Glycosylation of the human erythrocyte glucose transporter: a minimum structure is required for glucose transport activity.

The involvement of the carbohydrate moiety of the human erythrocyte glucose transporter in glucose transport activity was previously demonstrated (Feugeas et al. (1990) Biochim. Biophys. Acta 1030, 60-64): N-glycanase treatment of the transport glycoprotein reconstituted in proteoliposomes resulted in a dramatic decrease of the Vmax. In this study, kinetic measurements of glucose equilibrium influx confirm our previous results. In order to investigate that a minimum glycosidic structure is required to maintain glucose transport activity, proteoliposomes were respectively treated with either sialidase, or sialidase and endo-beta-galactosidase, or a pool of exo-glycosidases which allows the release of all the sugar residues, except the proximal N-acetylglucosamine. Kinetic measurements of zero-trans influx made on sialidase- and (sialidase + endo-beta-galactosidase)-treated proteoliposomes did not reveal any significant changes in the glucose transport activity. On the contrary, treatment of the same proteoliposomes by a pool of exoglycosidases led to a complete abolition of activity, suggesting that a minimum glycosidic structure is required for glucose transport activity.

Glycosylation of the human erythrocyte glucose transporter is essential for glucose transport activity.

The human erythrocyte glucose transporter is a fully integrated membrane glycoprotein having only one N-linked carbohydrate chain on the extracellular part of the molecule. Several authors have suggested the involvement of the carbohydrate moiety in glucose transport, but not definitive results have been published to date. Using transport glycoproteins reconstituted in proteoliposomes, kinetic studies of zero-trans influx were performed before and after N-glycanase treatment of the proteoliposomes: this enzymatic treatment results in a 50% decrease of the Vmax. The orientation of transport glycoproteins in the lipid bilayer of liposomes was investigated and it appears that about half of the reconstituted transporter molecules are oriented properly. Finally, it could be concluded that the release of the carbohydrate moiety from the transport glycoproteins leads to the loss of their transport activity.

Circulating malignant lymphocytes from Sezary syndrome express high level of glycoproteins carrying beta (1-6)N-acetylglucosamine-branched N-linked oligosaccharides.

The circulating forms of malignant cells from patients with Sezary syndrome exhibit on their glycoproteins a high level of beta (1-6)GlcNAc-branched N-linked oligosaccharides, a particular species of glycans related to the metastatic potential of several tumors and T lymphocytes activation. An increased activity of the N-acetylglucosaminyltransferase V and of the beta (1-4)galactosyltransferase, two enzymes implicated in beta (1-6)GlcNAc-branching is also found. Nevertheless, contrary to activated normal T lymphocytes, Sezary lymphocytes in agreement with their non-proliferating state, do not exhibit increased thymidine uptake. This result suggests that expression of the beta (1-6)GlcNAc-branched N-linked carbohydrates could be related to some of the malignant properties of Sezary lymphocytes.

Isolation and characterization of 1-O-alpha-2-acetamido-2-deoxy-D-galactopyranosyl-myo-inositol from pregnancy urine.

A new neutral glycoside of myo-inositol was isolated from the pregnancy urine of a single donor. Its structure was investigated by 1H-NMR spectroscopy and mass spectroscopy. It was identified as 1-O-alpha-2-acetamido-2-deoxy-D-galactopyranosyl-myo-inositol. No such structure or sequence has previously been reported in either myo-inositol or glucose glycosides.

T lymphocytes from Sézary syndrome patients express beta1 integrins whose beta(1-6)-branched N-linked oligosaccharides reflect their adhesive capacity.

Sézary syndrome (Sz), characterized by slowly progressing clonal proliferation of CD4+, CD45 RO+ T cells, has several forms that are distinguished according to the epidermotropic properties of the pathological cells. In a recent paper (Derappe C, Haentjens G, Lemaire S, Feugeas JP, Lebbe C, Pasqualetto V, Bussel A, Aubery M, Néel D. Leukemia 1996;10:138), we observed that T lymphocytes from most of the Sézary patients [Szbeta(1-6)+] expressed high levels of beta(1-6)-GlcNAc-branched N-linked oligosaccharides while T lymphocytes from other patients [Szbeta(1-6)-] did not. Because this observation suggests the possibility of two forms of Sz, distinguished according to the expression rate of these glycans, we looked for a possible relationship between this expression rate and T-cell adhesiveness. Using an original protocol (Braut-Boucher F, Pichon J, Rat P, Adolphe M, Aubery M, Font J. J Immunol Methods 1995;178:41), we observed that T lymphocytes obtained from the Szbeta(1-6)+ patients adhered less to normal keratinocyte monolayers than T lymphocytes from Szbeta(1-6)- patients and normal donors. As assessed by FACS analysis, all the integrin-subunits studied were more expressed on Szbeta(1-6)-, especially alpha4, alpha5, beta1 and beta2, than on Szbeta(1-6)+ and normal lymphocytes. Although these results suggest that beta1- and beta2-integrin expression is involved in the adhesive properties of these T-cells, other factors, such as glycosylation, may also contribute. To demonstrate this possibility, we sought the presence of beta(1-6)-GlcNAc-branched N-linked oligosaccharides on beta1 integrins expressed by T lymphocytes from Sz patients. Immunoblot experiments, performed using the specific lectin from Phaseolus vulgaris (Leukoagglutinin form), showed that only the beta1 integrin subunit expressed by T lymphocytes from Szbeta(1-6)+ patients carried these glycans, supporting the concept of the involvement of T-cell glycosylation in the evolution of Sz.

Activity of proximal promoter of the human beta(1)-integrin gene was increased in Sézary syndrome.

Changes in beta1-integrin expression have been involved in abnormal cellular interactions between malignant lymphocytes from Sézary (Sz) patients and keratinocytes. In this paper, we compare the activity of both distal and proximal promoters of the beta1-integrin gene in malignant lymphocytes from Sz patients with human normal lymphocytes. Activity of both beta1-integrin promoters was also analysed in human normal keratinocytes. Northern blot analysis shows that beta1-integrin mRNA expression is higher in malignant Sz lymphocytes than in normal lymphocytes. CAT assays show that the activity of proximal beta1-integrin promoter is markedly increased (up to 6-fold) in malignant lymphocytes from Sz patients, in comparison to normal lymphocytes. These results suggest that changes in activity of the proximal promoter of beta1-integrin subunit could be, in part, responsible for the abnormal cellular interactions between malignant lymphocytes and keratinocytes observed in Sz syndrome.

Structural analysis of the carbohydrate chain of glycopeptides isolated from Robinia pseudoacacia seed lectins.

Robinia pseudoacacia seeds contain lectins which are closely related. Pronase digestion of the dimeric and tetrameric lectins, RPA1 and RPA3, gave glycopeptides. The structure of the oligosaccharide was determined by 1H NMR spectroscopy and carbohydrate determination as alpha-D-Manp-(1-->3)-[beta-D-Xylp-(1-->2)]-[alpha-D-Manp+ ++-(1-->6)]-beta- D-Manp-(1-->4)-beta-D-GlcpNAc-(1-->4)-[alpha-L-Fucp-(1-->3)] -beta-D-GlcpNAc - (1-->4)-Asn. It appears that the 34-kDa constitutive polypeptide of RPA1 contains 4-5 carbohydrate chains whereas the 30.5-kDa and 29-kDa subunits of RPA3 contain two and one oligosaccharide chains, respectively.

Phorbol ester treatment of HL 60 leukemia cells results in increase of beta-(1 --> 4)-galactosyltransferase.

We previously showed that HL 60 leukemia cells exhibit various changes in their cellular glycans after phorbol 12-myristate 13-acetate (PMA) treatment. These changes could originate largely from changes in one or several glycosyltransferases. In this report, we show using enzymatic measures, fluorescence microscopy, immunoblotting and Northern blot that beta-(1 --> 4)-galactosyltransferase I (GalT I) activity was higher (> x 2) in PMA-treated compared with untreated HL 60 cells. Immunoblotting showed an increased intensity of the GalT I band at 49 kDa and Northern blot a weak increase of the GalT I transcript band, after PMA treatment. Moreover, Northern blot performed after actinomycin-D treatment of the cells, which inhibits transcription, suggests that the observed increase of GalT I expression could originate, in part, from increase of the stability of GalT I transcripts.

Determination of the structure of a fucose-containing trisaccharide monophosphate isolated from human pregnancy urine.

A new acidic oligosaccharide, isolated from the urine of a pregnant woman by gel filtration and ion-exchange chromatography, was shown on the basis of sugar analysis, methylation analysis, exo-glycosidase digestion, e.i.-m.s., f.a.b.-m.s., and n.m.r. spectroscopy to have the following structure: (Formula: see text).

Sialic acid-containing glycoconjugates in human lactation urine.

The excretion rate of neutral hexoses, fucose and sialic acid of oligosaccharide and glycopeptide fraction has been determined in lactation urine. Thin layer chromatography has been used to investigate the excretion pattern of oligosaccharides, specially sialyl-oligosaccharides, during the course of lactation and after weaning. Lactation urine contains sialyloligosaccharides which are not present in normal urine and their presence has been studied up to the 3rd week after weaning. These results indicate a mammary origin for most of these compounds and confirm the hypothesis of previous research.

Use of chemical ionization in gas chromatographic/mass spectrometric screening of human urine for disaccharides containing inositol.

Gas chromatography/mass spectrometry (GC/MS) in the positive chemical ionization (CI) mode was used to screen normal urine for inositol-containing disaccharides in the form of permethylated derivatives, after borodeuteride reduction of the reducing saccharides. Ammonia was the reactant gas. The results revealed the existence of deoxyhexosyl-inositol and hexosyl-inositol disaccharides, and of a new compound, N-acetylhexosaminyl-inositol disaccharide. Up to four isomers of deoxyhexosyl-inositol could be present in the same sample even though only one of them has so far been fully characterized in man. As regards the hexosyl-inositols, one to three isomers were present in the same sample and probably corresponded to the three isomers of galactosyl-inositol recently described in man. N-Acetylhexosaminyl-inositol (identified elsewhere by us as N-acetylgalactosaminyl-alpha (1-1)-myo-inositol) was seen in only a few samples. No relationship can be found between the excretion of all these inositol-containing disaccharides on the one hand, ABO(H) blood group and 'Secretor' status (Se or sese) of the donors on the other. The gas chromatographic CI mass spectrometric technique used here with various ammonia pressures can be applied to the screening of other biological fluids or tissues for inositol glycosides.

[Variability of biological glucose transport activity in human erythrocytes]. / Variabilité de l'activité biologique du transporteur du glucose dans les érythrocytes humains.

Glucose transport activity was determined on erythrocytes from healthy adult and children. A large variability of values was found between each donor, but transport rate was significantly higher in erythrocytes from adults than those from children. In opposite, the same number of glucose transport sites was found on erythrocytes from adult and children, suggesting that transporters from adult erythrocytes exhibited an higher own activity than transporters from child erythrocytes. Involvement of various factors in glucose transport activity was discussed.

Comparison of carbohydrate structure between human chorionic gonadotropin present in urine of patients with trophoblastic diseases and healthy individuals.

Human chorionic gonadotropin (hCG) highly purified from the urine of patients with trophoblastic diseases (choriocarcinoma and hydatidiform mole) and from healthy pregnant women contains four asparagine-linked sugar chains in one molecule. Comparative studies of the sugar chains released by hydrazinolysis revealed that the structures of the sugar chains of choriocarcinoma hCGs are quite different from those of mole and normal hCGs. The carbohydrate structures of mole hCGs are the same as those of normal hCG, while all choriocarcinoma hCGs examined contain two triantennary, two unusual biantennary, and one monoantennary complex-type sugar chains which are not found in normal hCGs. The triantennary and unusual biantennary sugar chains contain the +/- NeuAc alpha 2----Gal beta 1----4GlcNAc beta 1----4Man alpha 1----3 group in common. The total amounts of fucosylated sugar chains in choriocarcinoma hCGs are twice those found in the hCGs of other groups. Therefore, the appearance of the N-acetylglucosaminyl transferase responsible for the formation of the GlcNAc beta 1----4Man alpha 1----3 group and the increase of the fucosyl transferase responsible for the formation of the Fuc alpha 1----6 GlcNAc----Asn group appear to be specific characteristics of malignant transformation of the trophoblast, and the structural changes of sugar chains could serve as useful markers for the diagnosis of choriocarcinoma.

Expression of beta 1-6-branched N-linked oligosaccharides is associated with activation in human T4 and T8 cell populations.

Activation of human T lymphocytes by phorbol 12-myristate 13-acetate and leukoagglutinin from Phaseolus vulgaris (L-PHA) results in important changes in N-glycosylation. The most important event is the increase, in both T4 and T8 cells (especially the latter), of L-PHA+ structures characterized by beta 1-6-branching of complex-type oligosaccharides. Moreover, the existence of a CD4-mediated increase of these beta 1-6-branched structures on positively selected T4 cells, as compared with the negatively selected ones, suggests that the presence of these structures, not detectable on T8 resting cells, could be related to stimulation events triggered by both selection methods. This beta 1-6-branching on N-glycans, strongly associated with a metastatic phenotype in human and rodent tumors, is exhibited by numerous glycoproteins on stimulated cells, as shown by blot analysis.