Analysis of the oligosaccharides on androgen-binding proteins: Implications concerning their role in structure/function relationships

Rat androgen-binding protein (rABP), human testosterone-binding globulin (hTeBG) and rabbit (rb) TeBG are heterodimeric proteins. The source of the heterogeneity arises from the differential glycosylation of a common protein core. This glycosylation results in a heavy subunit (more glycosylation) and a light subunit (less glycosylation). Glycosylation is one factor responsible for multiple charged species seen when rABP, hTeBG, and rbTeBG are analyzed by two-dimensional gel electrophoresis. Enzymatic digestion with the endoglycosidase, peptide: N-glycosidase F indicated that all three proteins have asparagine (Asn)-linked oligosaccharides as their major glycan substituent. Treatment with exoglycosidases provided evidence for terminal sialic acid, galactose and mannose and N-acetylglucosamine residues. About 16–22% of the mass of the heavy subunit and about 8–14% of the mass of the light subunit is contributed by carbohydrate.

Serial lectin chromatography indicated that rABP is glycosylated differently from hTeBG and rbTeBG. About 40% of the rABP contains tri and tetraantennary complex oligosaccharides, while only about 20% of the hTeBG and TeBG from pregnant rabbits contains these types of glycans. About 9% of the TeBG from male rabbits bears these types of oligosaccharides. All of the biantennary complex oligosaccharides on rABP are fucosylated on the chitobiose core, but only 8% of those on hTeBG and none of those on rbTeBG are fucosylated in this manner. All three proteins are glycosylated at more than one site. The data indicate that the proteins may have more than one type of oligosaccharide on them. It is likely that differences in glycosylation are responsible for different physiological roles of the proteins.     

Analysis of the oligosaccharides on rat androgen-binding protein using serial lectin chromatography

The microheterogeneity seen when rat androgen-binding protein (rABP) is analyzed by two-dimensional polyacrylamide gel electrophoresis is attributable, at least in part, to the differential glycosylation of a single promoter. Further insight into the chemical nature of the oligosaccharide units on rABP was obtained by serial lectin chromatography. When rABP was chromatographed on immobilized Concanavalin A (Con-A), it was fractionated into three classes: (1) one that did not bind to the lectin (about 44% of the rABP), (2) one that was bound and could be eluted with 10 mM 1-O-methyl alpha-D-glucopyranoside (glucoside), about 34%, and (3) one that could be eluted with 0.5 M methyl alpha-D-mannopyranoside (mannoside), about 23%. Binding to Con-A indicates the presence of asparagine-linked oligosaccharides. Chromatography of the glucoside-eluted peak on lentil lectin (LcH) indicated that the rABP in that fraction contained a fucose residue on the chitobiose core. Chromatography of the mannoside-eluted peak on wheat germ agglutinin (WGA) indicated the presence of rABP with high mannose- (44%) and hybrid-type (56%) glycans attached. Chromatography on Ricinus communis I (RCA-I) lectin indicated a species containing galactosylated complex-type oligosaccharide chains. Treatment of rABP forms with exoglycosidases confirmed the presence of externally disposed fucose, sialic acid, mannose, and galactose residues. LcH chromatography indicated that about 30% of the rABP that did not bind to Con-A possessed triantennary oligosaccharides with fucose on the chitobiose core. About 28% of the rABP was retarded when it was chromatographed on Phaseolus vulgaris E lectin, suggesting the presence of bisected biantennary chains with terminal galactose residues. We were unable to detect rABP species with serine- or threonine-linked oligosaccharide chains in this fraction. Other forms of rABP in the nonretained fraction of Con-A were not resolved. Western blotting did not reveal major differences in relative molecular weight (Mr) among the rABP species; some differences in the ratio of the heavy to the light subunit of the molecule were detectable.     

Characterization of the oligosaccharides of plasma sex hormone binding globulin from noncirrhotic alcoholic patients

In previous reports we have demonstrated high plasma levels of sex hormone-binding globulin (SHBG) in asymptomatic alcoholic men. In the present work the physicochemical properties of SHBG from plasma of noncirrhotic alcoholic patients have been further compared with SHBG of control subjects. Steroid binding to SHBG was similar for the two groups: alcoholic men, K(d) of 0.62 +/- 0.07 nM and control individuals, K(d) of 0.70 +/- 0.10 nM. The structure of oligosaccharides attached to SHBG from controls and alcoholic men were determined by using serial chromatography. Our data indicated that 7% of SHBG of control individuals was not retarded by the Con-A column, whereas approximately 30% of SHBG of alcoholic men eluted in the void volume of Con A. Approximately 46% of SHBG of alcoholics applied to Con A, possessed biantennary complex oligosaccharides, as indicated by the fact that it could be eluted with methyl-alpha-D-glucopyranoside and by its retention on wheat germ agglutinin; in contrast, when SHBG from control men was analyzed, approximately 51% was eluted with methyl-alpha-D-glucopyranoside. Approximately 9% of the biantennary complex oligosaccharides on SHBG of control men and none of those on SHBG from alcoholic men were fucosylated on the chitobiose core, as determined by chromatography on Lenn culinaris lectin. Galactosylated oligosaccharides were also present on the SHBG fraction as indicated by its interaction with Ricinus communis-I. Approximately 24% of SHBG of alcoholic men and 39% of those on SHBG from control individuals applied to Con-A were retained and could be eluted with methyl-alpha-D-mannopyranoside. Evidence based on the binding on mannoside-eluted SHBG to Con-A, wheat germ agglutinin, and R. communis-I indicated that at least the SHBG in this fraction, from alcoholics or controls, contained two glycosylation sites and that the sites were differentially glycosylated.     

Lectin-binding patterns in the developing tooth

The lectin-binding patterns of the cells involved in amelogenesis and dentinogenesis in developing teeth of rats were studied. Undifferentiated odontogenic epithelia exhibited very slight staining with almost all of the lectins examined. The lectin-staining affinities of secretory ameloblasts could be divided into two categories: Concanavalin-A (Con-A), Wheat germ agglutinin (WGA) and Soybean agglutinin (SBA) binding occurred from the middle to apical cytoplasm, whereas Ricinus communis agglutinin-I (RCA-I) and Ulex europeus I (UEA-I) binding predominated in the basal regions. The cells of the stratum intermedium exhibited relatively strange lectin staining, which appeared to be dependent on ameloblastic maturation. The basement membranes in undifferentiated epithelia were markedly positive for lectin binding. Odontoblasts showed moderate Con-A staining on the apical side of the cells, as well as slight-to-moderate reactions with WGA and SBA. Pulp cells and dental papillae showed slight-to-moderate lectin staining, and predentin and dentin were also moderately positive for Con-A and RCA-I binding and slightly so for WGA and SBA. The lectin-binding affinities were enhanced during the formation of enamel and dentin, and appeared to be dependent on the degree of cellular differentiation in ameloblasts and odontoblasts.     

Lectin-binding patterns in the developing tooth

Summary The lectin-binding patterns of the cells involved in amelogenesis and dentinogenesis in developing teeth of rats, were studied. Undifferentiated odontogenic epithelia exhibited very slight staining with almost all of the lectins examined. The lectin-staining affinities of secretory ameloblasts could be divided into two categories: Concanavalin-A (Con-A), Wheat germ agglutinin (WGA) and Soybean agglutinin (SBA) binding occurred from the middle to apical cytoplasm, whereas Ricinus communis agglutinin-I (RCA-I) and Ulex europeus I (UEA-I) binding predominated in the basal regions. The cells of the stratum intermedium exhibited relatively stranges lectin staining, which appeared to be dependent on ameloblastic maturation. The basement membranes in undifferentiated epithelia were markedly positive for lectin binding. Odontoblasts showed moderate Con-A staining on the apical side of the cells, as well as slight-to-moderate reactions with WGA and SBA. Pulp cells and dental papillae showed slight-to-moderate lectin staining, and predentin and dentin were also moderately positive for Con-A and RCA-I binding and slightly so for WGA and SBA. The lectin-binding affinities were enhanced during the formation of enamel and dentin, and appeared to be dependent on the degree of cellular differentiation in ameloblasts and odontoblasts.     

Structure of the major concanavalin A reactive oligosaccharides of the extracellular matrix component laminin

Laminin, a high molecular weight (1,000,000) glycoprotein component of basement membranes, was isolated from the EHS murine tumor as a noncovalent complex with entactin by lectin affinity chromatography using the alpha-D-galactosyl binding lectin Griffonia simplicifolia I (GS I). Entactin was removed from this complex by passage over Sephacryl S-1000 in the presence of SDS. Compositional analysis showed that the affinity-purified laminin contained 25-30% carbohydrate by weight. Methylation analysis revealed that the oligosaccharides of laminin contained bi- and triantennary chains, the blood group I structure, and repeating sequences of 3Gal beta 1,4GlcNAc beta 1 units. Free oligosaccharides were derived from the asparagine-linked glycans of affinity-purified laminin by hydrazinolysis, re-N-acetylation, and reduction with NaB3H4. When fractionated by affinity chromatography on concanavalin A (Con A)-Sepharose, 80% of the oligosaccharides passed through the column unretarded and a single peak corresponding to 20% of the oligosaccharides was adsorbed and specifically eluted with a linear gradient of 0-30 mM methyl alpha-D-glucopyranoside. Further fractionation of the Con A reactive oligosaccharides on GS I-Sepharose demonstrated that 70% of these oligosaccharides possess at least one terminal nonreducing alpha-D-galactopyranosyl unit. The Con A reactive oligosaccharides were subjected to sequential digestion with endo- and exoglycosidases, and the reaction products were analyzed by gel filtration chromatography on a column of Bio-Gel P4. We thereby obtained evidence for a variety of structures not previously reported to exist on murine laminin including hybrid biantennary complex and biantennary complex structures containing poly(lactosaminyl) repeating units. The poly(lactosaminyl) units occur either on one or on both branches of the biantennary chains, as well as in more highly branched blood group I poly(lactosamine) structures. All sialic acid is present as N-acetylneuraminic acid linked alpha 2,3 to galactose.     

The microheterogeneity of rabbit testosterone-binding globulin is due to differential glycosylation of its single protomer

Affinity-purified rabbit testosterone-binding globulin (rbTeBG) is a homodimer with a molecular weight (Mr) of about 92,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the chemically cross-linked protein. When noncross-linked rbTeBG is subjected to SDS-PAGE, individual protomers (Mr approximately equal to 44,400 +/- 400 and Mr approximately equal to 42,000 +/- 1300) are resolved. The protomers are present in a ratio of approximately 2 (heavy):1 (light). Enzymatic deglycosylation of native rbTeBG or of rbTeBG that had been photoaffinity-labeled with [1,2-3H]17 beta-hydroxy-4,6-androstadien-3-one was conducted. The products were then identified on immunoblots using a monoclonal antibody that cross-reacts with rbTeBG, or by fluorography. These analyses indicated that rbTeBG contained sialic acid and asparagine (Asn)-linked oligosaccharides and provided evidence for the presence of serine/threonine (O)-linked glycans on the molecule. The presumptive removal of all oligosaccharides by enzymatic or chemical means resulted in the appearance of a single subunit (Mr approximately equal to 37,150 +/- 1200). On the basis of this monomeric molecular weight, carbohydrate would contribute 16% and 11% to the relative molecular mass of the nondeglycosylated heavy and light subunits, respectively. Therefore, the size heterogeneity of the nondeglycosylated rbTeBG subunits is a result of their differential glycosylation. In addition to size heterogeneity, the rbTeBG subunits are composed of multiple-charge variants. Although enzymatic and chemical methods of glycan removal altered the isoelectric points of the isoforms, none of the treatments yielded a single isoform. Thus, it is possible that moieties other than oligosaccharides are contributing charge to the isoelectric variants of rbTeBG.     

Precipitation of galactose-specific lectins by complex-type oligosaccharides and glycopeptides: studies with lectins from Ricinus communis (agglutinin I), Erythrina indica, Erythrina arborescens, Abrus precatorius (agglutinin), and Glycine max (soybean)

We have recently demonstrated that certain oligomannose and bisected hybrid type glycopeptides and bisected complex type oligosaccharides are bivalent for binding to concanavalin A and can precipitate the lectin [Bhattacharyya, L., Ceccarini, C., Lorenzoni, P., & Brewer, C.F. (1987) J. Biol. Chem. 262, 1288-1293; Bhattacharyya, L., Haraldsson, M., & Brewer, C.F. (1987) J. Biol. Chem. 262, 1294-1299]. The present results show that tri- and tetraantennary complex type oligosaccharides containing nonreducing terminal galactose residues, and a related triantennary glycopeptide, precipitate the D-galactose-specific lectins from Ricinus communis (agglutinin I) (RCA-I), Erythrina indica (EIL), Erythrina arborescens (EAL), and Glycine max (soybean) (SBA). Nonbisected and bisected biantennary complex type oligosaccharides can precipitate SBA, which is a tetrameric lectin, but not RCA-I, EIL, or EAL, which are dimeric lectins. The relative affinities of the oligosaccharides and glycopeptide were determined by hemagglutination inhibition measurements and their valencies by quantitative precipitin analyses. The equivalence points of the precipitin curves indicate that the tri- and tetraantennary oligosaccharides are tri- and tetravalent, respectively, for EIL, EAL, and SBA binding. However, the oligosaccharides are all trivalent for RCA-I binding due apparently to the larger size of the monomeric subunit of the lectin. The triantennary glycopeptide was also trivalent for RCA-I and EIL binding. Biantennary oligosaccharides with adequate chain lengths were found to be bivalent for binding to SBA; those with shorter chains did not precipitate the lectin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lectin histochemistry of rabbit nephron

In order to investigate the usefulness of lectin histochemistry to detail nephronal segmentation we used 12 different biotinylated lectins (Con-A, DBA, GS-I, LCA, PNA, PWN, RCA-I, RCA-II, SWGA, SBA, UEA-I, and WGA) and Avidin-Biotin-Peroxidase (ABC) system on formalin-fixed and paraffin-embedded rabbit kidney sections. Each lectin, except UEA-I which did not stain any nephron structure, shows a different staining pattern along the nephron. Con-A, LCA, and RCA-I display a diffuse staining, while BS-I, RCA-II, SWGA, PWN, DBA, SBA and PNA are selective markers for specific nephron tracts. Furthermore, it is possible, according to the WGA binding pattern, to differentiate the convoluted part of the proximal tubule into two parts, named Segment A and Segment B. Lectin histochemistry on formalin-fixed and paraffin-embedded rabbit kidney sections displays a specific binding pattern along the rabbit nephron and shows interesting morphofunctional correlations.     

Lectin-binding pattern on the surface epidermis of Ambystoma tigrinum larvae. A light- and electron-microscopic study

The surface epidermis of Ambystoma tigrinum larvae was examined at the light- and electron-microscope levels using five different lectin conjugates as probes for the detection of sugar residues on the cell membranes. Concanavalin A (Con-A), wheat-germ agglutinin (WGA), Ricinus communis agglutinin I (RCA-I), Dolichos biflorus agglutinin and soybean agglutinin (SBA) conjugates clearly labelled the surface cells, especially their apical surfaces. At electron microscopy, the labelling on plasma membranes was found to exhibit regional differences. Among the lectins tested WGA displayed a particularly characteristic binding pattern. WGA also bound to basolateral cell surfaces, including the tight-junction zone which was also stained by the RCA-I conjugate. The different labelling intensity and staining patterns obtained with the conjugates indicated the polarity of the cell surfaces. It is also assumed that the WGA staining of the basolateral membranes and intercellular spaces reflected transcellular transport, which is facilitated by acidic glycoconjugates. Other functional aspects of the polarized distribution of the lectin conjugates were also correlated with the receptor sites of certain sugar residues.     

Differential effects of oncogenic transformation on N-linked oligosaccharide processing at individual glycosylation sites of viral glycoproteins

Hamster sarcoma virus (HSV) transformation of Nil-8 fibroblasts is associated with an increase in the average size of N-acetyllactosamine (complex) type N-linked glycans due to an increase in both the average number of branches/chain and in the fraction of N-linked glycans containing poly(GlcNAc(beta 1,3) Gal-(beta 1,4)) (polylactosaminylglycan) chains. Analysis of glycopeptides from the envelope glycoproteins of Sindbis virus and vesicular stomatitis virus (VSV) grown in Nil-8 and Nil/HSV cells indicated that the transformation-associated shift to larger N-linked oligosaccharides selectively affects some glycosylation sites far more than others. Glycosylation of the Sindbis virus glycoproteins and of Asn-179 of VSV G was similar in Nil-8 and Nil/HSV cells; oligosaccharide processing generally did not proceed beyond the biantennary complex stage. In contrast, Asn-336 of VSV G carried primarily biantennary complex glycans in Nil-8-grown virus (ratio, triantennary, and larger to biantennary complex glycans (tri+/bi) = 0.5) but more highly branched structures in Nil/HSV-grown virus (tri+/bi = 8.1). All of the triantennary or larger oligosaccharides from Asn-336 of Nil/HSV-grown VSV G bound to leukoagglutinating phytohemagglutinin-agarose, indicating the presence of a branch attached to the Man3GlcNAc2 core via a beta 1,6-linked GlcNAc residue and suggesting that increased UDP-GlcNAc:alpha-D-mannoside beta 1,6-N-acetylglucosaminyl transferase V (GlcNAc transferase V) activity accompanied transformation. At least 20% of these leukoagglutinating phytohemagglutinin-binding oligosaccharides were sensitive to an enzyme specific for polylactosaminylglycan chains, Escherichia freundii endo-beta-galactosidase.     

Lectin-binding pattern on the surface epidermis of Ambystoma tigrinum larvae

Summary The surface epidermis of Ambystoma tigrinum larvae was examined at the light- and electron-microscope levels using five different lectin conjugates as probes for the detection of sugar residues on the cell membranes. Concanavalin A (Con-A), wheat-germ agglutinin (WGA), Ricinus communis agglutinin I (RCA-I), Dolichos biflorus agglutinin and soybean agglutinin (SBA) conjugates clearly labelled the surface cells, especially their apical surfaces. At electron microscopy, the labelling on plasma membranes was found to exhibit regional differences. Among the lectins tested WGA displayed a particularly characteristic binding pattern. WGA also bound to basolateral cell surfaces, including the tight-junction zone wich was also stained by the RCA-I conjugate. The different labelling intensity and staining patterns obtained with the conjugates indicated the polarity of the cell surfaces. It is also assumed that the WGA staining of the basolateral membranes and intercellular spaces reflected transcellular transport, which is facilitated by acidic glycoconjugates. Other functional aspects of the polarized distribution of the lectin conjugates were also correlated with the receptor sites of certain sugar residues.     

Characterization of the glycosylation state of a recombinant monoclonal antibody using weak cation exchange chromatography and mass spectrometry.

Recombinant monoclonal antibody heterogeneity is inherent due to various enzymatic and non-enzymatic modifications. In this study, a recombinant humanized monoclonal IgG1 antibody with different states of glycosylation on the conserved asparagine residue in the CH(2) domain was analyzed by weak cation exchange chromatography. Two major peaks were observed and were further characterized by enzymatic digestion and mass spectrometry. It was found that this recombinant monoclonal antibody contained three glycosylation states of antibody with zero, one or two glycosylated heavy chains. The peak that eluted earlier on the cation exchange column contained antibodies with two glycosylated heavy chains containing fucosylated biantennary complex oligosaccharides with zero, one or two terminal galactose residues. The peak that eluted later from the column contained antibodies with either zero, one or two glycosylated heavy chains. The oligosaccharide on the antibodies eluted in the later peak was composed of only two GlcNAc residues. These results indicate that conformational changes in large proteins such as monoclonal antibodies, caused by different types of neutral oligosaccharides as well as the absence of oligosaccharides, can be differentiated by cation exchange column chromatography.     

Oligosaccharide profiles of the prostate specific antigen in free and complexed forms from the prostate cancer patient serum and in seminal plasma: a glycopeptide approach

The oligosaccharide structures of prostate specific antigen (PSA) are expected to be useful in discriminating prostate cancer from benign conditions both accompanied by increased serum PSA levels. A large proportion of PSA forms a covalent complex with a glycoprotein, alpha(1)-antichymotrypsin, in human blood. In the present study, the glycan profiles of free and complexed forms of PSA from cancer patient serum and of seminal plasma PSA were compared by analyzing the glycopeptides obtained by lysylendopeptidase digestion of the electrophoretically separated PSA with mass spectrometry. The profiles of the PSA N-glycans from the free and complexed molecules were quite similar to each other and consisted of fucosylated biantennary oligosaccharides as the major class. They were mostly sialylated, and a considerable sialic acid fraction was alpha2,3-linked as determined by Streptococcus pneumoniae neuraminidase digestion of the glycopeptides. In the seminal plasma PSA, high-mannose and hybrid types of oligosaccharides were predominant, and the sialic acids attached to the latter as well as to biantennary oligosaccahrides were exclusively alpha2,6-linked because they were removed by Arthrobacter ureafaciens neuraminidase but resistant to S. pneumoniae neuraminidase. Complex-type oligosaccharides from other sources were found in the seminal plasma sample, indicating that analysis of released glycans carries a risk of being misleading. The results suggest that identification of alpha2,3-linked sialic acids on PSA potentially discriminates malignant from benign conditions, if the analysis is applied to oligosaccharides specifically attached to the N-glycosylation site of PSA in either a free or a complexed form in the serum.     

3'-Azidothymidine potently inhibits the biosynthesis of highly branched N-linked oligosaccharides and poly-N-acetyllactosamine chains in cells

Previous studies in our laboratory have characterized 3'-azido-3'-deoxythymidine (AZT) as a potent inhibitor of glycosphingolipid biosynthesis in cultured cells (Steet, R., Alizadeh, M., Melan?on, P., and Kuchta, R. D. (1999) Glycoconj. J. 16, 237-245; Yan, J.-P., Ilsley, D. D., Frohlick, C., Steet, R., Hall, E. T., Kuchta, R. D., and Melan?on, P. (1995) J. Biol. Chem. 270, 22836-22841). Here, we report that AZT treatment of K562 cells results in significant alterations in the profile of N-linked oligosaccharides. Fractionation of [(3)H]mannose-labeled oligosaccharides from AZT-treated K562 cells using lectin affinity chromatography revealed striking changes in the branching and processing of N-linked glycoconjugates. AZT treatment resulted in the production of fewer highly branched complex glycans (60% of control at 20 micrometer AZT) and a significant accumulation of core-fucosylated biantennary oligosaccharides. In addition, extension of branched oligosaccharides with multiple poly-N-acetyllactosamine repeats is nearly abolished by AZT concentrations as low as 2 micrometer. A shift from multiantennary to moderately branched oligosaccharides was also apparent in the melanoma cell line SK-MEL-30 upon AZT treatment. N-Linked glycans from both cell lines exhibited increased affinity for the beta-galactoside-binding lectin RCA-I in the presence of AZT, suggesting that the addition of terminal sialic acid is sensitive to the drug. These results demonstrate the ability of AZT to modulate strongly the processing of asparagine-linked glycoconjugates in whole cells and reveal a novel mechanism by which AZT treatment may cause anemia.     

Characterisation of N-glycans bound to IGFBP-3 in sera from healthy adults

Human IGFBP-3 contains three potential N-linked glycosylation sites. Published data concerning the type and saccharide composition of the N-glycans is scarce. The aim of this study was to characterise N-glycans covalently attached to IGFBP-3 from sera of healthy adults (men and women). In order to do that a panel of eight lectins covering broad saccharide specificity was used: agarose-immobilised SNA (Sambucus nigra agglutinin), Con A (lectin from Canavalia ensiformis), RCA I (Ricinus communis agglutinin I), PHA-E (Phaseolus vulgaris erythroagglutinin), PHA-L (P. vulgaris leukoagglutinin), succinylated WGA (wheat germ agglutinin), ECL (Erythrina cristagalli lectin) and UEA (Ulex europaeus agglutinin). IGFBP-3 interacted with SNA, Con A, RCA I, PHA-E and, to a much lesser extent, with PHA-L. These results indicate that human IGFBP-3 bears mostly biantennary complex type N-glycans with a very high content of α-2,6-linked Sia at their termini. Hybrid type and high-mannose type N-glycans are present, as well as a bisecting GlcNAc residue, which may be core fucosylated. N-glycosylation of IGFBP-3 follows the N-glycosylation pattern of major serum proteins. This study represents a ground for the future research of glycosylation pattern of IGFBP-3 from the circulation of men and women diagnosed with different illnesses.     

N-glycosylation site mapping of human serotransferrin by serial lectin affinity chromatography, fast atom bombardment-mass spectrometry, and 1H nuclear magnetic resonance spectroscopy.

This report describes the N-glycosylation site mapping of human serotransferrin (h-STF). Reduced and S-carboxymethylated h-STF was digested with trypsin or chymotrypsin. Glycopeptides in the proteolytic digests were isolated by serial concanavalin A (Con A), Sambucus nigra agglutinin (SNA), and Phaseolus vulgaris leukoagglutinin (LPHA) affinity chromatography and subjected to preliminary analysis by 1H NMR spectroscopy. The glycopeptide fractions were then individually digested with N-glycanase. One part of the digest of each fraction was analyzed by fast atom bombardment-mass spectrometry (FAB-MS) to identify the peptide sequences of the glycosylation sites. The other part was used to isolate the oligosaccharide by the corresponding lectin affinity chromatography and to characterize the structures of the isolated oligosaccharides by 1H NMR spectroscopy and FAB-MS. The oligosaccharides in the Con A-bound fraction were shown to have bi-alpha(2-->6)-sialyl, diantennary structures. The SNA-bound fraction was shown to contain trisialyl, triantennary structures. Di- and triantennary oligosaccharides were found to occur on each of the two N-glycosylation sites of h-STF (Asn413 and Asn611) in the ratio of approximately 85:15. The SNA-bound glycopeptides were further fractionated by LPHA affinity chromatography. Two different oligosaccharides were characterized, namely, a trisialyl 2,4-triantennary and a trisialyl 2,6-triantennary glycan. The ratio of 2,4-triantennary vs 2,6-triantennary oligosaccharides attached to glycosylation site Asn413 was found to be approximately 5:1, whereas the two isomeric triantennary oligosaccharides were found to be attached to glycosylation site Asn611 in the ratio approximately 1:1.     

Rapid and simple preparation of N-linked oligosaccharides by cellulose-column chromatography.

As a means of preparing N-linked oligosaccharides from hydrazinolysates of glycoproteins in a rapid and simple manner, a method has been developed using cellulose-column chromatography. Hydrazinolysates of human IgG, containing a series of biantennary complex type oligosaccharides, were applied to a cellulose column equilibrated with (4:1:1, v/v) 1-butanol-ethanol-water. The N-linked oligosaccharides were eluted with (1:1, v/v) ethanol-water, and analyzed by HPLC in combination with sequential glycosidase digestion. The oligosaccharides, with or without sialic acid, were quantitatively recovered in the fraction eluted with (1:1, v/v) ethanol-water without UV-detectable contamination by impurities derived from protein or the cellulose. Other types of N-linked oligosaccharides of alpha1-acid glycoprotein (tetraantennary complex-type), ovalbumin (hybrid-type), and ribonuclease B (high mannose-type) were also quantitatively prepared from the hydrazinolysates by elution of the cellulose column with (1:1, v/v) ethanol-water and these had as high a quality as those prepared by conventional paper chromatography.     

N-linked oligosaccharides of the murine transferrin receptor from a plasmacytoma cell line. Comparison with total cellular N-glycans

The N-linked oligosaccharides synthesised by the murine plasmacytoma cell line NS-1 have been analysed by lectin affinity chromatography on columns of immobilised concanavalin A (Con A), Lens culinaris (lentil), Ricinus communis agglutinin (RCA) and leuko-phytohemagglutinin (L-PHA). The majority of complex N-glycans in this transformed cell line were branched structures with only a low level of biantennary complex chains detected. The analysis showed the major complex N-glycan fraction consisted of a minimum sialylated triantennary structure. [3H]Mannose-labelled transferrin receptor was isolated from NS-1 cells by immunoprecipitation followed by electroelution from SDS polyacrylamide gels. The isolated receptor was digested with Pronase and the 3H-labelled glycopeptides analysed by lectin affinity chromatography. Analysis by Con A-Sepharose indicated that approx. 50% of the labelled glycopeptides were branched complex N-glycans (unbound fraction) while the remainder were oligomannose structures (strongly bound). The presence of tri and/or tetraantennary structures in the Con A unbound fraction was further suggested by the interaction of 61% of the fraction with L-PHA. The lectin profiles obtained for the complex N-glycans of the transferrin receptor glycopeptides were similar to those for the total cellular glycopeptides of NS-1 cells. Reverse-phase HPLC analysis of tryptic glycopeptides of the isolated [3H]mannose-labelled transferrin receptor gave three 3H-labelled peaks, indicating that all three potential N-glycosylation sites on the receptor are utilised. The Con A-Sepharose profiles of the three fractions indicated the presence of branched complex N-glycans and high mannose chains at each site. The profiles of two of the tryptic glycopeptide fractions were very similar, while the third had a higher content of oligomannose oligosaccharides.     

Interaction with lectin of kappa opioid binding sites solubilized from human placenta

Kappa opioid binding sites from human placenta, prelabeled with 3H-etorphine and solubilized, were retained on wheat germ agglutinin (WGA) agarose and specifically eluted with N-acetylglucosamine. No significant retention was found with other immobilized lectins, including Concanavalin A (Con A), soybean seed lectin (SBA), Pisum sativum lectin (PsA), Lens culinaris Medik. lectin (LcA), and Lathyrus tingitanus lectin(LtA). About 23% of applied kappa sites were specifically eluted from WGA agarose, less than half of the proportion of rat brain opioid binding sites eluted from the same lectin (55%). Receptors from placental extracts were compared with those from other tissues enriched in either kappa or mu sites. The proportion of applied kappa sites from guinea pig cerebellum eluted specifically from WGA agarose was 36%, whereas elution of binding sites from rat thalamus and rabbit cerebellum (enriched in mu sites) was at a level of 55%. This difference in the level of retention on and elution from WGA may reflect differences in the sugar composition of the glycoproteins of the two types of receptors. Succinylation of WGA abolished its ability to retain opioid binding sites, consistent with involvement of sialic acid. However, currently available evidence suggests that differences in retention on WGA between kappa and mu sites may be due to differences in either sialic acid or N-acetylglucosamine content or both.