled substructure of chromatids is also enriched in sialylated proteins. In Western blot analysis 11 MAA-binding protein bands with molecular weights ranging from 54 to 215kDa were identified, while SNA only bound to one protein band of 67kDa. MAA and SNA are specific for α (2|ad3)- and α (2|ad6)-linked sialic acid residues, respectively. Thus, it is likely that α (2|ad3)-linked sialic acid residues are more common in chromosomal proteins than α(2|ad6)-linked sialic acid residues. These data suggest that Gal/GalNAc and sialic acid-containing glycoproteins exist in metaphase chromosomes and that these proteins may have a role in the formation of higher order metaphase chromosome structures.     

Characterization of the carbohydrate moiety of Clerodendron trichotomum lectins. Its structure and reactivity toward plant lectins

Lectins were isolated from fruits and leaves of Clerodendron trichotomum by affinity chromatography on lactamyl-Sepharose. The purified lectins (C. trichotomum agglutinin: CTA) were homogeneous on SDS/polyacrylamide gel electrophoresis, and the carbohydrate moiety was characterized by physicochemical and immunochemical methods. The asparagine-linked oligosaccharides were released by treatment with N-oligosaccharide glycopeptidase (almond, EC 3.5.1.52) of peptic glycopeptides obtained from fruit CTA, and separated by gel filtration and thin-layer chromatography. The structure of the predominant oligosaccharide was determined as Xyl beta 1----2 (Man alpha 1----6)(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4(Fuc alpha 1----3)GlcNAc by high-performance liquid chromatography, sugar analysis and 1H-NMR spectroscopy. The reactivity of the carbohydrate moiety of CTA toward various lectins was studied. Fruit and leaf CTAs were applied to polyacrylamide gel electrophoresis, transferred to nitrocellulose sheets and detected with horseradish-peroxidase-conjugated lectins. Concanavalin A, lentil lectin, pea lectin, Vicia faba lectin and Ulex europeus agglutinin I, but not wheat germ lectin, bound to fruit CTA. The results indicate new binding properties of these plant lectins: a beta-xylosyl residue substituted at C-2 of the beta-mannosyl residue of N-linked oligosaccharide does not affect the binding with mannose-specific lectins, lentil, pea and Vicia faba lectins can bind to N-linked oligosaccharides containing an alpha-L-fucosyl residue attached to C-3 of the asparagine-linked N-acetyl-D-glucosamine residue, and Ulex europeus agglutinin I can bind to the (alpha 1----3)-linked fucose residue of the N-linked oligosaccharide.     

Lectin Binding to Radopholus citrophilus and R. similis Proteins

Lectin-binding glycoproteins in seven populations of two burrowing nematode sibling species were probed with five different biotinylated lectins on Western blots, and differences were correlated with nematode ability to parasitize citrus and to overcome citrus rootstock resistance. Banding patterns of molecular weight standards were fit best by an exponential decay function, and a predictive equation was used to estimate molecular weights (r² = 0.999). A band (131 kDa) that labeled with the lectin Concanavalin A (Con A) occurred in extracts from cuticles and egg shells of populations of Radopholus citrophilus that parasitize citrus. Wheat germ agglutin labeled a band (58 kDa) in aqueous homogenates of populations that reproduce in roots of citrus rootstock normally resistant to burrowing nematodes. The two sibling species R. citrophilus and R. similis were distinguished by a high molecular weight Con A-labeled band (608 kDa) from cuticle and egg shells. Probing blots with the lectin Limulus polyphemus agglutinin indicated that each population contained a band (12-16 kDa) specifically inhibited by the addition of 25 mM neuraminic acid, suggesting that glycoproteins with sialic acid moieties are present in burrowing nematodes.     

Isolation and characterization of gastric microsomal glycoproteins. Evidence for a glycosylated beta-subunit of the H+/K(+)-ATPase.

Detergent-solubilization of hog gastric microsomal membrane proteins followed by affinity chromatography using wheat germ agglutinin or Ricinus communis I agglutinin resulted in the isolation of five glycoproteins with the apparent molecular masses on sodium dodecyl sulfate polyacrylamide gels of (in kDa): 60-80 (two glycoproteins sharing this molecular mass); 125-150; and 190-210. In the nonionic detergent Nonidet P-40 (NP-40), the 94 kDa H+/K(+)-ATPase was recovered exclusively in the lectin-binding fraction; however, in the cationic detergent dodecyltrimethylammonium bromide, most of the ATPase was recovered in the nonbinding fraction. Detection of glycoproteins either by periodic acid-dansyl hydrazine staining of carbohydrate in polyacrylamide gels or by Western blots probed with lectins indicated that the majority of the ATPase molecules are not glycosylated. In addition, in the absence of microsomal glycoproteins, the NP-40-solubilized ATPase does not bind to a lectin column. Taken together, these results suggest that the recovery of NP-40-solubilized ATPase in the lectin-binding fraction is due to its noncovalent interaction with a gastric microsomal glycoprotein. Immunoprecipitation of the ATPase from NP-40-solubilized microsomal membrane proteins resulted in the co-precipitation of a single 60-80 kDa glycoprotein. Characterization of the 60-80 kDa glycoprotein associated with the ATPase revealed that: it is a transmembrane protein; it has an apparent core molecular mass of 32 kDa; and, it has five asparagine-linked oligosaccharide chains. Given its similarity to the glycosylated beta-subunit of the Na+/K(+)-ATPase, this 60-80 kDa gastric microsomal glycoprotein is suggested to be a beta-subunit of the H+/K(+)-ATPase.     

A lectin-based gold nanoparticle assay for probing glycosylation of glycoproteins

We report a glycoanalysis method in which lectins are used to probe the glycans of therapeutic glycoproteins that are adsorbed on gold nanoparticles. A model mannose-presenting glycoprotein, ribonuclease B (RNase B), and the therapeutic monoclonal antibody (mAb) rituximab, were found to adsorb spontaneously and non-specifically to bare gold nanoparticles such that glycans were accessible for lectin binding. Addition of a multivalent binding lectin, such as concanavalin A (Con A), to a solution of the modified gold nanoparticles resulted in cross-linking of the nanoparticles. This phenomenon was evidenced within 1 min by a change in the hydrodynamic diameter, D(H), measured by dynamic light scattering (DLS) and a shift and increase in absorbance of the plasmon resonance band of the gold nanoparticles. By combining the sugar-binding specificity and the cross-linking capabilities of lectins, the non-specific adsorption of glycoproteins to gold surfaces, and the unique optical reporting properties of gold nanoparticles, a glycosylation pattern of rituximab could be generated. This assay provides advantages over currently used glycoanalysis methods in terms of short analysis time, simplicity of the conjugation method, convenience of simple spectroscopic detection, and feasibility of providing glycan characterization of the protein drug product by using a variety of binding lectins.     

Nerve-induced secretion of glycoconjugates from cat submandibular glands: a correlative study with lectin probes on tissues and saliva.

Horseradish peroxidase-conjugated lectins were used on tissue sections to localize the main secretory glycoproteins in cat submandibular glands and on Western blots to evaluate their movement into saliva with selective nerve stimulation. Central acinar cells bound lectins from Arachis hypogaea (PNA) specific for the terminal disaccharide Gal beta 1, 3GalNac, Griffonia simplifolia (GSA I-B4) specific for terminal alpha Gal, and Lotus tetragonolobus (LTA) specific for fucose. Lectins from Limax flavus (LFA) specific for sialic acid and Dolichos biflorus (DBA) specific for terminal alpha GalNac reacted preferentially with demilunar cells, whereas apical granules in striated ducts were recognized principally by LTA. Parasympathetic stimulation promoted the release of lectin-reactive glycoconjugates from both central and demilunar cells. In contrast, sympathetic stimulation caused almost complete release of LTA-reactive granules in striated ducts and only moderate secretion from demilunar cells. Lectin blots of stimulated saliva discriminated many of the constituent bands, providing information about their glycosylation. Several bands were common to both parasympathetic and sympathetic saliva, and many bands gave wider ranges of lectin binding than anticipated from the histochemistry. The major component in parasympathetic saliva was a glycoconjugate of less than 12 KD which reacted with every lectin tested. Lectin blots of sympathetic saliva showed a prominent diffuse LTA-reactive band around 33 KD, which was attributed to tissue kallikrein. The identity and cellular origin of most bands in stimulated submandibular saliva are still unclear but the technique shows considerable promise for improving the recognition and characterization of individual glycoconjugates.     

Glycotope sharing between snail hemolymph and larval schistosomes: larval transformation products alter shared glycan patterns of plasma proteins

Recent evidence supports the involvement of inducible, highly diverse lectin-like recognition molecules in snail hemocyte-mediated responses to larval Schistosoma mansoni. Because host lectins likely are involved in initial parasite recognition, we sought to identify specific carbohydrate structures (glycans) shared between larval S. mansoni and its host Biomphalaria glabrata to address possible mechanisms of immune avoidance through mimicry of elements associated with the host immunoreactivity. A panel of monoclonal antibodies (mABs) to specific S. mansoni glycans was used to identify the distribution and abundance of shared glycan epitopes (glycotopes) on plasma glycoproteins from B. glabrata strains that differ in their susceptibilities to infection by S. mansoni. In addition, a major aim of this study was to determine if larval transformation products (LTPs) could bind to plasma proteins, and thereby alter the glycotopes exposed on plasma proteins in a snail strain-specific fashion. Plasma fractions (< 100 kDa/> 100 kDa) from susceptible (NMRI) and resistant (BS-90) snail strains were subjected to SDS-PAGE and immunoblot analyses using mAB to LacdiNAc (LDN), fucosylated LDN variants, Lewis X and trimannosyl core glycans. Results confirmed a high degree of glycan sharing, with NMRI plasma exhibiting a greater distribution/abundance of LDN, F-LDN and F-LDN-F than BS-90 plasma (< 100 kDa fraction). Pretreatment of blotted proteins with LTPs significantly altered the reactivity of specific mABs to shared glycotopes on blots, mainly through the binding of LTPs to plasma proteins resulting in either glycotope blocking or increased glycotope attachment to plasma. Many LTP-mediated changes in shared glycans were snail-strain specific, especially those in the < 100 kDa fraction for NMRI plasma proteins, and for BS-90, mainly those in the > 100 kDa fraction. Our data suggest that differential binding of S. mansoni LTPs to plasma proteins of susceptible and resistant B. glabrata strains may significantly impact early anti-larval immune reactivity, and in turn, compatibility, in this parasite-host system.     

Isolation and characterization of BanLec-I, a mannoside-binding lectin from Musa paradisiac (banana).

A lectin (BanLec-I) from banana (Musa paradisiac) with a binding specificity for oligomannosidic glycans of size classes higher than (Man)6GlcNAc was isolated and purified by affinity chromatography on a Sephadex G-75 column. It did not agglutinate untreated human or sheep erythrocytes, but it did agglutinate rabbit erythrocytes. BanLec-I stimulated T-cell proliferation. On size-exclusion chromatography, BanLec-I has a molecular mass of approx. 27 kDa, and on SDS/PAGE the molecular mass is approx. 13 kDa. The isoelectric point is 7.2-7.5. BanLec-I was found to be very effective as a probe in detecting glycoproteins, e.g. on nitrocellulose blots.     

Biochemical and functional characterization of the Tn-specific lectin from Salvia sclarea seeds.

SSL, the lectin isolated from Salvia sclarea seeds, recognizes the Tn antigen (GalNAcalpha-O-Ser/Thr), a specific marker of many human carcinomas. Two-dimensional electrophoresis, amino-acid and amino-sugar analysis, and MALDI-TOF MS showed that SSL is an acidic (pI 5.5), 60-61-kDa dimeric glycoprotein composed of apparently identical subunits linked by a single disulfide bond. The apparent molecular mass of SSL in solution determined by equilibrium sedimentation analytical ultracentrifugation was 59 +/- 9 kDa. This value did not change in the pH range 2.5-8.5, indicating that SSL does not associate into higher order structures. Tandem mass spectrometry and methylation analysis of N-glycans released from SSL by hydrazinolysis indicated that SSL possesses 2-3 glycosylation sites occupied with the typical plant glycans Manalpha1-6[(Manalpha1-3)(Xylbeta1-2)]Manbeta1-4 -GlcNAcbeta1-4(Fucalp ha1-3)GlcNAc and [(Manalpha1-3/6)(Xylbeta1-2)]Manbeta1-4-GlcNAcbeta1 -4(Fucalpha1-3)Glc NAc. The influence of adjacent Tn structures on the binding of two Tn-specific lectins (SSL and the isolectin B4 from Vicia villosa) and an anti-Tn monoclonal antibody (mAb 83D4) was evaluated using synthetic Tn glycopeptides. The binding of both lectins to the synthetic Tn glycopeptides was independent of the density of Tn structures. On the other hand, mAb 83D4 only reacted with glycopeptides displaying two or three consecutive Tn structures.     

The use of lectin microarray for assessing glycosylation of therapeutic proteins

Glycans or carbohydrates attached to therapeutic glycoproteins can directly affect product quality, safety and efficacy, and therefore must be adequately analyzed and controlled throughout product life cycles. However, the complexity of protein glycosylation poses a daunting analytical challenge. In this study, we evaluated the utility of a lectin microarray for assessing protein glycans. Using commercial lectin chips, which contain 45 lectins toward distinct glycan structures, we were able to determine the lectin binding patterns of a panel of 15 therapeutic proteins, including 8 monoclonal antibodies. Lectin binding signals were analyzed to generate glycan profiles that were generally consistent with the known glycan patterns for these glycoproteins. In particular, the lectin-based microarray was found to be highly sensitive to variations in the terminal carbohydrate structures such as galactose versus sialic acid epitopes. These data suggest that lectin microarray could be used for screening glycan patterns of therapeutic glycoproteins.     

Isolation and characterization of gastric microsomal glycoproteins. evidence for a glycosylated β-subunit of the H+/K−-ATPase

Detergent-solubilization of hog gastric microsomal membrane proteins followed by affinity chromatography using wheat germ agglutinin or Ricinus communis I agglutinin resulted in the isolation of five glycoproteins with the apparent molecular masses on sodium dodecyl sulfate polyacrylamide gels of (in kDa): 60–80 (two glycoproteins sharing this molecular mass); 125–150; and 190–210. In the nonionic detergent Nonidet P-40 (NP-40), the 94 kDa H⁺/K⁺-ATPase was recovered exclusively in the lectin-binding fraction; however, in the cationic detergent dodecyltrimethylammonium bromide, most of the ATPase was recovered in the nonbinding fraction. Detection of glycoproteins either by periodic acid-dansyl hydrazine staining of carbohydrate in polyacrylamide gels or by Western blots probed with lectins indicated that the majority of the ATPase molecules are not glycosylated. In addition, in the absence of microsomal glycoproteins, the NP-40-solubilized ATPase does not bind to a lectin column. Taken together, these results suggest that the recovery of NP-40-solubilized ATPase in the lectin-binding fraction is due to its noncovalent interaction with a gastric microsomal glycoprotein. Immunoprecipitation of the ATPase from NP-40-solubilized microsomal membrane proteins resulted in the co-precipitation of a single 60–80 kDa glycoprotein. Characterization of the 60–80 kDa glycoprotein associated with the ATPase revealed that: it is a transmembrane protein; it has an apparent core molecular mass of 32 kDa; and, it has five asparagine-linked oligosaccharide chains. Given its similarity to the glycosylated β-subunit of the Na⁺/K⁺-ATPase, this 60–80 kDa gastric microsomal glycoprotein is suggested to be a β-subunit of the H⁺/K⁺-ATPase.     

Comparison of the carbohydrate-binding specificities of seven N-acetyl-D-galactosamine-recognizing lectins

Seven plant lectins, Dolichos biflorus agglutinin (DBA), Griffonia simplicifolia agglutinin (GSA, isolectin A4), Helix pomatia agglutinin (HPA), soybean (Glycine max) agglutinin (SBA), Salvia sclarea agglutinin (SSA), Vicia villosa agglutinin (VVA, isolectin B4) and Wistaria floribunda agglutinin (WFA), known to be specific for N-acetyl-D-galactosamine-(GalNAc) bearing glycoconjugates, have been compared by the binding of their radiolabelled derivatives, to eight well-characterized synthetic oligosaccharides immobilized via a spacer on an inert silica matrix (Synsorb). The eight oligosaccharides included the Forssman, the blood group A and the T antigens, as well as alpha GalNAc coupled directly to the support (Tn antigen) and also structures with GalNAc linked alpha or beta to positions 3 or 4 of an unsubstituted Gal. The binding studies clearly distinguished the lectins into alpha GalNAc-specific agglutinins like DBA, GSA and SSA, and lectins which recognize alpha- as well as beta-linked GalNAc residues like HPA, VVA, WFA and SBA. HPA was the only lectin which bound to the beta Gal1----3 alpha GalNAc-Synsorb adsorbent (T antigen) indicating that it also recognizes internal GalNAc residues. Among the alpha GalNAc-specific lectins, DBA strongly recognized blood group A structures while GSA displayed weaker recognition, and SSA bound only slightly to this affinity matrix. In addition, DBA and SSA were able to distinguish between GalNAc linked alpha 1----3 and GalNAc linked alpha 1----4, to the support, the latter being a much weaker ligand. These results were corroborated by the binding of the lectins to biological substrates as determined by their hemagglutination titers with native and enzyme-treated red blood cells carrying known GalNAc determinants, e.g. blood group A, and the Cad and Tn antigens. For SSA, the binding to the alpha GalNAc matrix was inhibited by a number of glycopeptides and glycoproteins confirming the strong preference of this lectin for alpha GalNAc-Ser/Thr-bearing glycoproteins.     

Maturation-dependent glycoproteins containing both N- and O-linked oligosaccharides in epididymal sperm plasma membrane of rhesus monkeys (Macaca mulatta)

Glycosylation is one of the important post-translational modifications of sperm plasma membrane proteins during the maturation of epididymal spermatozoa that results in the development of motility and fertilizing capability. The aim of the present study was to identify and characterize the maturation-dependent asparagine-linked (N-linked) and serine- and threonine-linked (O-linked) glycoproteins of the epididymal spermatozoa of rhesus monkeys. The presence of N- and O-linked glycoproteins was confirmed by treatment of sperm membranes with N-glycosidase F and O-glycosidase. The major maturation-dependent sperm membrane glycoproteins identified on blots of SDS-PAGE-fractionated proteins of purified sperm plasma membranes from five segments of epididymis, probed with biotinylated lectins and Vectastain-ABC reagent included O-linked 170, 150, 86 and 60/58 kDa glycoproteins; N-linked 68, 56, 48 and 38 kDa glycoproteins and N- and O-linked 116 kDa glycoprotein, all of which exhibited marked differences in the degree of glycosylation between immature and mature sperm surfaces. These glycoproteins can be used as markers of sperm maturation in the epididymis of rhesus monkeys, during the screening of antifertility agents acting at the epididymis, or may be developed as potential sperm antigens. The 100% inhibition of fertility in female rats and rabbits immunized with major maturation-dependent 116 kDa glycoprotein showed the significance of glycosylation changes in the maturation status of epididymal spermatozoa. This 116 kDa protein can be used as a marker parameter of sperm maturation in the rhesus monkey, which is often the preferred animal model for preclinical studies. These results will contribute to the identification of an appropriate animal model for the development of male contraceptives in humans.     

A two-site lectinoenzymatic assay for determination of tumour marker glycoproteins in rectal secretions

A method is described for a titre-tray based two-site lectinoenzymatic assay of glycoproteins. WGA lectin, reacting with the core-part of glycans, was combined with lectins PNA and DBA, the latter two reacting with terminal parts of glycans. A standard curve was obtained with bovine submaxillary gland asialomucin, and measurements of human rectal secretion were calibrated against this curve. The assay showed an intra-assay reproducibility of 2.4–7.5%, and inter-assay reproducibility of 3.9–20.8% Recovery tests showed a linearity close to predicted values. The selected standard was ideal as inhibition of lectin binding by monosaccharides showed similar inhibition profiles for human rectal secretion and for asialomucin standard. Neuraminidase treatment dramatically increased the PNA binding to human rectal secretion immobilized on WGA. Western blotting of human rectal secretion demonstrated a large range of lectin-reactive glycoproteins, the main fraction reacting with all lectins being approximately 250 kDa. The assay described is well suited for studies of the glycan part of tumour marker glycoproteins, and changes occurring in these. It has a high sensitivity by ignoring that the glycans may be present on different molecules. Examination of rectal secretions from various cancer patients showed significantly increased PNA binding, as well as an increased PNA/DBA binding ratio, in patients with colorectal cancer (p<3×10-3) and, unexpectedly, in patients with other cancers (p<5×10-3). Abbreviations: HRS, human rectal secretion; PNA, peanut agglutinin; DBA, dolichos biflorus agglutinin; WGA, wheat germ agglutinin; BSA, bovine serum albumin; ELLSA, enzyme linked lectino-solid-phase assay; HRP, horseradish peroxidase; HRS: human rectal secretion; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; prot., protein; kDa, kilodalton; OPD, Ortho-phenylen-diamine; SA, Sialic acid; Gal, Galactose; GlcNAc, N-acetyl-D-glucosamine; GalNAc, N-acetyl-D-galactosamine; Fuc, fucose; Man, mannose; Glc, glucose This revised version was published online in November 2006 with corrections to the Cover Date.