Analysis of asparagine-linked oligosaccharides by sequential lectin affinity chromatography

Lectins are proteins that specifically bind to a particular carbohydrate structure. Affinity chromatography with immobilized lectins is a quite effective technique not only for the fractionation of glycoproteins or oligosaccharides but also their structural assessment. In this article, we focus on the separation of glycopeptides and oligosaccharides derived from glycoproteins by affinity chromatography on immobilized lectin columns.     

Systematic fractionation of oligosaccharides of human immunoglobulin G by serial affinity chromatography on immobilized lectin columns

Human immunoglobulin G is known to contain 16 different biantennary complex-type asparagine-linked sugar chains, each of which occurs in a nonsialylated, monosialylated, or disialylated form. These oligosaccharides can be separated into 14 fractions by sequential affinity chromatography with Aleuria aurantia lectin (AAL)-Sepharose, RCA120-WG003, and E4-phytohemagglutinin-agarose columns. Twelve of them were found to contain a single oligosaccharide, while the fraction which passed through all three columns was shown to contain two oligosaccharides, GlcNAc beta 1----2Man alpha 1----6(+/- GlcNAc beta 1----4) (GlcNAc beta 1----2Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4GlcNAcOT. The fraction, which bound to the AAL-Sepharose column and passed through the remaining two lectin columns, also contained two oligosaccharides, GlcNAc beta 1----2Man alpha 1----6(+/- GlcNAc beta 1----4) (GlcNAc beta 1----2Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4 (Fuc alpha 1----6)GlcNAcOT. These results indicated that serial affinity chromatography with the three lectin columns can be used effectively to detect changes in the sugar chains of IgG resulting from diseases such as rheumatoid arthritis.     

Purification of androgen-binding protein from rat testis using high-performance liquid chromatography and physicochemical properties of the iodinated molecule

The androgen-binding protein (ABP) has been purified 87,500-fold from rat testis using 4 steps of HPLC, with a yield of 14%. The molecule was 99% pure with a specific activity estimated to 16,600 pmol/mg protein. The iodinated molecule was eluted in 2 peaks in Sephacryl S300 gel filtration with a molecular mass estimated to be 92,600 +/- 3300 and 50,300 +/- 4000 Da. The column isoelectrofocusing of 125I-ABP demonstrated 3 isoproteins isoelectric at pH 4.7, 4.9 and 5.3 and the sedimentation coefficient was estimated to be 4.7 S in sucrose gradient ultracentrifugation. The 125I-ABP had similar physiochemical properties to the non-labelled ABP of epididymis.     

Characterization of oligosaccharides by lectin affinity high-performance liquid chromatography

Alterations of the oligosaccharide structures of glycoproteins are associated with differentiation, malignant transformation, and expression of the same protein in different cell types. The potential biological importance of oligosaccharides has resulted in a growing need for detailed structural information. When glycoproteins are available in limited quantities and/or bear highly heterogeneous oligosaccharides, characterization of their oligosaccharides is difficult. We have developed an efficient approach for obtaining detailed information about oligosaccharides by determining structural 'fingerprints' using lectin affinity high-performance liquid chromatography.     

Androgens and androgen-binding protein in the rat epididymis

The levels of testosterone, dihydrotestosterone (DHT), 5alpha-androstan-3alpha,17beta-diol and androgen-binding protein (ABP) were measured in various segments of the epididymis from adult rats which had been unilaterally orchidectomized for 4 weeks. On the 'intact' side, ABP concentrations were highest in the caput region. The segmental distribution of DHT closely followed that of ABP with the highest concentration in the caput (40 ng/g tissue) and lowest in the cauda (10 ng/g tissue) epididymidis. There was a high degree of correlation (r = 0.98) between the concentration of DHT in the epididymis and ABP levels. 'Castration' completely abolished the DHT gradient. The levels of testosterone and androstanediol were lower than those of DHT; most was present in the corpus epididymidis. The relative differences were reduced after 'castration'. It is concluded that ABP in the rat epididymis is the primary factor for determining the concentration of DHT in the epididymal fluid.     

Isolation of glycopeptides containing O-linked oligosaccharides by lectin affinity chromatography on jacalin-agarose

Jacalin is a lectin which has high specificity and affinity for the core disaccharide, 1-beta-galactopyranosyl-3-(alpha-2-acetamido-2-deoxygalactopyranoside ), in O-linked oligosaccharides. Here, it is shown that this lectin can be used for isolation of glycopeptides bearing O-linked oligosaccharides. Peptides produced by digestion of reduced and carboxamidomethylated human plasminogen or of bovine protein Z were chromatographed on a column of jacalin-agarose. Reverse-phase high-performance liquid chromatography revealed that two peptides from plasminogen and one from protein Z were eluted from the jacalin-agarose column by alpha-methylgalactopyranoside. Amino acid sequence and compositional analysis showed that both of the peptides from plasminogen consisted of residues 330-357 and that the single peptide from protein Z represented residues 385-396. These sequences contain the single known site of attachment of O-linked oligosaccharides to these proteins. The present analysis suggested that there may be a fraction of plasminogen with two sites of O-linked glycosylation. The two tryptic peptides isolated from plasminogen represented the same segment of the protein but sequence analysis showed that one peptide was modified only at Thr346, the known site of glycosylation, and the other peptide contained a modification of Ser339 as well. Results of the present study indicate that lectin affinity chromatography using jacalin-agarose can be a useful technique for isolating glycopeptides containing O-linked oligosaccharides and thereby localizing sites of attachment of these oligosaccharides.     

Analysis of glycoprotein oligosaccharides using high-pH anion exchange chromatography

The natural heterogeneity of glycoprotein glycans requires that chromatography be an essential part of structural elucidation. The isomeric nature of oligosaccharide structure requires chromatography which is selective for not only composition, size and anomerity, but also ring substitutions and branching configurations. HPAEC is sensitive to these structural features and thus, has become an important new method for understanding the elusive function of glycoprotein glycans.     

Characterization studies of a rat hepatic cytosolic androgen-binding protein

A rat hepatic cytosolic [3H]methyltrienolone (R1881) binding protein was studied under various conditions. This protein was also compared with the male-specific high capacity--low affinity estrogen-binding protein derived from the same cytosolic fraction. Analysis of the R1881 binding protein in adult (60-85 days old) male rat liver cytosol indicated the presence of a high affinity--low capacity binding site (Kd = 0.3 nM; Bmax = 5.9 fmol/mg) and a lower affinity--higher capacity component (Kd = 10.4 nM; Bmax = 131 fmol/mg). The latter component was eliminated by addition of triamcinolone or cortisol to the assay mixture. Steroid binding to the high affinity R1881 site was specific for testosterone, dihydrotestosterone, androstenedione, and mibolerone, with a moderate specificity to cyproterone acetate, flutamide hydroxide, and estradiol. Saturation studies indicated that these steroids were binding to the same or a similar high affinity component except for flutamide hydroxide which produced nonsaturable displacement. The high affinity site had no specificity for progesterone, diethylstilbestrol, or cortisol. Like the high capacity--low affinity protein, this protein was not present in the immature, adult, or 10-day ovariectomized adult female. However, unlike the high capacity--low affinity protein, it was present in low quantities in the immature male. In addition, castration of the adult for 18 h, 4 days, or 10 days or hypophysectomy for 10-17 days did not have a significant effect on the high affinity component compared with the controls. Testosterone administration to these animals did not alter this protein binding. These studies indicate that a specific, high affinity--low capacity androgen-binding protein exists in rat hepatic cytosol. Furthermore, this protein shows age and sex dependency, but its presence is not affected by altering gonadal or hypophyseal factors in the adult male.     

Immunohistochemical demonstration of androgen-binding protein in the rat prostatic gland.

Androgen-binding protein (ABP) is one of the best-characterized products of synthesis by the Sertoli cells in the rat. Although the exact physiological role of ABP remains to be determined, it has been widely used to study Sertoli cells and testicular function in this species. Since this protein is the principal carrier for testosterone in rat testis and epididymis, we decided to investigate ABP immunoreactivity (ABP-I) in androgen-dependent organs, including testicle, epididymides, prostate, and seminal vesicles. The location of ABP was investigated by immunohistochemistry using specific antisera against rat ABP. As previously described in the testis, rat ABP-I was identified in the seminiferous tubules within the cytoplasm of the Sertoli cells and the tubular luminae. The epididymis showed ABP-I only in epithelial cells of the proximal caput. We demonstrated ABP-I in the apical portions of epithelial cells of the rat prostate. Short-term castration and/or ligation of the efferent ducts did not suppress prostatic ABP-I. ABP-I was not present in seminal vesicles of control rats nor under any of the experimental conditions used throughout this study. The results also indicate the presence of ABP-I in prostatic epithelium, probably because of a mechanism similar to that described in epididymis. Our data support and enhance the concept that ABP may serve as a transmembrane carrier protein for androgens in androgen target organs in the male reproductive tract.     

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.     

Purification and immunochemical characterization of the cytoplasmic androgen-binding protein of rat liver

The cytoplasmic androgen-binding (CAB) protein of the male rat liver has been implicated to play a role in the androgen-dependent regulation of alpha 2u-globulin synthesis. The liver of the adult male rat contains about 50 fmol of specific high-affinity androgen-binding activity per milligram of total cytosolic protein. Photoaffinity labeling with [3H]R-1881 followed by SDS-polyacrylamide gel electrophoresis and autoradiography shows that the CAB is a 31-kilodalton protein. By means of DEAE-cellulose chromatography and preparative SDS-polyacrylamide gel electrophoresis, we have purified the CAB protein to electrophoretic homogeneity and have raised polyclonal rabbit antiserum that is monospecific to this protein. In the sucrose density gradient, the antiserum reacted with the androgen-binding component of the male liver cytosol prelabeled with tritiated dihydrotestosterone. Western blot analysis of the liver cytosol showed that the antiserum recognizes only the 31-kDa androgen-binding component. Such immunoblotting also showed that unlike the young adult, the androgen-insensitive states during prepuberty and senescence are associated with a marked reduction in the hepatic concentration of the immunoreactive CAB protein. No immuno-chemical cross-reactivity between CAB and another androgen-binding component of Mr 29K (which is associated with androgen insensitivity during prepuberty and senescence) was observed. The latter finding favors the possibility that 31- and 29-kDa androgen-binding components may have distinct sequence structure.     

The role of asparagine-linked oligosaccharides in the subunit structure, steroid binding, and secretion of androgen-binding protein.

Testicular androgen-binding protein (ABP) and liver sex hormone-binding globulin are encoded by the same gene. These proteins have the same primary amino acid sequences, but they differ in attached oligosaccharides; the differences are presumably due to cell-specific glycosylation mechanisms. To investigate the role of oligosaccharides in ABP/sex hormone-binding globulin subunit structure, secretion, and steroid binding, mutant rat ABP proteins were constructed that eliminated one or both of the two potential sites of asparagine (Asn)-linked glycosylation. Immunoblot analysis of wild type recombinant ABP yielded the typical heterogeneous banding pattern. Secreted ABP was composed of two protomers of M(r) 46,000 and M(r) 43,000, while cellular ABP yielded three mol wt species (M(r) 43,000, 41,000, and 39,000). Substitution of the Asn residue in either consensus sequence for Asn-linked glycosylation with an Ile residue resulted in increased mobility of the immunoreactive ABP species. These changes are consistent with the loss of an Asn-linked oligosaccharide. Substitution of both Asn residues yielded a single immunoreactive species in the medium and cell extracts that migrated as a M(r) 39,000 protein. These results demonstrate that the mol wt heterogeneity of ABP is due to differential Asn-linked glycosylation of both potential sites. All three mutant forms of ABP were secreted by the COS cells. However, the amount of immunoreactive ABP and [3H]5 alpha-dihydrotestosterone binding in the medium was lower than wild type (100%) in one of the single mutants (65%) and in the double mutant (29%). Unlike the glycosylation mutants, alteration of other residues, not involved in glycosylation, yielded cellular ABP and no detectable medium ABP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of the glycoproteome of Toxoplasma gondii using lectin affinity chromatography and tandem mass spectrometry

Glycoproteins are involved in many important molecular recognition processes including invasion, adhesion, differentiation, and development. To identify the glycoproteins of Toxoplasma gondii, a proteomic analysis was undertaken. T. gondii proteins were prepared and fractioned using lectin affinity chromatography. The proteins in each fraction were then separated using SDS-PAGE and identified by tryptic in gel digestion followed by tandem mass spectrometry. Utilizing these methods 132 proteins were identified. Among the identified proteins were 17 surface proteins, 9 microneme proteins, 15 rhoptry proteins, 11 heat shock proteins (HSP), and 32 hypothetical proteins. Several proteins had 1–5 transmembrane domains (TMD) with some being as large as 608.3 kDa. Both lectin-fluorescence labeling and lectin blotting were employed to confirm the presence of carbohydrates on the surface or cytoplasm of T. gondii parasites. PCR demonstrated that selected hypothetical proteins were expressed in T. gondii tachyzoites. This data provides a large-scale analysis of the T. gondii glycoproteome. Studies of the function of glycosylation of these proteins may help elucidate mechanism(s) involved in invasion improving drug therapy as well as identify glycoproteins that may prove to be useful as vaccine candidates.     

Fractionation of L-fucose-containing oligosaccharides on immobilized Aleuria aurantia lectin

The carbohydrate-binding specificity of Aleuria aurantia lectin was investigated by analyzing the behavior of a variety of fucose-containing oligosaccharides on an A. aurantia lectin-Sepharose column. Studies with complex-type oligosaccharides obtained from various glycoproteins by hydrazinolysis and their partial degradation fragments indicated that the presence of the alpha-fucosyl residue linked at the C-6 position of the proximal N-acetylglucosamine moiety is indispensable for binding to the lectin column. Binding was not affected by the structures of the outer chain moieties nor by the presence of the bisecting N-acetylglucosamine residue. These results indicated that A. aurantia lectin-Sepharose is useful for the group separation of mixtures of complex-type asparagine-linked sugar chains. Studies of glycosylated Bence Jones proteins indicated that this procedure is also applicable to intact glycoproteins. The behavior of oligosaccharides isolated from human milk and the urine of patients with fucosidosis indicated that the oligosaccharides with Fuc alpha 1----2Gal beta 1----4GlcNAc and Gal beta 1----4(Fuc alpha 1----3)GlcNAc groups interact with the lectin, but less strongly than complex-type sugar chains with a fucosylated core. Lacto-N-fucopentaitol II, which has a Gal beta 1----3(Fuc alpha 1----4)GlcNAc group, interacts less strongly than the above two groups with the matrix. Oligosaccharides with Fuc alpha 1----2Gal beta 1----3GlcNAc and Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4(Fuc alpha 1----3)GlcNAc groups showed almost no interaction with the matrix.     

Analysis of glycosaminoglycan-derived oligosaccharides using reversed-phase ion-pairing and ion-exchange chromatography with suppressed conductivity detection

Oligosaccharides prepared from glycosaminoglycans (GAGs) including heparin, heparan sulfate, chondroitin sulfates, dermatan sulfate, and keratan sulfate were analyzed using reverse-phase ion-pairing HPLC and ion-exchange HPLC with suppressed conductivity detection. The results were compared with those obtained by strong anion-exchange HPLC using uv detection. These oligosaccharides were first prepared by enzymatically depolymerizing the GAGs with enzymes including heparin lyase (EC 4.2.2.7), heparan sulfate lyase (EC 4.2.2.8), chondroitin ABC lyase (EC 4.2.2.4), and keratan sulfate hydrolase (EC 3.2.1.103). Analysis was then performed without derivitization under isocratic conditions with a limit of sensitivity in the picomole range. Preliminary studies suggest that this approach may be particularly useful in examining oligosaccharides having no uv chromophore such as those prepared from keratan sulfate.     

Modification of the N-linked oligosaccharides in cell surface glycoproteins during chick embryo development. A using lectin affinity and a high resolution chromatography study

Important differences in asparagine-linked glycopeptides were observed in vitro cultured fibroblasts derived from chick embryo at different stages of development. Cells from 8-day and 16-day embryos were labeled metabolically with [3H]mannose. Cell surface glycopeptides obtained after mild trypsin treatment were extensively digested with pronase and then chromatographed on concanavalin-A-Sepharose and other immobilized lectins. The most important changes concerned the complex type chains. The ratio between triantennary plus tetraantennary and biantennary chains increased about 2.5-fold from the 8th to the 16th day of development. In the same way, complex chains with bisecting N-acetylglucosamine increased from 8-day to 16-day cells as shown by Phaseolus-vulgaris-erythroagglutinin--agarose chromatography. In 16-day cells, the majority of triantennary chains (60%) with alpha-linked mannose substituted at C2 and C6 positions and biantennary chains (50%) were shown to contain fucosyl (alpha 1----6)N-acetylglucosaminyl structure in the core region by their ability to bind to a lentil lectin affinity column. Similarly, in 8-day cells, triantennary chains (50%) were more fucosylated than biantennary chains (35%). Thus, complex structures exhibited an increased fucosylation of their invariable core from the 8th to the 16th day of development, except for fucosylated triantennary chains which were retained on Phaseolus vulgaris Leucoagglutin and on lentil lectin. These latter structures were present at the surface of 8-day cells and absent at the surface of 16-day cells. After chromatography on Bio-Gel P6 and treatment with endo-beta-N-acetylglucosaminidase H, the [3H]-mannose-labeled glycopeptides were separated by high resolution chromatography into glycopeptides with complex chains and glycopeptides with high-mannose chains. Analysis of the high-mannose oligosaccharides released after endo-beta-N-acetylglucosaminidase H treatment by chromatography on Bio-Gel P4 indicated that the same type of high-mannose chains were present at the surface of 8-day and 16-day cells. Quantification of mannose, galactose and sialic acid residues using gas liquid chromatography was consistent with a decrease of the relative amount of oligomannose chains and an increase of the relative amount of complex type chains in 16-day cells compared to 8-day cells. Thus N-linked oligosaccharides derived from cell surface glycoproteins undergo changes during embryo development resulting in greater complexity of carbohydrate chains.     

Characterization of the human submandibular/sublingual saliva glycoproteome using lectin affinity chromatography coupled to multidimensional protein identification technology

In-depth analysis of the salivary proteome is fundamental to understanding the functions of salivary proteins in the oral cavity and to reveal disease biomarkers involved in different pathophysiological conditions, with the ultimate goal of improving patient diagnosis and prognosis. Submandibular and sublingual glands contribute saliva rich in glycoproteins to the total saliva output, making them valuable sources for glycoproteomic analysis. Lectin-affinity chromatography coupled to mass spectrometry-based shotgun proteomics was used to explore the submandibular/sublingual (SM/SL) saliva glycoproteome. A total of 262 N- and O-linked glycoproteins were identified by multidimensional protein identification technology (MudPIT). Only 38 were previously described in SM and SL salivas from the human salivary N-linked glycoproteome, while 224 were unique. Further comparison analysis with SM/SL saliva of the human saliva proteome, revealed 125 glycoproteins not formerly reported in this secretion. KEGG pathway analyses demonstrated that many of these glycoproteins are involved in processes such as complement and coagulation cascades, cell communication, glycosphingolipid biosynthesis neo-lactoseries, O-glycan biosynthesis, glycan structures-biosynthesis 2, starch and sucrose metabolism, peptidoglycan biosynthesis or others pathways. In summary, lectin-affinity chromatography coupled to MudPIT mass spectrometry identified many novel glycoproteins in SM/SL saliva. These new additions to the salivary proteome may prove to be a critical step for providing reliable biomarkers in the diagnosis of a myriad of oral and systemic diseases.     

Thin-layer chromatography of hyaluronate oligosaccharides

Odd- and even-numbered hyaluronate oligosaccharides with N-acetylglucosamine, glucuronic acid, or 4,5-unsaturated glucuronic acid at their nonreducing ends were separated by thin-layer chromatography, using silica gel and a solvent system of isopropanol-water (66 : 34) containing 0.05 M NaCl. In the isopropanol system, small amounts of electrolytes were necessary for the resolution of each oligosaccharide.