Enzymatic synthesis of natural and 13C enriched linear poly-N-acetyllactosamines as ligands for galectin-1.

As part of a study of protein-carbohydrate interactions, linear N-acetyl-polyllactosamines [Galbeta1,4GlcNAcbeta1,3]nwere synthesized at the 10-100 micromol scale using enzymatic methods. The methods described also provided specifically [1-13C]-galactose-labeled tetra- and hexasaccharides ([1-13C]-Galbeta1,4GlcNAcbeta1,3Galbeta1,4Glc and Galbeta1, 4GlcNAcbeta1,3[1-13C]Galbeta1,4GlcNAcbeta1,3Galbeta 1,4Glc) suitable for NMR studies. Two series of oligosaccharides were produced, with either glucose or N-acetlyglucosamine at the reducing end. In both cases, large amounts of starting primer were available from human milk oligosaccharides (trisaccharide primer GlcNAcbeta1,3Galbeta1, 4Glc) or via transglycosylation from N-acetyllactosamine. Partially purified and immobilized glycosyltransferases, such as bovine milk beta1,4 galactosyltransferase and human serum beta1,3 N- acetylglucosaminyltransferase, were used for the synthesis. All the oligo-saccharide products were characterized by1H and13C NMR spectroscopy and MALDI-TOF mass spectrometry. The target molecules were then used to study their interactions with recombinant galectin-1, and initial1H NMR spectroscopic results are presented to illustrate this approach. These results indicate that, for oligomers containing up to eight sugars, the principal interaction of the binding site of galectin-1 is with the terminal N-acetyllactosamine residues.

Induction of histamine release from non-immunized guinea pigs: a possible involvement of lectin-like factor(s) in pollinosis.

To investigate nonimmune pathogenic functions of pollens, vascular permeability enhancement (VPE) activity of pollen extracts was examined using guinea pigs nonimmunized against pollens. Ryegrass, ragweed, mesquite and almond, but not common cattail and sumac, induced VPE which was inhibited primarily by an anti-histamine drug. Ryegrass pollen VPE activity was extracted more at pH 7.3 than at pH 6.5 or 8.0 and the maximal activity was extracted in 30 min. Interestingly, more than 60% of the maximal activity was extracted in 5 min. The maximal VPE activity had a dose-dependency similar to histamine (3 x 10(-5) M) but lasted longer than the histamine activity. The VPE activity was inhibited by oligomannose-glycosylated ovalbumin or avidin, as well as the oligosaccharides but not by the deglycosylated proteins. These results indicate that some pollens contain lectin-like, histamine-releasing factor(s), which may be involved in part in pollinosis, by inducing mast cell degranulation through a nonimmune mechanism and resulting in allergy-like symptoms.

The effects of monensin on blood-borne arrest and glycosylation of BL/VL3 lymphoma cells.

We have previously shown that inhibitors of N-glycan processing alter both the cell surface carbohydrates and the homing properties in lymphoid cells. We have now studied the effects of the ionophore monensin (MON) on these parameters. Arrest in the spleen of [111In]-labelled BL/VL3 murine T lymphoma cells, injected intravenously was clearly reduced if the cells had been cultured for 24 h in the presence of monensin (0.1-1.0 microgram ml-1). We have characterized glycopeptides from BL/VL3 murine T lymphoma cells. Following labelling with tritiated precursors (fucose, mannose, galactose, glucosamine), surface glycopeptides from BL/VL3 murine T lymphoma cells, were released by trypsin and separated by gel filtration on Bio-Gel P6 and by affinity chromatography on immobilized lectins. After treatment with MON, a class of high molecular mass glycopeptides was no longer found. There were less complex and more high mannose glycans, as a consequence of a reduction of terminal glycosylation (sialylation, fucosylation or incorporation of N-acetyl-glucosamine). Similar findings were obtained with immunoprecipitated Thy-1 antigen. However, as estimated by flow cytometry analysis, the cell surface expression of Thy-1 was not reduced in MON-treated cells. Taken together our results show that cell surface oligosaccharides are modified dramatically, but that at least, certain cell surface antigens are present in normal amounts. It is tempting to speculate that changes in glycosylation account for the abnormal homing properties of MON-treated cells.

Comparison of the N- and O-linked glycopeptides of lymph node cells from C57 BL/6 lpr/lpr and C57 BL/6 mice.

We have tested the hypothesis that some phenotypic characteristics of the lymphocytes from mice with lymphoproliferative disease (lpr) could be explained by abnormal glycosylation of membrane proteins. Lymph node cells from normal C57 BL/6 and from C57 BL/lpr mice were labelled with tritiated sugars. Membrane proteins were released with trypsin, then with pronase. After complete pronase digestion, glycopeptides were first separated on Bio Gel P-6 and then on Con A-Sepharose. Fractions not binding to Con A (Con A negative) were also separated on Lens culinaris agglutinin-Sepharose. Marked differences between normal and lpr cells were noticed. First, there were more glucosamine-labelled peptides with very high molecular weight (eluting fast on Bio Gel P-6) on lpr cells than on normal lymphocytes. Second, the proportion of mannose-labelled peptides binding to Con A was smaller in the lpr cells. Third, among the Con A negative peptides, the proportion binding to Lens culinaris agglutinin was higher in lpr cells. Thus, lpr cells seem to carry more alpha 1-6 fucosylated chains and larger size carbohydrates. These alterations were also confirmed by gel electrophoresis of lectin-selected iodinated cell surface antigens and seem to be restricted to a very limited number of peptides. Thus, there may be primary changes in glycosylation in lpr cells. Alternatively, the glycosylation pattern of lpr cells may be characteristic for a subpopulation of T-lymphocytes that is expanded in this disease, or for a certain stage of activation. A large proportion of Con A-negative, Lens culinaris-positive peptides is a rather unusual feature in murine cells and requires further investigation.

Multiple differences between wild-type B16 melanoma cells and a wheat germ agglutinin resistant clone.

A low-metastatic, glycosylation-defective variant of the B16 murine melanoma was obtained by Tao and Burger (1977) through selection with wheat germ agglutinin. We found that variant and parental (wild-type) cell lines were equally invasive when confronted with precultured embryonic chick heart fragments in vitro. Also, a short-term in vivo arrest assay showed no significant differences. After intravenous injection, wild-type cells killed the recipient mice faster than did the variant cells. We were able to confirm the changes in glycosylation at the enzyme level. In addition, we showed that the pattern of endogenous lectins was strikingly different, at least at the quantitative level. We also looked at another set of receptor proteins, namely receptors for neurotransmitters coupled to adenylate cyclase. The response to the vasoactive intestinal peptide and prostaglandins was lower in the variant cells, which also had a delayed response to cholera toxin. Although most of the data can be explained by altered glycosylation in the variant cells, the large number of differences between variant and parent cells makes it difficult to identify the biochemical basis of altered metastatic behaviour. This might also be the case with other pairs of cells differing in metastatic activity.

Effect of glycosylation inhibitors on N-glycosylpeptides and on invasion of malignant mouse MO4 cells in vitro.

Cell surface glycans are believed to play a role in tumour invasion and metastasis. Yet, we have previously shown that the inhibitors of N-linked glycan processing swainsonine (SW) and 1-deoxynojirimycin (dNM) did not prevent invasion of chick heart fragments by MO4 murine fibrosarcoma cells in organ culture. We now present biochemical evidence that these and other inhibitors of processing were indeed effective in remodeling glycans, including those expressed at the cell surface. After metabolic labeling with tritiated mannose or fucose, glycosylpeptides were obtained by Pronase treatment of material released from intact cells by trypsin. Glycosylpeptides were separated by Biogel P-10 chromatography. With all drugs tested, there was a shift towards lower molecular weight of the glycan chains. There were, however, major quantitative differences between the different drugs and also, for monensin (MON; 0.1 microgram ml-1), between fucose-labeled and mannose-labeled chains. The shift in apparent molecular weight affected mainly fucose-labeled peptides after treatment of MO4 cells with SW (0.4 microgram ml-1). The shift induced by dNM (10 mM) + SW (0.4 microgram ml-1) in both fucosylated and mannosylated chains was much larger than that induced by SW given alone. 1-Deoxymannojirimycin (dMM; 1 mM) had major effects on both mannose and fucose-labeled structures and so did N-methyl-1-deoxynojirimycin (MdNM; 2 mM) and castanospermine (CS; 100 micrograms ml-1). With the latter drugs, incorporation of fucose in complex-type glycosylpeptides was dramatically reduced. The effect of SW on fucose-labeled glycosylpeptides of embryonic chick heart was similar to that observed on MO4 cells.(ABSTRACT TRUNCATED AT 250 WORDS)