Hybrid-type Glycans Research Articles

Structural assessment of β-glucuronidase carbohydrate chains by lectin affinity chromatography

Rat liver beta-glucuronidase was studied by sequential lectin affinity chromatography. beta-Glucuronidase glycopeptides were obtained by extensive Pronase digestion followed by N-[14C]acetylation and desialylation by neuraminidase treatment. According to the distribution of the radioactivity in the various fractions obtained by chromatography on different lectins, and on the assumption that all glycopeptides were acetylated to the same specific radioactivity, a relative distribution of glycan structure types is proposed. The presence of complex biantennary and oligomannose type glycans (56.8% and 42.7%, respectively) was indicated by Concanavalin A-Sepharose chromatography. Ulex europaeus agglutinin-agarose chromatography revealed the presence of alpha(1-3)linked fucose in some of the complex biantennary type glycans (16.6% of the total glycopeptides). Wheat germ agglutinin chromatography indicated that the minority (0.5%) were hybrid or poly (N-acetyllactosamine) type glycans. Furthermore, the absence of O-glycans, tri-, tetra- and bisected biantennary type glycans was demonstrated by analysis of Concanavalin A-Sepharose unbound fraction by chromatography on immobilized soybean agglutinin, Ricinus communis agglutinin and Phaseolus vulgaris erythroagglutinin.

Modification of blood group A antigen expression in a pancreatic cancer cell line (PC-1) by inhibitors of N-glycan processing.

Pancreatic adenocarcinomas induced in Syrian hamsters by treatment with N-nitrosobis(2-oxopropyl) amine express blood group A antigen, which is absent in normal pancreatic cells. On membrane glycoproteins purified from tumors, blood group A antigen has been found to be expressed on multiantennary Asn-linked complex glycans. In this study, we investigated the effect of inhibitors of Asn-glycan processing on blood group A antigen bearing glycan structures in a cell line (PC-1) established from a primary induced pancreatic cancer. Expression of blood group A antigen on cells and in membrane preparations was blocked by treatment with 1-deoxymannojirimycin, an inhibitor of mannosidase I, but was retained after treatment with swainsonine, an inhibitor of mannosidase II. However, swainsonine treatment altered the glycan structure associated with blood group A antigen from an endoglycosidase H resistant type to a sensitive type, indicating that the blood group A structure might shift from a complex type to a hybrid type glycan by this treatment. These results demonstrate that Asn-linked glycans carry the major blood group A antigens in PC-1 cells.

Interactions of Bowringia mildbraedii agglutinin with complex- and hybrid-type glycans

Affinity chromatography on Bowringia mildbraedii agglutinin (BMA) Sopharose of glycopeptides confirmed a previous report using oligo-saccharides (Animashaun, T. and Hughes, R.C. (1989) J. Biol. Chem. 264,4657–4663) that high affinity binding requires the sequence Manα1→2 Manα1→6 Manα1→6 Manβ1→4. However, moderate binding was still exhibited by structures lacking this sequence provided the oligosaccharide core sequence Manα1→3[Manα1→6]Manβ1→4GlcNAc was present. This moderate binding was not affected by substitution with N-acetylgluco-samine at C2 and C4, respectively, of the Manα1→3 and Manβ1→4 residues and BMA Sepharose should prove to be a useful tool for the isolation of bisected or non-bisected hybrid-type glycans.

Effects of brefeldin A on oligosaccharide processing. Evidence for decreased branching of complex-type glycans and increased formation of hybrid-type glycans

Brefeldin A (BFA), a drug that induces redistribution of Golgi-apparatus proteins into the endoplasmic reticulum, was used to determine the role of subcellular compartmentalization in the processing of asparagine-linked oligosaccharides. Baby-hamster kidney cells were pulse-labelled with [3H]mannose for 30-60 min and chased for up to several hours in the presence or in the absence of BFA or labelled continuously for several hours with and without the drug. Cellular glycoproteins were digested to glycopeptides with Pronase and either fractionated into glycan classes by lectin affinity chromatography or digested further by endoglycosidase H and endoglycosidase D. Released oligosaccharides obtained in the latter procedure were then separated from each other and from endoglycosidase-resistant glycopeptides by paper chromatography. The results show that BFA induces a very fast processing of protein-linked Glc3Man9GlcNAc2 oligosaccharide down to man5GlcNAc2 and conversion into complex-type and hybrid-type glycans. The major difference between untreated and BFA-treated cells is a large increase in bi-antennary and hybrid-type glycans in the latter cells. These results indicate that galactosylation of a mono-antennary GlcNAcMan5GlcNAc2 hybrid blocks subsequent action by mannosidase II and N-acetylglucosaminyl transferase II, producing galactosylated hybrid-type glycans. Similarly, galactosylation of the product of N-acetylglucosaminyltransferases I and II, i.e. a Man3GlcNAc2 core substituted with GlcNAc beta 1----2 on both alpha 1----3- and alpha 1----6-linked mannose residues, blocks branching N-acetylglucosaminyltransferases IV and V, thereby causing an increase in bi-antennary glycans and a decrease in tri- and tetra-antennary glycans.

Carbohydrate-binding specificity of Tetracarpidium conophorum lectin

The carbohydrate-binding specificity of a novel plant lectin isolated from the seeds of Tetracarpidium conophorum (Nigerian walnut) has been studied by quantitative hapten inhibition assays and by determining the behavior of a number of oligosaccharides and glycopeptides on lectin-Sepharose affinity columns. The Tetracarpidium lectin shows preference for simple, unbranched oligosaccharides containing a terminal Gal beta 1----4GlNAc sequence over a Gal beta 1----3GlcNAc sequence and substitution by sialic acid or fucose of the terminal galactose residue, the subterminal N-acetylglucosamine or more distally located sugar residues of oligosaccharides reduce binding activity. Branched complex-type glycans containing either Gal beta 1----4GlcNAc or Gal beta 1----3GlcNAc termini bind with higher affinity than simpler oligosaccharides. The lectin shows highest affinity for a tri-antennary glycan carrying Gal beta 1----4GlcNAc substituents on C-2 and C-4 of Man alpha 1----3 and C-2 of Man alpha 1----6 core residues. Bi- and tri-glycans lacking this branching pattern bind more weakly. Tetra-antennary glycans and mono- and di-branched hybrid-type glycans also bind weakly to the immobilized lectin. Therefore, Tetracarpidium lectin complements the binding specificities of well-known lectins such as Datura stramonium agglutinin, Phaseolus vulgaris agglutinin, and lentil lectin and will be a useful additional tool for the identification and separation of complex-type glycans.

Analysis of the Oligosaccharides on Rat Androgen-Binding Protein Using Serial Lectin Chromatography1

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.

Carbohydrate-binding specificity of the daffodil ( Narcissus pseudonarcissus) and amaryllis ( Hippeastrum hybr.) bulb lectins

The carbohydrate binding specificity of the daffodil ( Narcissus pseudonarcissus; NPA) and amaryllis ( Hippeastrum hybr.; HHA) lectins, isolated from extracts of their bulbs by affinity chromatography on immobilized mannose, was studied by quantitative precipitation, sugar hapten inhibition, and affinity chromatography on the immobilized lectins. These lectins gave strong precipitation reactions with several yeast mannans, but did not precipitate with α- d-glucans (e.g., dextrans and glycogen). Interestingly, both lectins reacted strongly with yeast galactomannans having multiple nonreducing terminal α- d-galactosyl groups, a synthetic linear α-1,6-mannan, and an α-1,3-mannan (DP = 30). Treatment of the linear α-1,3-mannan with periodate, resulting in oxidation of the terminal, nonreducing mannosyl group, did not reduce its reactivity with NPA or HHA. Taken together, these observations suggest that NPA and HHA react not only with terminal but also with internal α- d-mannosyl residues. Sugar hapten inhibition studies showed these lectins to possess the greatest specific activity for α- d-mannosyl units whereas d-Glc and d-GlcNAc did not inhibit either lectin precipitation system. Of the oligosaccharides tested, the best inhibitor of NPA interaction was α-1,6-linked mannotriose, which was twice as good an inhibitor as Manα 1,6Manα- O-Me and 10 times better than methyl α- d-mannoside. On the other hand, oligosaccharides containing either 1,3- or 1,6-linked mannosyl units were good inhibitors of the HHA-mannan precipitation system (6- to 20-fold more active than d-Man). These results indicate that both lectins appear to possess an extended binding site(s) complementary to at least three 1,6-linked α-mannosyl units. Various glycosylasparagine glycopeptides which contain α-1,6-Man units were retarded on the immobilized NPA column. On the other hand, those containing either α-1,3- or α-1,6-mannosyl residues were retarded on the immobilized HHA column; Man 5-GlcNAc 2-Asn [containing two Manα 1,3 (Manα 1,6) units] bound to the HHA column. On the contrary, glycopeptides with hybrid type glycan chains were not retarded on either column. These two new lectins which differ in their fine sugar binding specificity from each other, and also from the snowdrop lectin, should prove to be useful probes for the detection and preliminary characterization of glycoconjugates on cell surfaces and in solution.

Lectin-resistant variants of mouse Lewis lung carcinoma cells. II. Altered glycosylation of membrane glycoproteins

Lectin-resistant variants of mouse Lewis lung carcinoma LL2 cell line, selected with wheat germ agglutinin (WGAR), Ricinus communis agglutinin II (RCA IIR) and Aleuria aurantia agglutinin (AAAR) were studied. Total cellular glycopeptides of the parent LL2 line and of the five lectin-resistant variants were analyzed by gel filtration and affinity chromatography on immobilized concanavalin A and Lens culinaris agglutinin. The results revealed that low-metastatic WGAR and RCA IIR variants possessed less highly branched tri- and tetra-antennary N-acetyllactosaminic type glycans with a simultaneous increase in biantennary N-acetyllactosaminic type, oligomannosidic type or hybrid type glycans, as compared to the parent metastasizing LL2 cell line. These findings imply that cell surface carbohydrate changes may possibly be relevant for metastasis. However, the AAAR variant, which possessed reduced spontaneous metastatic ability after s.c. administration, but increased experimental metastatic ability after i.v. inoculation, exhibited apparently the same glycan pattern than the parent LL2 line. This particular variant is under investigation in order to find specific modification(s) of glycan(s) which could play a specific role in the metastatic process.

Specificity towards oligomannoside and hybrid type glycans of the endo-?-N-acetylglucosaminidase B from the basidiomy ceteSporotrichum dimorphosporum

We have previously shown that an endo-beta-N-acetylglucosaminidase (EC 3.2.1.96) named "Endo B", isolated from culture filtrates of the basidiomycete Sporotrichum dimorphosporum cleaves asialo-, and to some extent, monosialylated bi-antennary glycans of the N-acetyllactosamine type linked to the asparagine residue of peptide or protein moieties [Bouquelet S, Strecker G, Montreuil J, Spik G (1980) Biochimie 62:43-49]. In the present paper, the substrate specificity of the enzyme towards oligomannoside and hybrid type glycans has been analyzed. The results obtained indicate that ovalbumin glycopeptides containing four to seven mannose residues and bovine lactotransferrin glycopeptides containing four to nine mannose residues were completely hydrolyzed by the enzyme. The degree of cleavage was variable among hybrid type structures, since glycopeptides containing the following glycans: (Gal)1(GlcNAc)3(Man)5(GlcNAc)2; (GlcNAc)3(Man)5(GlcNAc)2;(GlcNAc)3(Man)4(GlcNAc)2 were not hydrolyzed by the enzyme while the percentage of hydrolysis of a glycopeptide containing (GlcNAc)2(Man)5(GlcNAc)2 glycan reached 90%. The bovine lactotransferrin was partially deglycosylated (40%) in the absence of non-ionic detergent while native ovalbumin glycoprotein was not hydrolyzed by the enzyme. The oligomannoside- and the N-acetyllactosamine-type degrading activities present in the culture filtrates were not separated at any step of the purification procedure. Both activities were eluted as a single component with an apparent molecular mass of 89 kDa suggesting that they are located on the same enzyme molecule. Endo B represents a powerful tool for removing oligomannoside- and N-acetyllactosamine-type glycans from N-glycopeptides and N-glycoproteins. Moreover, advantages in the use of Endo B in a soluble form as well as in an immobilized form result in its high activity and in its stability to heat denaturation and storage.

Binding properties of a mannose-specific lectin from the snowdrop (Galanthus nivalis) bulb.

Carbohydrate binding properties of a new plant lectin (GNA) isolated from snowdrop bulbs were studied using the technique of quantitative precipitation, hapten inhibition, and affinity chromatography on immobilized lectin. Purified GNA precipitated highly branched yeast mannans but did not react with most glucans. Hapten inhibition experiments showed that D-mannose is an inhibitor of GNA-mannan interaction but neither N-acetyl-D-mannosamine nor D-glucose is an inhibitor. Hapten inhibition with various sugars showed that GNA requires the presence of equatorial hydroxyl groups at the C-3 and C-4 positions and an axial group at the C-2 position of the D-pyranose ring. A nonreducing terminal D-mannose residue is necessary for the interaction of oligosaccharides, and oligosaccharides with terminal Man(alpha-1-3)Man units showed the highest inhibitory potency (10-30 times greater than D-mannose) among the manno-oligosaccharides tested. The presence of the hydrophobic p-nitrophenyl aglycone increased the affinity of D-mannose only slightly. Immobilized GNA bound yeast mannan but did not bind glycogen. The behavior of glycoproteins with high mannose type glycan chains depended on the density and the structure of their glycan chains. Glycopeptides which carry Man(alpha 1-3)Man units were retarded on the immobilized GNA column whereas those lacking this unit or with hybrid type glycan chains were not retarded on the column.

Variation in the core and branch carbohydrate sequences of serum glycoprotein hormone alpha subunit as determined by lectin affinity chromatography.

Serum alpha subunits from patients with pituitary tumours and from normal controls were studied for their ability to bind to Lens culinaris agglutinin-Sepharose (LCA), L-phytohaemagglutinin-agarose (L-PHA) and soybean agglutinin-Sepharose (SBA). Serum alpha subunits from normal controls which had previously been shown to bind to Concanavalin A-Sepharose (Con A) were not retained by LCA. In contrast, Con A-reactive alpha subunits from patients with pituitary tumours bound specifically to LCA. Non-Con A-reactive alpha subunits from patients with pituitary tumours were also largely not bound to LCA, but were retained by L-PHA. No alpha subunits from any source bound to SBA. These results indicate that the structural alterations resulting in non-Con A-reactive serum alpha subunits include highly branched complex oligosaccharides in addition to the hybrid-type glycans previously described. The increased branching appears to be associated with fucosylation in the core region of the oligosaccharides. Serum alpha subunit from any source appears to be devoid of terminal N-acetylgalactosamine residues. These structural modifications may be related to the variable biological activity of alpha subunit which has been reported.