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J Biol Chem, Vol. 273, Issue 50, 33644-33651, December 11, 1998


Characterization of a UDP-Gal:Galbeta 1-3GalNAc alpha 1,4-Galactosyltransferase Activity in a Mamestra brassicae Cell Line*

Michel LopezDagger , Maud Gazon§, Sylvie Juliant§, Yves PlanckeDagger , Yves LeroyDagger , Gérard StreckerDagger , Jean-Pierre Cartron, Pascal Bailly, Martine Cerutti§, André VerbertDagger , and Philippe DelannoyDagger parallel

From the Dagger  Laboratoire de Chimie Biologique, Unité Mixte de Recherche du CNRS 111, Université des Sciences et Technologies de Lille, F-59655 Villeneuve d'Ascq, France, § Station de Pathologie Comparée INRA/Unité de Recherche Associée du CNRS 2209, route d'Alès, F-30380 Saint Christol-les-Alès, France, and  INSERM U-76, Glycoconjugués des Cellules Sanguines, Institut National de Transfusion Sanguine, 6 rue Alexandre Cabanel, F-75739 Paris, France

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The binding of Bandeiraea simplicifolia lectin-I isolectin B4 on the endogenous glycoproteins of different insect cell lines led us to characterize for the first time a UDP-Gal:Galbeta 1-3GalNAc alpha 1,4-galactosyltransferase in a Mamestra brassicae cell line (Mb). The study of the acceptor specificity indicated that the Mb alpha -galactosyltransferase prefers Galbeta 1-3-R as acceptor, and among such glycans, the relative substrate activity Vmax/Km was equal to 20 µl·mg-1·h-1 for Galbeta l-3GlcNAcbeta 1-O-octyl and to 330 µl·mg-1·h-1 for Galbeta 1-3GalNAcalpha -1-O-benzyl, showing clearly that Galbeta 1-3GalNAc disaccharide was the more suitable acceptor substrate for Mb alpha -galactosyltransferase activity. Nuclear magnetic resonance and mass spectrometry data allowed us to establish that the Mb alpha -galactosyltransferase synthesizes one unique product, Galalpha 1-4Galbeta 1-3GalNAcalpha 1-O-benzyl. The Galbeta 1-3GalNAc disaccharide is usually present on O-glycosylation sites of numerous asialoglycoproteins and at the nonreducing end of some glycolipids. We observed that Mb alpha 1,4-galactosyltransferase catalyzed the transfer of galactose onto both natural acceptors. Finally, we demonstrated that the trisaccharide Galalpha 1-4Galbeta 1-3GalNAcalpha 1-O-benzyl was able to inhibit anti-PK monoclonal antibody-mediated hemagglutination of human blood group PK1 and PK2 erythrocytes.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

In recent years, the development of therapeutic glycoprotein production using the baculovirus expression system in insect cells (1-4) has stimulated concerns to determine the post-translational modifications capacity, in particular the glycosylation potential of insect cells. In fact, it is now well established that protein-bound carbohydrate side chains play important roles on the physicochemical properties and functions of glycoproteins such as antigenicity, immunogenicity, and metabolic clearance (5-8).

The N-glycosylation pattern of recombinant glycoproteins expressed by lepidopteran cells using the baculovirus vector is now well documented and revealed that the N-linked oligosaccharides found are essentially of high mannose type (Man9-5GlcNAc2) and short truncated structures (Man3-2(Fuc0-1)GlcNAc2)1 frequently substituted by a Fuc residue alpha 1,6-linked to the asparagine-bound GlcNAc residue (9-24). Only a few studies have shown complex N-linked glycans in insect cells. Recent data suggest that the insect cell lines Estigmena acrea and Trichoplusia ni clone Tn-5-B14 (Tn) can add some terminal galactose residues to the recombinant human interferon gamma  and to the heavy chain of a heterologous murin IgG (25, 26), respectively. Only one group has reported that recombinant human plasminogen carries complex type N-glycans with terminal sialic acid residues when expressed in Spodoptera frugiperda clone Sf-21 (Sf-21) or Mamestra brassicae clone SPCMb-92-C6 (Mb) (27-29).

As mainly observed on recombinant glycoproteins expressed by the baculovirus/insect cell system, endogenous N-glycan structures found in insect cell glycoproteins have no complex type glycans (16, 30-32), but a small amount of oligosaccharides contain terminal GlcNAc or GalNAc residues (33-35). Activity measurements of several glycosyltransferases involved in the addition of terminal sugars to N-linked oligosaccharides have demonstrated that lepidopteran cells contain alpha 1,6-fucosyltransferase (36) and a low level of beta 1,2-N-acetylglucosaminyltransferase I (37, 38) but lack beta 1,4-galactosyltransferase and sialyltransferase activities.

Compared with the information available on the structure and biosynthesis of lepidopteran N-glycans, our knowledge about the synthesis of O-linked oligosaccharides remains rather poor. A comparison of O-glycans on recombinant pseudorabies virus envelope protein gp50 expressed in S. frugiperda clone Sf-9 (Sf-9), Vero and Chinese hamster ovary cells has shown that the protein synthesized by both mammalian cell lines contained exclusively the core 1 disaccharide Galbeta 1-3GalNAc (T-antigen) mainly substituted by one or two sialic acid residues. On the other hand, most of the O-glycosylation sites of insect-produced protein were only substituted by GalNAc residues, forming Tn-antigen. The Sf-9 cell line also synthesized Galbeta 1-3GalNAc but in very low amounts, and none of these O-glycans are substituted by sialic acid residues (39). A characterization of the O-linked oligosaccharidic structures on a chimeric respiratory syncytial virus protein as well as on a human interleukin-2 with artificially introduced O-glycosylation sites, expressed in Sf-9 and Sf-21 cells, respectively, yielded similar results; only GalNAc and Galbeta 1-3GalNAc were found (15, 18). Measurement of the two relevant O-glycosyltransferase activities performed in Sf-9, Vero, and Chinese hamster ovary cells has revealed that, even if the three cell lines expressed a comparable level of polypeptide N-acetylgalactosaminyltransferase activity, there were high variations in core 1 beta 1,3-Gal-T activity, with Sf-9 cells containing the lowest level (39).

In summary, the determination of the glycosylation potential of lepidopteran cells achieved through the measurement of the different glycosyltransferase activities, as well as through the identification of the N- and O-glycan structures present on endogenous insect cell glycoproteins or on recombinant glycoproteins expressed by the baculovirus/insect cell system, showed that the glycosylation potential of insect cell lines is deficient in comparison with mammalian cells. Since N- and O-glycan structures found on the insect recombinant glycoproteins are often different from those occurring in the corresponding natural glycoproteins, the aberrant glycosylation pattern can limit the development of the therapeutic glycoprotein production in lepidopteran cells using the baculovirus expression system. Indeed, these changes in glycosylation can cause a decrease of the half-life of the recombinant glycoproteins after blood injection (8, 40) or induce an immunogenic response against normally cryptic peptide epitopes (41-43) but also against glycan epitopes normally absent in humans.

It is clear that some oligosaccharide epitopes present on glycoproteins or glycolipids expressed at the cell surface of most mammalian species cause a rapid rejection after exposure to human blood. The major element responsible for such human rejection has been identified as a unique carbohydrate structure, the alpha -galactosyl epitope Galalpha 1-3Gal (44-45). Indeed, as much as 1% of the circulating human IgG were found to interact with Galalpha 1-3Gal epitopes, both on glycoproteins (46-47) and on glycosphingolipids (48-50).

Because the alpha -galactosyl epitope is strongly antigenic in humans, it was particularly interesting to look for such carbohydrate epitopes on endogenous insect cell glycoproteins, since the glycosylation patterns of the expressed glycoproteins are determined to a large extent by the host cell glycosylation machinery. For that purpose, after electrophoresis and Western blotting of noninfected insect cell homogenates, we have tested the affinity of the insect glycoproteins for an alpha Gal-specific lectin, B. simplicifolia lectin-I isolectin B4 (BSI-B4), which strongly recognizes Galalpha 1-3/4Gal sequences (51-53); demonstrated the binding of BSI-B4 to insect cell glycoproteins, especially to Mb endogenous glycoproteins; and characterized a new UDP-Gal:Galbeta 1-3GalNAc alpha 1,4-galactosyltransferase (alpha 1,4-Gal-T) activity specific to Galbeta 1-3GalNAc acceptor substrates in these cells.

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- All reagents were of analytical grade. UDP-[6-3H]Gal (650 GBq·mmol-1) was purchased from Amersham Pharmacia Biotech and used after dilution with nonradioactive nucleotide sugar from Sigma. Peroxidase-labeled BSI-B4, 3,3'-diaminobenzidine tetrahydrochloride tablets, insoluble peroxidase substrate, BSI-B4, GM1a, GA1, GD1b, Galbeta l-3GalNAcalpha 1-O-bn, lactose (Galbeta 1-4Glc), Gal, Galalpha 1-p-nitrophenol, Galbeta 1-p-nitrophenol, Galalpha 1-O-methyl, fetuin, angiotensin I, and Triton X-100 were purchased from Sigma. N-Acetyllactosamine (Galbeta 1-4GlcNAc) and N-acetyl-isolactosamine (Galbeta 1-3GlcNAc) were from Calbiochem. Lacto-N-tetraose (Galbeta 1-3GlcNAcbeta 1-3Galbeta 1-4Glc) and lacto-N-neotetraose (Galbeta 1-4GlcNAcbeta 1-3Galbeta 1-4Glc) were prepared from bovine colostrum (54). Lactosylceramide was the generous gift of Dr. J.-F. Bouhours (INSERM U-437, Nantes, France). Galbeta l-3GalNAc, Galbeta l-3GalNAcbeta 1-O-bn, Galbeta l-3(2-O-Ac)Galbeta 1-O-methyl, Galbeta l-3GlcNAcbeta 1-O-octyl, and Galbeta l-4GlcNAcbeta 1-O-bn were generous gifts of Dr. C. Augé (URA CNRS 462, Orsay, France). Asialofetuin was prepared by mild acid hydrolysis of fetuin. The sugar composition of desialylated compounds was controlled by gas chromatography after methanolysis and trimethylsililation. Dowex 1 × 2 (Cl- form, 100-200 mesh), Bio-Gel P-2 (200-400 mesh), and P-4 (200-400 mesh) were purchased from Bio-Rad (Ivry-sor-Seine, France). The Sep-Pak C18 cartridge was purchased from Waters Corp. (Milford, MA). Nitrocellulose membrane BioTrace NT was from Gelman Sciences. Murine monoclonal antibodies anti-P1, anti-Pk, and anti-P and red blood cells from individuals typed in the P/P1 blood group system were from the Institut National de la Transfusion Sanguine (Paris, France).

Insect Cell Cultures and Preparation of Cellular Homogenates-- The S. frugiperda clone Sf-9 of IPLB-Sf-21-AE cells (55) was obtained from ATCC (CRL 1711), and the T. ni clone Tn-5-B14 was provided by A. Bernard (Glaxo Institute). The M. brassicae clone SPCMb-92-C6 was established by J. M. Quiot at the Station de Pathologie Comparée INRA/CNRS URA 2209.2 The three cell lines were adapted, maintained, and cultivated at 28 °C in EX-CELL 401TM (Sera-Lab, Blackthorn, United Kingdom), a semidefined medium for the serum-free culture of lepidopteran insect cells.

Cells were harvested at confluence, and cell homogenates were prepared by lysing the cells at 0 °C with 10 mM sodium cacodylate buffer, pH 6.5, containing 1% Triton X-100, 20% glycerol, 0.5 mM dithiothreitol, and 5 mM MnCl2 (1 ml/3 × 107 cells). After a 10-min incubation under continuous stirring, cell homogenates were centrifuged at 10,000 × g for 15 min, and the supernatants were used for SDS-polyacrylamide gel electrophoresis. Protein concentration was determined according to the method described by Peterson (56) using bovine serum albumin as a standard.

Electrophoresis and Western Blotting-- SDS-polyacrylamide gel electrophoresis was performed under reducing conditions on 4-20% gradient polyacrylamide gel (57) using 40 µg of protein/lane for each cell homogenates. After migration, proteins were transferred to a nitrocellulose membrane as described by Vaessen et al. (58). The blot was then separated in two parts and treated with polyvinylpyrolidone (2% in Tris-buffered saline (Tris/HCl 10 mM, NaCl 0.15 M, pH 7.4)) prior the incubation with the peroxidase-labeled BSI-B4 (2 µg·ml-1 in Tris-buffered saline containing 1 mM MgCl2, 1 mM MnCl2, 1 mM CaCl2) with or without 0.5 M Galalpha 1-O-methyl used as a competitor. After washing, labeled glycoproteins were revealed according to the Sigma Fast Staining protocol using 3,3'-diaminobenzidine tetrahydrochloride and urea/H2O2.

Enzymatic Assay-- 2 × 108 Mb cells cultured in EX-CELL 401TM were lysed by ultrasonication (5-s pulsed at 0 °C in 10 ml 0.25 M sucrose, 0.2 M NaCl solution). Microsome suspensions were prepared by centrifugation as described previously (59) and stored at -80 °C in NaCl/sucrose solution until use.

Assays were performed at least in duplicate. The incorporation of [6-3H]Gal was determined by subtraction of the radioactivity measured in the absence of exogenous acceptors, and results were expressed as average values as nmol of Gal residues transferred/mg of protein/h.

Microsomal fractions (35 µg of protein) were brought to a final volume of 50 µl with 0.125 M Mes, pH 7.0, 0.15% Triton X-100, 5 mM gamma -galactonolactone, 10 mM ATP, 12.5 mM MnCl2, 1 mM UDP-[6-3H]Gal (235 MBq·mmol-1; 11.75 kBq/50 µl), containing 4 mM of oligosaccharide or aryl glycoside, 1 mM of ganglioside, or 2 mg·ml-1 of glycoprotein acceptor and incubated for 1 h at 37 °C.

Reaction products were separated from UDP-[3H]Gal depending on the acceptor substrate. For oligosaccharide acceptors, assay mixtures were diluted to 0.5 ml with H2O to stop the reaction, and the amount of incorporated [3H]Gal was determined by ion exchange chromatography on a 1-ml Dowex 1 × 2 column (Cl- form, 100-200 mesh) equilibrated in water. The radioactivity of the corresponding products was detected by scintillation counting (60). For glycoproteins, reactions were stopped by adding 1 ml of ice-cold phosphotungstic acid (5% in 2 M HCl). Precipitates were collected on glass fiber filters, washed extensively with 5% trichloroacetic acid, distilled water, and ethanol, and processed for scintillation counting (60). For aryl glycosides and glycolipids, the reactions were stopped by adding 1 volume of ethanol. Samples were centrifuged at 3000 × g for 5 min, and supernatants were applied to a Sep-Pak C18 cartridge. After washing with 5 ml of water, aryl glycosides were eluted with 5 ml of 30% acetonitrile in water and glycolipids with 2 ml of CH3OH, followed by a 5-ml elution with CHCl3/CH3OH (1:1, v/v) and processed for scintillation counting. The rates of all reactions were linear with time, at least for 1 h. For kinetic analysis, incubations were performed for 1 h using various concentrations (0.125-4 mM) of Galbeta l-3GalNAcalpha 1-O-bn and Galbeta l-3GlcNAcbeta 1-O-octyl.

Large Scale Preparation of the Galactosylated Product of Galbeta 1-3GalNAcalpha 1-O-bn-- Mb microsomal fractions (850 µg of protein) were brought to a final volume of 600 µl in conditions described above with 4 mM UDP-[6-3H]Gal (62 MBq·mmol-1; 0.148 MBq/600 µl), 4 mM of Galbeta l-3GalNAcalpha 1-O-bn and incubated for 24 h at 37 °C. The incubation mixture was applied to a Sep-Pak C18 cartridge, eluted as described previously, and lyophilized. Aliquots of the incubation mixture (5 µl) were collected after 0, 2, 4, 8, and 24 h and assayed for radioactivity (data not shown). The yield of transfer after 24 h of incubation was 66% (~970 µg of galactosylated product). The purified product was further analyzed by NMR and mass spectrometry (MS).

For the immunoreactivity study, we produced 4 mg of that compound with 90% of purity. That large scale preparation was performed as described above, in a final volume of 1.85 ml (1.4 mg of Mb microsomal proteins) and incubated for 48 h at 37 °C.

Two-dimensional Homonuclear and Heteronuclear NMR Spectroscopy-- 1H-NMR spectroscopy was performed on a Brüker ASX 400WB spectrometer. Chemical shifts are expressed in ppm downfield from internal sodium 4,4'-dimethyl-4-silapentane-1-sulfonate but were actually measured by reference to internal acetone (delta  = 2.225 ppm in D2O at 25 °C). The two-dimensional homonuclear correlation spectroscopy (COSY), with simple and double relay transfer, and the heteronuclear multiple-quantum coherence spectroscopy (HMQC) spectroscopy experiments were performed using Brüker standard pulse-sequences.

Matrix-assisted Laser Desorption Mass Spectrometry (MALD-MS) and Gas Chromatography-Mass Spectrometry (GC-MS)-- 100 µg of the galactosylated product were permethylated according to Ciucanu and Kerek (61) prior to MALD-MS analysis. The methylated compound was then methanolyzed, and the formed methylglycosides were analyzed by GC-MS after peracetylation as described by Fournet et al. (62).

Molecular weight of the permethylated galactosylated product was measured by MALD-MS on a Vision 2000 time-of-flight mass spectrometer (Finnigan MAT, Bremen, Germany) equipped with a 337-nm UV laser. The mass spectra were acquired in reflectron mode under 6-kV acceleration voltage and positive detection. The sample was prepared by mixing directly onto the target 1 µl of the analyte solution (typically 50 pmol) and 1 µl of a 2,5-dihydroxybenzoic acid matrix solution (12 mg·ml-1 in CH3OH/H2O, 70:30) and then allowed to crystallize at room temperature. External calibration was performed using an angiotensin I standard (Mr = 1296.7). 10 shots were accumulated for the mass spectrum.

GC separation of partially methylated and acetylated methylmonosaccharides from galactosylated product was performed on a fused silica capillary column from SGE (0.32 mm x 25 m) using helium as carrier gas at pressure of 0.5 bar with the following temperature program (110-240 °C at 3 °C/min). The GC-MS analyzer (Delsi DI 700 gas chromatograph) was coupled with a Nermag R10-10 mass spectrometer (Rueil Malmaison, France) using an electron energy of 70 eV and an ionizing current of 0.2 mA.

Release of [6-3H]Gal-labeled Oligosaccharides from Asialofetuin-- Asialofetuin (1.25 mg, 5 mg·ml-1 final concentration) was incubated for 24 h at 37 °C under the conditions described above with Mb microsomal fraction (210 µg of protein) and 0.5 mM of UDP-[6-3H]Gal (0.57 GBq·mmol-1; 74 kBq·µl-1). The sample was made free of UDP-[6-3H]Gal by desalting on Bio-Gel P2 column (200-400 mesh) equilibrated in 0.1 M pyridine acetate, pH 5.6. Further, 3H-labeled asialofetuin sample was submitted to alkaline treatment under reducing conditions in 0.1 M NaOH containing 1 M NaBH4 for 48 h at 45 °C (63). At the end of the incubation time, the sample was acidified at pH 6 by addition of Dowex 50 × 8 (H+ form, 20-50 mesh) and evaporated. The sample was then resuspended in methanol and evaporated three times, dissolved in water, and applied to a Bio-Gel P4 column (80 × 1.2 cm, 200-400 mesh) equilibrated in 0.1 M pyridine acetate pH 5.6, to separate released O-glycans from N-glycopeptides.

Immunoreactivity of Galalpha 1-4Galbeta 1-3GalNAcalpha -O-bn-- Monoclonal antibodies anti-P1, anti-Pk, anti-P, and BSI-B4 were previously tested by agglutination assays using 0.7% (by volume) suspensions of papain-treated P1, Pk1, Pk2, and p (Tja-) human red cells, for 7 min at 37 °C, in sodium phosphate buffer, pH 6.8, containing 150 mM NaCl. This agglutinant system titration allowed to control the specificity of reagents and to determine the correct concentration needed for each antibody or lectin, in order to perform the inhibition assays with both aryl glycosides Galbeta 1-3GalNAcalpha 1-O-bn and Galalpha 1-4Galbeta 1-3GalNAcalpha 1-O-bn. Then the monoclonal antibodies and the BSI-B4 at the appropriate dilution were incubated for 4 h at 22 °C in a final volume of 20 µl with a serial dilution of Galbeta 1-3GalNAcalpha 1-O-bn or Galalpha 1-4Galbeta 1-3GalNAcalpha 1-O-bn. The mixtures were further tested as described above by agglutination assays against 20 µl of papain-treated P1, Pk1, or Pk2 erythrocytes.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

BSI-B4 Binds to Mb Glycoproteins-- 40 µg of cellular glycoproteins from Sf-9, Mb, or Tn cultured in EX-CELL 401TM medium were fractionated by SDS-polyacrylamide gel electrophoresis and revealed with peroxidase-labeled BSI-B4. As indicated in Fig. 1, the three lepidopteran cell lines expressed glycoproteins recognized by BSI-B4. The specificity of the binding of the lectin was shown by the total absence of staining observed when BSI-B4 was incubated in the presence of 0.5 M Galalpha 1-O-methyl used as a competitor.


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  		Fig. 1.   Binding of BSI-B4 onto endogenous glycoproteins of Sf-9, Mb, and Tn cell lines. After fractionation on a 4-20% SDS-polyacrylamide gel electrophoresis gradient gel and Western blotting of the glycoproteins from Sf-9 (lanes 1 and 4), Mb (lanes 2 and 5), and Tn (lanes 3 and 6) cells, the blot was separated into two parts. On the left (lanes 1-3), glycoproteins were detected with BSI-B4. On the right (lanes 4-6), negative controls were performed by competition with 0.5 M Galalpha 1-O-methyl. The position of the prestained molecular mass markers is indicated on the left (in kDa).

The pattern of expression of revealed bands was dependent on the cell line, showing that the higher staining intensity was obtained with Mb glycoproteins. In Mb, a 100-110-kDa band was strongly revealed by BSI-B4 compared with Sf-9 or Tn in which the staining was of a lower intensity. In Sf-9, BSI-B4 revealed a large 95-105-kDa band with two other weak signals at 85 and 190 kDa. Tn glycoprotein staining was observed between 95 and 120 kDa with a main signal at 100-110 kDa. According to the specificity of BSI-B4, which recognizes Galalpha 1-3/4Gal sequences (51-53), these data indicated the presence of alpha -Gal residues in terminal position of endogenous high molecular weight glycoproteins of Sf-9, Mb, and Tn, reflecting the activity of an alpha -galactosyltransferase in these cell lines. Because the strongest binding was observed on Mb glycoproteins, we have focused our interest on this cell line in order to characterize the activity of the galactosyltransferase responsible for the biosynthesis of these structures.

Mb Preferentially Transfers Galactose onto the Terminal Position of Galbeta 1-3GalNAc Sequence-- To characterize the substrate specificity of the putative alpha -galactosyltransferase activity, we incubated microsomes with various potential acceptors containing a terminal galactose residue. As indicated in Table I, the galactosyltransferase activity expressed in the microsomal fraction of Mb transferred preferentially galactose residue onto Galbeta 1-3GalNAcalpha 1-O-bn, Galbeta l-3GalNAcbeta 1-O-bn, Galbeta 1-3GalNAc, or Galbeta l-3-(2-O-Ac)-Galbeta 1-O-methyl, a synthetic substrate that was previously shown to be a good acceptor for the transfer of sialic acid residues in alpha 2,3-linkage to terminal Gal (64). The microsomal fraction of Mb also transferred galactose residue onto N-acetylisolactosamine (Galbeta l-3GlcNAc) or Galbeta l-3GlcNAcbeta 1-O-octyl but with a lower yield (36.9 and 39.9%, respectively) compared with Galbeta 1-3GalNAcalpha 1-O-bn. As indicated in Table I, the transfer of galactose residues to N-acetyllactosamine (Galbeta l-4GlcNAc) or to lacto-N-neotetraose (Galbeta l-4GlcNAcbeta 1-3Galbeta 1-4Glc) was 2.7- or 1.5-fold lower than for N-acetylneolactosamine (Galbeta l-3GlcNAc) or lacto-N-tetraose (Galbeta l-3GlcNAcbeta 1-3Galbeta 1-4Glc), respectively. A similar difference was observed between Galbeta l-4GlcNAcbeta 1-O-bn and Galbeta l-3GlcNAcbeta 1-O-octyl, showing that Mb alpha -galactosyltransferase preferred Galbeta 1-3-R over Galbeta 1-4-R acceptor substrates. A small amount of galactose residues was transferred to lactose (Galbeta l-4Glc), but Gal or alpha - and beta -arylglycosides of Gal were not acceptor substrates for that enzymatic activity. According to these data, it appeared that the substrate specificity of Mb alpha -galactosyltransferase activity completely differed from UDP-Gal:Galbeta 1-4GlcNAc alpha 1,3-galactosyltransferase previously purified and cloned from different cell lines (65-67).

                              
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Table I
Determination of the substrate specificity of M. brassicae cells UDP-Gal:Galbeta 1-3GalNAc alpha 1,4-galactosyltransferase toward oligosaccharidic acceptors
Shown are the relative activities to the incorporation of galactose onto Galbeta 1-3GalNAcalpha 1-O-bn as an acceptor substrate.

Since both Galbeta 1-3GalNAc and Galbeta 1-3GlcNAc sequences appeared to be acceptor substrates for Mb alpha -galactosyltransferase activity, we further compared the kinetic parameters of transfer of galactose residues by Mb microsomal fraction using Galbeta 1-3GalNAcalpha -1-O-bn and Galbeta l-3GlcNAcbeta 1-O-octyl as acceptors. As shown on Table II, the Km value for Galbeta 1-3GalNAcalpha -1-O-bn was 11-fold lower compared with Galbeta l-3GlcNAcbeta 1-O-octyl. In addition, the relative substrate activity Vmax/Km was equal to 20 µl·mg-1·h-1 for Galbeta l-3GlcNAcbeta 1-O-octyl and to 330 µl·mg-1·h-1 for Galbeta 1-3GalNAcalpha -1-O-bn, showing undoubtedly that Galbeta 1-3GalNAc disaccharide is the most suitable acceptor substrate for Mb alpha -galactosyltransferase activity.

                              
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Table II
Comparison of the kinetic properties of M. brassicae cells UDP-Gal:Galbeta 1-3GalNAc alpha 1,4-galactosyltransferase toward Galbeta 1-3GalNAc and Galbeta 1-3GlcNAc sequences

Galactose Residue Is alpha 1,4-Linked at the Nonreducing Position of Galbeta 1-3GalNAc-- In order to precisely determine the linkage (anomery and substitution) of Gal transferred onto Galbeta 1-3GalNAc by the Mb alpha -galactosyltransferase activity, NMR analysis was performed on the product of the transfer reaction onto Galbeta 1-3GalNAcalpha -1-O-bn (compound A) as acceptor (Table III and Fig. 2). The main characteristics of the NMR spectrum of the acceptor are the signals relative to the terminal beta -Gal unit, observed at delta  = 4.454 ppm (H-1), delta  = 3.620 ppm (H-3), and delta  = 3.900 ppm (H-4). By integration of the signals relative to terminal beta -GalIIA (delta H-1 = 4.454 ppm) and substituted beta -GalIIB (delta H-1 = 4.521 ppm) a ratio of 3:7 was measured, corresponding to an effective transfer of galactose reaching up to 70%. The new anomeric proton resonance observed at delta  = 4.949 ppm (J1,2 ~3 Hz) is characteristic of the alpha -form of galactose, the conformation of which was confirmed by the set of its vicinal coupling constants JH1,H2, JH2,H3, and JH3,H4. The downfield shift that affects the beta -GalIIB H-3 (Delta delta  = +0.072 ppm) and H-4 (Delta delta  = +0.125 ppm) resonance is in favor of an O-4 substitution of this sugar unit. This interpretation was confirmed by examination of the HMQC spectrum, which clearly shows two C-4 resonances at delta  = 70.20 ppm (terminal alpha -GalIIIB) and delta  = 78.47 ppm (O-4-substituted beta -GalIIB).

                              
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Table III
1H- and 13C-chemical shifts from Galbeta 1-3GalNAcalpha -1-O-bn (compound A) and Galalpha 1-4Galbeta 1-3GalNAcalpha -1-O-bn (compound B)


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  		Fig. 2.   1H- and 13C-NMR analysis of the material resulting from the transfer of galactose to the acceptor Galbeta 1-3GalNAcalpha -1-O-bn. Left panel, two-step relayed COSY; right panel, HMQC spectrum. Compound A, Galbeta 1-3GalNAcalpha -1-O-bn (acceptor); compound B, Galalpha 1-4Galbeta 1-3GalNAcalpha -1-O-bn (formed).

On the basis of these observations, the structure of the neosynthesized compound was established as follows: Galalpha 1-4Galbeta 1-3GalNAcalpha -1-O-bn (compound B).

The NMR result was confirmed by the MS analysis. The MALD-MS spectrum of the permethylated aryl glycosides (Fig. 3) showed only two predominant salted (Na+ or K+) molecules. The first pseudomolecular ions masses corresponded to the expected mass of the permethylated acceptor Galbeta 1-3GalNAcalpha -1-O-bn ([M + Na]+ = 594.4 m/z or [M + K]+ = 610.7 m/z/compound A). The second pseudomolecular masses observed corresponded to the theoretical mass of the permethylated compound B ([M + Na]+ = 798.9 m/z or [M + K]+ = 814.7 m/z). The signal integration allowed to determine the intensity ratio of 20:80 between the compound A and B. Moreover, the acetylated methylgalactosides identified by GC-MS, the methyl-2,3,6-tri-O-methyl-4-mono-O-acetyl-galactopyrannoside and the methyl-2,3,6-tri-O-methyl-5-mono-O-acetyl-galactofurannoside, confirmed that the transferred galactose residue was linked to the galactose residue acceptor through a 1,4-linkage (Table IV).


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  		Fig. 3.   MALD-MS spectrum of the permethylated material resulting from the transfer of galactose to Galbeta 1-3GalNAcalpha -1-O-bn. Compound A, Galbeta 1-3GalNAcalpha -1-O-bn (acceptor); compound B, Galalpha 1-4Galbeta 1-3GalNAcalpha -1-O-bn (formed); MA, observed mass of the permethylated compound A; MB, observed mass of the permethylated compound B.

                              
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Table IV
Methylated and acetylated methyl monosaccharide methyl ethers identified by GC-MS after acetylation of the methanolysate permethylated material resulting from the transfer of galactose to the acceptor Galbeta 1-3GalNAcalpha -1-O-bn
Compound A is Galbeta 1-3GalNAcalpha -1-O-bn (acceptor); compound B is Galalpha 1-4Galbeta 1-3GalNAcalpha -1-O-bn (formed).

Activity of the UDP-Gal:Galbeta 1-3GalNAc alpha 1,4-Galactosyltransferase toward Glycolipids and Asialoglycoproteins-- We have determined the activity of Mb alpha 1,4-Gal-T toward asialofetuin and several glycolipids. The concentration for asialofetuin was fixed to 2 mg·ml-1, which corresponds to a final concentration in Galbeta 1-3GalNAc equal to 0.13 mM, and the final concentration used for glycolipid assays was 1 mM. As indicated in Table V, among the five tested glycolipids, GM1a and GA1 were acceptors of Mb alpha 1,4-Gal-T. A low transfer was also observed onto lactosylceramide, which can be correlated with the transfer obtained onto free lactose, and almost no transfer was observed onto GM2, which possesses a GalNAc residue in the terminal position. Surprisingly, GD1b which has a Galbeta 1-3GalNAc terminal sequence, was a poor acceptor substrate. This is probably due to a steric incompatibility between the alpha 1,4-Gal-T and the alpha 2,8-linked disialylated chain present on this glycolipid. In all cases, the relative activity of Mb alpha 1,4-Gal-T toward glycolipids containing Galbeta 1-3GalNAc terminal sequences was low compared with Galbeta l-3GalNAcalpha 1-O-bn (less than 10%). This weak activity could reflect that the transfer onto glycolipids requires specific incubation conditions different from those applied for oligosaccharides and aryl-glycosides.

                              
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Table V
Determination of the substrate specificity of M. brassicae cells UDP-Gal:Galbeta 1-3GalNAcalpha 1,4-galactosyltransferase toward glycoproteins and glycolipids
Shown are the relative activities to the incorporation of galactose onto Galbeta 1-3GalNAcalpha 1-O-bn as an acceptor substrate.

As related in Table V, despite the fact that terminal Galbeta 1-3GalNAc was low in concentration in the asialofetuin assay (0.13 mM), the activity of transfer onto asialofetuin was 6.2 nmol·h-1·mg-1. At the same concentration, the transfer onto Galbeta l-3GalNAcalpha 1-O-bn was approximately 30 nmol·h-1·mg-1 and indicated that under these incubation conditions asialofetuin was a good acceptor substrate for the Mb alpha 1,4-Gal-T.

Release of [6-3H]Gal-labeled Oligosaccharides from Asialofetuin-- Asialofetuin (Mr 45,500) contains three triantennary complex-type N-glycan chains with Galbeta 1-4GlcNAc terminal sequence and three O-linked Galbeta 1-3GalNAcalpha 1-O-Ser/Thr chains (68). Therefore, 5 mg·ml-1 of asialofetuin in the reaction mixture allowed one to reach a concentration of terminal galactose acceptor sites equal to ~206 µM for Galbeta 1-3GalNAc-R and equal to ~618 µM for Galbeta 1-4GlcNAc-R terminal sequences. After 24 h of incubation at 37 °C, 36.2% of the terminal galactose residues of asialofetuin were substituted with [3H]Gal. In order to discriminate between [3H]Gal added onto N- or O-glycans of asialofetuin, respectively, a reductive beta -elimination was performed, and released O-oligosaccharides were separated from N-glycopeptides by fractionation on a Bio-Gel P4 column previously calibrated (Fig. 4). Gel permeation resolved two main peaks, appearing at the elution volume of N-glycopeptides (arrow at position 1) and of O-linked glycans of asialofetuin (arrow at position 2), respectively.


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  		Fig. 4.   Fractionation by gel permeation of the [6-3H]Gal-labeled O-oligosaccharides and N-glycopeptides from asialofetuin using Mb microsomal fraction as enzyme source. Reduced O-oligosaccharides and N-glycoproteins were separated on a Bio-Gel P4 column (80 × 1.2 cm) in pyridine acetate pH 5.6 and eluted with the same buffer at a flow rate of 10 ml·h-1. Fractions of 1 ml were collected and assayed for radioactivity. The column was previously calibrated with N-glycopeptides (1) and O-linked chains of fetuin (2).

This experiment confirmed that alpha 1,4-Gal-T activity expressed in the microsomal fraction of Mb transferred galactose onto glycoproteins containing terminal galactose residues. We observed that the transfer of [3H]Gal was mainly onto O-oligosaccharides (74.7% of the transferred radioactivity) rather than onto the N-glycopeptides (25.3% of the transferred radioactivity). This result also indicated that 65.5% of the Galbeta 1-3GalNAc terminal sequences were substituted by a Gal residue, while only 7.9% of the Galbeta 1-4GlcNAc terminal sequences were occupied. These data confirmed that the Mb alpha 1,4-Gal-T formed preferentially Galalpha 1-4Galbeta 1-3GalNAcalpha 1-O-Ser/Thr on asialofetuin.

Immunoreactivity of Galalpha 1-4Galbeta 1-3GalNAcalpha -O-bn-- The human blood group P system consists of five phenotypes, P1, P2, Pk1, Pk2, and p, depending on the presence or absence of the three antigens P (GalNAcbeta 1-3Galalpha 1-4Galbeta 1-4Glcbeta 1-ceramide), Pk (Galalpha 1-4Galbeta 1-4Glcbeta 1-ceramide), and P1 (Galalpha 1-4Galbeta 1-4GlcNAcbeta 1-3Galbeta 1-4Glcbeta 1-ceramide). The red cells P1 express P and P1 antigens; the erythrocytes Pk1 express Pk and P1 antigens; and red cells Pk2 express only Pk antigens. We have determined the minimum concentration of Galalpha 1-4Galbeta 1-3GalNAcalpha 1-O-bn able to inhibit human blood group P erythrocyte agglutination (Table VI). Negative controls were previously performed using Galbeta 1-3GalNAcalpha 1-O-bn, and as expected, no inhibition was observed. In contrast, Galalpha 1-4Galbeta 1-3GalNAcalpha 1-O-bn was able to inhibit the agglutination mediated by anti-Pk mAbs (0.4 mM minimum concentration) and by BSI-B4 (0.08 mM minimum concentration). However, the agglutination of P1 erythrocytes mediated by both anti-P or anti-P1 mAbs was not inhibited by the Galalpha 1-4Galbeta 1-3GalNAcalpha 1-O-bn.

                              
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Table VI
Inhibition of human blood group P erythrocyte agglutinations by Galalpha 1-4Galbeta 1-3GalNAcalpha -O-bn
Minimum concentration (mM) of Galalpha 1-4Galbeta 1-3GalNAcalpha -O-bn for inhibition of papain-treated erythrocytes agglutination caused by monoclonal antibodies or BSI-B4 is shown.


    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

In this paper, we demonstrated for the first time the occurrence of a UDP-Gal:Galbeta 1-3GalNAc alpha 1,4-galactosyltransferase, in a lepidopteran cell line. This observation is supported by several lines of evidence.

Western blot analysis of endogenous glycoproteins from Sf-9, Mb, and Tn have shown specific binding of BSI-B4 onto high molecular weight glycoproteins, which differed from one cell line to another. The staining was particularly intense for a 100-110-kDa band in Mb cells, and, according to the specificity of BSI-B4 (51-53), that fitted well with the presence of Galalpha 1-3/4Gal terminal sequences in the glycoproteins of the different examined lepidopteran cell lines.

Based on that first observation, we focused our experiments on the characterization of an alpha -galactosyltransferase activity in Mb. The specificity of the glycosyltransferase activity responsible for the biosynthesis of the Mb alpha -Gal terminal sequences was studied using several acceptor substrates containing a terminal galactose residue. That choice took into account the specificity of BSI-B4 and also the fact that the different alpha -galactosyltransferases characterized so far transferred Gal residue in alpha -linkage onto a terminal galactose to form the terminal disaccharides Galalpha 1-3Gal (65-67) or Galalpha 1-4Gal (69-71). Oligosaccharide substrates revealed that the Mb alpha -galactosyltransferase preferred the Galbeta 1-3-R unit as acceptor (Table I), and among such glycans, the relative substrate activity Vmax/Km shows that the Galbeta 1-3GalNAc is the most suitable acceptor (Table II).

To determine the nature of the linkage of the transferred Gal residue, we have performed a large scale preparation using Galbeta l-3GalNAcalpha 1-O-bn acceptor. NMR and MS data allowed us to establish that the Mb alpha -galactosyltransferase synthesized one unique product: Galalpha 1-4Galbeta 1-3GalNAcalpha 1-O-bn. The fact that only one product was formed, without any trace of beta -linkage or of other substitution than onto the C-4 position of the galactose acceptor, leads us to conclude that Mb microsomes contained essentially a galactosyltransferase activity able to catalyze the addition of galactose residues alpha 1,4-linked to Galbeta 1-3GalNAc terminal sequences.

Galbeta 1-3GalNAc disaccharide is usually present on O-glycosylation sites of numerous asialoglycoproteins but also on glycolipids, including GA1, GM1a, or GD1b. We determined that Mb alpha 1,4-Gal-T acted both on glycoproteins and glycolipids, although in our enzymatic assay the transfer onto glycolipids was low. Furthermore, we have confirmed that the Gal residue was added preferentially onto Galbeta 1-3GalNAcalpha 1-O-Ser/Thr rather than onto Galbeta 1-4GlcNAc sequence present in the terminal position of N-glycans of asialofetuin. Such a result is in agreement with the first observation of the binding of BSI-B4 onto endogenous Mb glycoproteins.

Using a microsomal fraction as enzyme source, the presence of other galactosyltransferases using UDP-Gal as a donor substrate could not be excluded. Only one core 1 beta 1,3-galactosyltransferase (UDP-Gal:GalNAcalpha 1-O-Ser/Thr beta 1,3-galactosyltransferase) has been described in lepidopteran cells (39), and the beta 1,4-galactosyltransferase (UDP-Gal:GlcNAc beta 1,4-galactosyltransferase) that adds a Gal residue onto N-glycan structures has never been detected in insect cells. Moreover, both enzymes did not use terminal Gal oligosaccharides as acceptors. Consequently, interference in alpha -galactosyltransferase assays was rather improbable, even if Mb microsomes may contain numerous different glycosyltransferases.

The high activity of the Mb alpha 1,4-Gal-T allowed us to synthesize sufficient amounts of Galalpha 1-4Galbeta 1-3GalNAcalpha 1-O-bn to study the possible applications of that trisaccharidic epitope. Galalpha 1-4Gal-specific mAbs are important tools for the characterization of tissues or isolated glycoconjugates (73). We examined, therefore, the capacity of Galalpha 1-4Galbeta 1-3GalNAcalpha 1-O-bn to inhibit the agglutination of P1, Pk1, or Pk2 red blood cells mediated by anti-Pk, anti-P1, anti-P mAbs, or BSI-B4 (Table VI). Inhibition of hemagglutination assays showed that Galalpha 1-4Galbeta 1-3GalNAcalpha -O-bn was recognized as a good competitor for anti-Pk mAb, as well as for BSI-B4. As expected, Galalpha 1-4Galbeta 1-3GalNAcalpha 1-O-bn could not inhibit either the anti-P-mediated agglutination of P1 erythrocytes or the anti-P1-mediated agglutination of Pk1 red blood cells. Such results were in agreement with previous results indicating that the anti-Pk mAb recognized mainly the terminal disaccharide Galalpha 1-4Gal, whereas anti-P1 was specific for the terminal P1 trisaccharide Galalpha 1-4Galbeta 1-4GlcNAc (74-76). In addition, the observation that Galalpha 1-4Galbeta 1-3GalNAcalpha -O-bn strongly inhibited the BSI-B4-mediated hemagglutination fitted well with the binding of that lectin on Mb glycoproteins observed by Western blotting (Fig. 1) and favors the presence of such glycans in that insect cell line.

Interestingly, chemical and immunochemical studies have indicated that galabiosylceramide and P1 and Pk antigens are expressed in many pig and human tissues (73, 77-79). In addition, several studies have shown that Galalpha 1-4Gal-containing glycolipids act as receptors for the binding of pathogenic bacteria, such as Escherichia coli to human uroepithelial cells (79) and Streptococcus suis, which is responsible for meningitis in pigs and humans (80, 81). Galalpha 1-4Gal-mediated binding is also displayed by bacterial toxins, such as E. coli verotoxin (82, 83), Shiga toxin produced by Shigella dysenteriae type 1 (84), and staphylococcal enterotoxin-B (85). Galalpha 1-4Galbeta 1-3GalNAc, free or linked onto an O-glycoprotein, could be a good candidate to inhibit pathogen attachment (bacteria or toxins) to host cells.

Finally, this work shows the presence of a new alpha 1,4-Gal-T activity in Mb cells responsible for the presence of the disaccharide epitope Galalpha 1-4Gal onto endogenous Mb O-glycoproteins. Our results can be related to studies showing new glycosyltransferase activities in lepidopteran cells, as the beta 1,4-N-acetylgalactosaminyltransferase enzymatic activity in Tn, Sf-9, and Mb catalyzing the transfer of GalNAc residues to oligosaccharides carrying a terminal beta -linked GlcNAc residue (86) or the alpha 1,3-fucosyltransferase activity in Mb, which is able to transfer fucose into alpha 1,3-linkage to the asparagine-bound GlcNAc residue of N-glycans (36). Knowledge of these new insect glycosyltransferase activities will allow selection of the lepidopteran cell clones deficient in such activities for use in the production of recombinant glycoproteins. In addition, these new insect glycosylation potentials could represent an abundant source of enzymes for the biosynthesis of epitopes of potential therapeutic value.

    ACKNOWLEDGEMENTS

We are grateful to Prof. Gérard Devauchelle and to Dr. Jean-Marie Quiot (Station de Recherche de Pathologie Comparée INRA/Unité de Recherche Associée du CNRS 2209) for constant interest and support; to Prof. Jean Montreuil and Dr. Jean-Claude Michalski (members of the Unité Mixte de Recherche du CNRS 111) for helpful discussions; and to Dr. Claudine Augé (URA CNRS 462, Orsay, France) and Dr. J.-F. Bouhours (INSERM U-437, Nantes, France) for kindly providing acceptor substrates.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

parallel To whom correspondence should be addressed: Laboratoire de Chimie Biologique, Unité Mixte de Recherche du CNRS 111, Université des Sciences et Technologies de Lille, F-59655 Villeneuve d'Ascq, France. Tel.: 33-320-43-69-23; Fax: 33-320-43-65-55; E-mail: pdelanno{at}chouia.univ-lille1.fr.

The abbreviations used are: Fuc, fucose; alpha 1, 4-Gal-T, UDP-Gal:Galbeta 1-3GalNAc alpha 1,4-galactosyltransferase; bn, benzyl; BSI-B4, B. simplicifolia lectin-I isolectin B4; COSY, two-dimensional homonuclear correlation spectroscopy; GC, gas chromatography; MS, mass spectrometry; HMQC, two-dimensional heteronuclear multiple-quantum coherence spectroscopy; MALD-MS, matrix-assisted laser desorption; Mb, M. brassicae clone SPCMb-92-C6; Sf-9, S. frugiperda clone ATCC CRL 1711; Sf-21, S. frugiperda cell line IPLB-Sf-21-AE; Tn, T. ni clone Tn-5-B14; Mes, 4-morpholineethanesulfonic acid; mAb, monoclonal antibody; GM1a, Galbeta 1-3GalNAcbeta 1-4[NeuAcalpha 2-3]Galbeta 1-4Glc-ceramide; GA1, Galbeta 1-3GalNAcbeta 1-4Galbeta 1-4Glc-ceramide; GD1b, Galbeta 1-3GalNAcbeta 1-4[NeuAcalpha 2-8NeuAcalpha 2-3]Galbeta 1-4Glc-ceramide; GM2, GalNAcbeta 1-4[NeuAcalpha 2-3]Galbeta 1-4Glc-ceramide.

2 J. M. Quiot, unpublished data.

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Abstract
Introduction
Procedures
Results
Discussion
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