Glycoengineering of therapeutic glycoproteins: in vitro galactosylation and sialylation of glycoproteins with terminal N-acetylglucosamine and galactose residues.

Therapeutic glycoproteins produced in different host cells by recombinant DNA technology often contain terminal GlcNAc and Gal residues. Such glycoproteins clear rapidly from the serum as a consequence of binding to the mannose receptor and/or the asialoglycoprotein receptor in the liver. To increase the serum half-life of these glycoproteins, we carried out in vitro glycosylation experiments using TNFR-IgG, an immunoadhesin molecule, as a model therapeutic glycoprotein. TNFR-IgG is a disulfide-linked dimer of a polypeptide composed of the extracellular portion of the human type 1 (p55) tumor necrosis factor receptor (TNFR) fused to the hinge and Fc regions of the human IgG(1) heavy chain. This bivalent antibody-like molecule contains four N-glycosylation sites per polypeptide, three in the receptor portion and one in the Fc. The heterogeneous N-linked oligosaccharides of TNFR-IgG contain sialic acid (Sia), Gal, and GlcNAc as terminal sugar residues. To increase the level of terminal sialylation, we regalactosylated and/or resialylated TNFR-IgG using beta-1,4-galactosyltransferase (beta1,4GT) and/or alpha-2,3-sialyltransferase (alpha2,3ST). Treatment of TNFR-IgG with beta1,4GT and UDP-Gal, in the presence of MnCl(2), followed by MALDI-TOF-MS analysis of PNGase F-released N-glycans showed that the number of oligosaccharides with terminal GlcNAc residues was significantly decreased with a concomitant increase in the number of terminal Gal residues. Similar treatment of TNFR-IgG with alpha2,3ST and CMP-sialic acid (CMP-Sia), in the presence of MnCl(2), produced a molecule with an approximately 11% increase in the level of terminal sialylation but still contained oligosaccharides with terminal GlcNAc residues. When TNFR-IgG was treated with a combination of beta1,4GT and alpha2,3ST (either in a single step or in a stepwise fashion), the level of terminal sialylation was increased by approximately 20-23%. These results suggest that in vitro galactosylation and sialylation of therapeutic glycoproteins with terminal GlcNAc and Gal residues can be achieved in a single step, and the results are similar to those for the stepwise reaction. This type of in vitro glycosylation is applicable to other glycoproteins containing terminal GlcNAc and Gal residues and could prove to be useful in increasing the serum half-life of therapeutic glycoproteins.

Chinese hamster ovary (CHO) cells may express six beta 4-galactosyltransferases (beta 4GalTs). Consequences of the loss of functional beta 4GalT-1, beta 4GalT-6, or both in CHO glycosylation mutants.

Six beta4-galactosyltransferase (beta4GalT) genes have been cloned from mammalian sources. We show that all six genes are expressed in the Gat(-)2 line of Chinese hamster ovary cells (Gat(-)2 CHO). Two independent mutants termed Pro(-)5Lec20 and Gat(-)2Lec20, previously selected for lectin resistance, were found to have a galactosylation defect. Radiolabeled biantennary N-glycans synthesized by Pro(-)5Lec20 were proportionately less ricin-bound than similar species from parental CHO cells, and Lec20 cell extracts had a markedly reduced ability to transfer Gal to GlcNAc-terminating acceptors. Northern blot analysis revealed a severe reduction in beta4GalT-1 transcripts in Pro(-)5Lec20 cells. The Gat(-)2Lec20 mutant expressed beta4GalT-1 transcripts of reduced size due to a 311-base pair deletion in the beta4GalT-1 gene coding region. Northern analysis with probes from the remaining five beta4GalT genes revealed that Gat(-)2 CHO and Gat(-)2Lec20 cells express all six beta4GalT genes. Unexpectedly, the beta4GalT-6 gene is not expressed in either Pro(-)5 or Pro(-)5Lec20 cells. Thus, in addition to a deficiency in beta4GalT-1, Pro(-)5Lec20 cells lack beta4GalT-6. Nevertheless, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry data of N-glycans released from cellular glycoproteins showed that both the beta4GalT-1(-) (Gat(-)2Lec20) and beta4GalT-1(-)/beta4GalT-6(-) (Pro(-)5Lec20) mutants have a similar Gal deficiency, affecting neutral and sialylated bi-, tri-, and tetraantennary N-glycans. By contrast, glycolipid synthesis was normal in both mutants. Therefore, beta4GalT-1 is a key enzyme in the galactosylation of N-glycans, but is not involved in glycolipid synthesis in CHO cells. beta4GalT-6 contributes only slightly to the galactosylation of N-glycans and is also not involved in CHO cell glycolipid synthesis. These CHO glycosylation mutants provide insight into the variety of in vivo substrates of different beta4GalTs. They may be used in glycosylation engineering and in investigating roles for beta4GalT-1 and beta4GalT-6 in generating specific glycan ligands.

Characterization of EDTA-soluble polysaccharides from the scape of Musa paradisiaca (banana).

The polysaccharide components present in the scape of Musa paradisiaca (banana) were fractionated into water-soluble (WSP), EDTA-soluble (EDTA-SP), alkali-soluble (ASP) and alkali-insoluble (AISP) polysaccharide fractions [Anjaneyalu, Jagadish and Raju (1997) Glycoconj. J. 14, 507-512]. The EDTA-SP was further fractionated by iso-amyl alcohol into EDTA-SP-A and EDTA-SP-B. The homogeneity of these two polysaccharides was established by repeated precipitation with iso-amyl alcohol, gel-filtration chromatography and sedimentation analysis. The polysaccharides were characterized by monosaccharide composition analysis, methylation linkage analysis, iodine affinity, ferricyanide number, blue value, hydrolysis with alpha-amylase, gold-electron microscopy and X-ray diffraction spectroscopy. Data from all of these studies suggest that EDTA-SP-A is a branched amylose-type alpha-D-glucan and that EDTA-SP-B is a highly branched amylopectin-type polymer. The nature of the branching patterns of these polysaccharides suggests that they are unique to M. paradisiaca.

Species-specific variation in glycosylation of IgG: evidence for the species-specific sialylation and branch-specific galactosylation and importance for engineering recombinant glycoprotein therapeutics.

Immunoglobulins (IgG) are soluble serum glycoproteins in which the oligosaccharides play significant roles in the bioactivity and pharmacokinetics. Recombinant immuno-globulins (rIgG) produced in different host cells by recombinant DNA technology are becoming major therapeutic agents to treat life threatening diseases such as cancer. Since glycosylation is cell type specific, rIgGs produced in different host cells contain different patterns of oligosaccharides which could affect the biological functions. In order to determine the extent of this variation N-linked oligosaccharide structures present in the IgGs of different animal species were characterized. IgGs of human, rhesus, dog, cow, guinea pig, sheep, goat, horse, rat, mouse, rabbit, cat, and chicken were treated with peptide-N-glycosidase-F (PNGase F) and the oligosaccharides analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for neutral and acidic oligosaccharides, in positive and negative ion modes, respectively. The data show that for neutral oligosaccharides, the proportions of terminal Gal, core Fuc and/or bisecting GlcNAc containing oligosaccharides vary from species to species; for sialylated oligosaccharides in the negative mode MALDI-TOF-MS show that human and chicken IgG contain oligosaccharides with N-acetylneuraminic acid (NANA), whereas rhesus, cow, sheep, goat, horse, and mouse IgGs contain oligosaccharides with N-glycolylneuraminic acid (NGNA). In contrast, IgGs from dog, guinea pig, rat, and rabbit contain both NANA and NGNA. Further, the PNGase F released oligosaccharides were derivatized with 9-aminopyrene 1,4,6-trisulfonic acid (APTS) and analyzed by capillary electrophoresis with laser induced fluorescence detection (CE-LIF). The CE-LIF results indicate that the proportion of the two isomers of monogalactosylated, biantennary, complex oligosaccharides vary significantly, suggesting that the branch specificity of beta1, 4-galactosyltransferase might be different in different species. These results show that the glycosylation of IgGs is species-specific, and reveal the necessity for appropriate cell line selection to express rIgGs for human therapy. The results of this study are useful for people working in the transgenic area.

A convenient microscale colorimetric method for terminal galactose on immunoglobulins.

A new approach for quantitative determination of terminal galactose (Gal) residues of immunoglobulins was developed by combining exoglycosidase digestion with the classical colorimetric estimation of reducing sugars. The ferricyanide colorimetric method was modified to increase the stability of the chromophore (Prussian blue) and adapted to determine the amount of terminal Gal residues present in immunoglobulins. The method involves the release of covalently bound Gal from immunoglobulins by Diplococcus pneumoniae beta-D-galactosidase (specific for beta(1,4) linked galactose), removal of the glycoprotein and enzyme from the reaction mixture by heat denaturation or ethanol precipitation, followed by colorimetric measurement of the released sugar using the ferricyanide assay. The ferricyanide method was modified to enhance the solubility and stability of the chromophore by increasing the concentration of aqueous sulfuric acid and sodium dodecyl sulfate (SDS). The linear range of the modified method was from approximately 11 to 111 microM Gal. Typical variation in assay results was on the order of 5%. Using the modified method, the terminal Gal content of a recombinant chimeric monoclonal antibody (anti-CD20, rIgG) expressed in Chinese hamster ovary (CHO) cells was determined and evaluated for batch-to-batch consistency. The method was used to optimize pH, time, temperature, and enzyme concentration for beta-galactosidase digestion for maximal release of terminal Gal residues from rIgG.

Gain-of-function Chinese hamster ovary mutants LEC18 and LEC14 each express a novel N-acetylglucosaminyltransferase activity.

LEC18 and LEC14 cells are gain-of-function glycosylation mutants isolated from Chinese hamster ovary cells for resistance to pea lectin. Structural studies have shown that LEC18 cells synthesize complex N-glycans with a GlcNAc residue linked at the O-6 position of the core GlcNAc (Raju, T. S., Ray, M. K., and Stanley, P. (1995) J. Biol. Chem. 270, 30294-30302), whereas LEC14 cells synthesize complex N-glycans with a GlcNAc residue linked at the O-2 position of the core beta-linked Man residue (Raju, T. S., and Stanley, P. (1996) J. Biol. Chem. 271, 7484-7493). Both modifications are novel and have not been reported in glycoproteins from any other source. We now show that, in both LEC18 and LEC14 cells, GlcNAc transfer is mediated by a distinct N-acetylglucosaminyltransferase (GlcNAc-T) activity. The LEC18 activity, termed GlcNAc-TVIII, transfers GlcNAc to GlcNAcbeta1-O-pNP and to a GlcNAc-terminating, biantennary, complex N-glycan, with or without a core fucose. By contrast, the LEC14 transferase, termed GlcNAc-TVII, does not have significant activity with simple acceptors, and transfers GlcNAc preferentially to a GlcNAc-terminating biantennary glycopeptide that contains a core fucose residue. The acceptor specificities and other biochemical properties of GlcNAc-TVII and GlcNAc-TVIII differ from previously characterized GlcNAc-transferases including GlcNAc-TIII, indicating that they represent new members of the mammalian GlcNAc-T group of transferases.

Polysaccharide components from the scape of Musa paradisiaca: main structural features of water-soluble polysaccharide component.

Polysaccharide components present in the pseudo-stem (scape) of M. paradisiaca were purified from acetone powder of the scape by delignification followed by extraction with aqueous solvents into water soluble polysaccharide (WSP), EDTA-soluble polysaccharide (EDTA-SP), alkali-soluble polysaccharide (ASP) and alkali-insoluble polysaccharide (AISP) fractions. Sugar compositional analysis showed that WSP and EDTA-SP contained only D-Glc whereas ASP contained D-Glc, L-Ara and D-Xyl in approximately 1:1:10 ratio, respectively, and AISP contained D-Glc, L-Ara and D-Xyl in approximately 10:1:2 ratio, respectively. WSP was further purified by complexation with iso-amylalcohol and characterized by specific rotation, IR spectroscopy, Iodine affinity, ferricyanide number, blue value, hydrolysis with alpha-amylase and glucoamylase, and methylation linkage analysis, and shown to be a amylopectin type alpha-D-glucan.

A comparison of the fine saccharide-binding specificity of Dioclea grandiflora lectin and concanavalin A.

The lectin from the seeds of Dioclea grandiflora (DGL) is a Man/Glc-specific tetrameric protein with physical and saccharide-binding properties reported to be similar to that of the jack bean lectin concanavalin A (ConA). Unlike other plant lectins, both DGL and ConA bind with high affinity to the core trimannoside moiety, 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside, which is present in all asparagine-linked carbohydrates. In the present study, hemagglutination inhibition techniques have been used to investigate binding of DGL and ConA to a series of mono- and dideoxy analogs of methyl 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside and to a series of asparagine-linked oligomannose and complex oligosaccharides and glycopeptides. The results indicate that both DGL and ConA recognize epitopes on all three residues of the trimannoside: the 3-, 4-, and 6-hydroxyl groups of the alpha(1-6)Man residue, the 3-hydroxyl group of the alpha(1-3)Man residue, and the 2- and 4-hydroxyl groups of the central Man residue of the core trimannoside. However, unlike ConA, DGL does not bind to biantennary complex carbohydrates. This was confirmed by showing that biantennary complex glycopeptides do not bind to a DGL-Sepharose affinity column. Unlike ConA, DGL does not show enhanced affinity for a large N-linked oligomannose carbohydrate (Man9 glycopeptide) relative to the trimannoside. Thus, DGL and ConA share similar epitope recognition of the core trimannoside moiety. However, they exhibit differences in their fine specificities for larger N-linked oligomannose and complex carbohydrates.

CHO cells provide access to novel N-glycans and developmentally regulated glycosyltransferases.

Chinese hamster ovary (CHO) cells express only a subset of the glycosyltransferases activities known to exist. They do not express several fucosyltransferases, galactosyltransferases, sialyltransferases or N-acetylglucosaminyltransferases. However, following mutagenesis or transfection with large amounts of DNA, rare mutants that express a transferase activity de novo have been obtained. The first CHO mutant of this type was LEC10, which expresses the N-acetylglucosaminyltransferase, GlcNAc-TIII, that adds the bisecting GlcNAc to complex N-glycans. Several analogous gain-of-function mutants have now been characterized and, all express a new glycosyltransferase activity. In several cases, expression is known to reflect gene activation at the transcriptional level. Thus, CHO cells contain quiescent glycosyltransferase genes that may be activated by mutational events. Several of these transferases have properties distinct from previously described enzymes. In fact, the most recently characterized dominant CHO mutants, LEC14 and LEC18, each express a GlcNAc-T activity that creates novel N-glycans never before observed in glycoproteins from any other source. In these and possibly other cases, it appears the CHO genome has provided access to new GlcNAc-Ts that may be difficult to identify by conventional methods.

LEC14, a dominant Chinese hamster ovary glycosylation mutant expresses complex N-glycans with a new N-acetylglucosamine residue in the core region.

The Chinese hamster ovary cell (CHO) glycosylation mutant, LEC14, was previously selected for resistance to pea lectin (Pisum sativum agglutinin) and shown to behave dominantly. The lectin resistance properties of LEC14 cells are related to, but distinct from, those of LEC18, a dominant Chinese hamster ovary mutant that synthesizes complex N-glycans with a novel O-6-linked GlcNAc residue in the core region (Raju, T.S., Ray, M., and Stanley, P. (1995) J. Biol. Chem. 270, 30294-30302). Detailed structural studies of a complex N-glycan fraction from LEC14 cells have revealed yet another novel modification of the core region. [3H]Glc-labeled LEC14 cellular glycopeptides were desialylated, and the fraction that did not bind to concanavalin A-Sepharose was found to have an increased proportion of species that bound to tomato-agarose, and to ricin-agarose. 1H NMR spectroscopy and methylation linkage analysis of the tomato and ricin-bound fractions purified from approximately 10(10) LEC14 cells showed they were complex N-glycans containing a 2,3,6-trisubstituted core Man residue. To examine the core region more closely, these N-glycans were digested with mixtures of beta-D-galactosidases and N-acetyl-beta-D-glucosaminidases to obtain core glycopeptides. The latter were largely unbound by concanavalin A-Sepharose or pea lectin-agarose. 1H NMR spectroscopy and electrospray ionization-mass spectrometry showed that the LEC14 core glycopeptides contain a new GlcNAc residue that substitutes the core beta(1-4)-Man residue at O-2 to give the following novel, N-linked core structure. [structure: see text]

LEC18, a dominant Chinese hamster ovary glycosylation mutant synthesizes N-linked carbohydrates with a novel core structure.

The dominant Chinese hamster ovary cell glycosylation mutant, LEC18, was selected for resistance to pea lectin (Pisum sativum agglutinin (PSA)). Lectin binding studies show that LEC18 cells express altered cell surface carbohydrates with markedly reduced binding to 125I-PSA and increased binding to 125I-labeled Datura stramonium agglutinin (DSA) compared with parental cells. Desialylated [3H]Glc-labeled LEC18 cellular glycopeptides that did not bind to concanavalin A-Sepharose exhibited an increased proportion of species that were bound to DSA-agarose. Most of these glycopeptides bound to ricin-agarose and were unique to LEC18 cells. This fraction was purified from approximately 10(10) cells and shown by 1H NMR spectroscopy and methylation linkage analysis to contain novel N-linked structures. Digestion of these glycopeptides with mixtures of beta-D-galactosidases and N-acetyl-beta-D-glucosaminidases gave core glycopeptides that, in contrast to cores from parental cells, were mainly not bound to concanavalin A-Sepharose or to PSA-agarose. 1H NMR spectroscopy, matrix-assisted laser desorption ionization/time of flight mass spectrometry, electrospray mass spectrometry, and collision-activated dissociation mass spectrometry showed that the LEC18 core glycopeptides contained a new GlcNAc residue that substitutes the core GlcNAc residues. Methylation linkage analysis of the parent compound provided evidence that the GlcNAc is linked at O-6 to give the following novel, N-linked core structure. [formula: see text]

Lec32 is a new mutation in Chinese hamster ovary cells that essentially abrogates CMP-N-acetylneuraminic acid synthetase activity.

LEC29.Lec32 is a glycosylation mutant that was isolated from a selection of mutagenized Chinese hamster ovary (CHO) cells for lectin resistance. Compared with LEC29 CHO cells, the double mutant exhibited an unusually high sensitivity to the toxic lectin, ricin, indicating increased exposure of galactose residues on cell surface carbohydrates. Structural analysis of LEC29.Lec32 cellular glycoproteins showed a nearly complete lack of sialic acid residues. Genetic analysis demonstrated that the lec32 mutation is recessive and novel. Biochemical analysis showed that the mutant cells contained less than 5% of the cytidine 5'-monophosphate N-acetylneuraminic acid (CMP-NeuAc) present in parental CHO cells (1.6 nmol/mg of cell protein). A sensitive radiochemical assay used to measure CMP-NeuAc synthetase activity showed that the properties of this enzyme in parental CHO cells were essentially identical to those of CMP-NeuAc synthetase in various mammalian tissues. However, no CMP-NeuAc synthetase activity was detected in LEC29.Lec32 extracts. Mixing experiments provided no evidence for an inhibitor in the mutant CHO cells, and two revertants, which expressed only the LEC29 phenotype, had normal CMP-NeuAc synthetase levels. The combined evidence indicates that the lec32 mutation resides in either the structural gene encoding CMP-NeuAc synthetase or in a gene that regulates the production of active enzyme.

New approach towards deglycosylation of sialoglycoproteins and mucins.

A modified procedure for chemical deglycosylation of glycoproteins containing sialylated and/or O-linked oligosaccharides, using anhydrous trifluoromethane sulfonic acid (TFMSA) is described. Although sialic acid residues are acid labile, it has been known that anhydrous TFMSA does not effectively remove carbohydrate side chains from glycoproteins if they are sialylated. In this procedure, sialic acid residues were removed by mild acid hydrolysis and the desialylated glycoprotein was treated with anhydrous TFMSA reagent under conditions which remove all the carbohydrate residues except the core D-GalNAc linked to serine/threonine. The core D-GalNAc residues were removed by reacting the glycoprotein with periodate followed by a second treatment with anhydrous TFMSA; this procedure gave a completely deglycosylated protein. The protein thus obtained was soluble in aqueous buffers and useful for biochemical and biophysical studies. The method was successfully employed to isolate polypeptides from alpha 1-acid glycoprotein (N-linked), fetuin, canine tracheal mucin and gastric mucin.

Role of sialic acid on the viscosity of canine tracheal mucin glycoprotein.

The role of sialic acid on the viscosity of canine tracheal mucin (CTM) was investigated. The mucin glycoprotein, purified from canine tracheal mucus, was subjected to mild acid hydrolysis with aqueous acetic acid and autohydrolysis in water, in which approximately 50% drop in the relative viscosity (nr) occurred. Carbohydrate compositional analysis before and after mild acid hydrolysis and autohydrolysis showed the complete removal of glycosidically bound sialic acid residues while all other sugar residues (i.e. galactose, N-acetyl galactosamine and N-acetyl glucosamine) remained unaltered, indicating that sialic acid residues are contributing towards the viscosity of CTM to a greater extent.

Structural features of water-soluble novel polysaccharide components from the leaves of Tridax procumbens Linn.

Two water-soluble polysaccharide fractions, WSTP-IA and WSTP-IB were purified from the leaves of Tridax procumbens Linn. with graded ethanol precipitation followed by mild delignification and size-exclusion chromatography. WSTP-IA contained L-Araf and D-Galp in approximately 1:3 molar proportions, and WSTP-IB contained only D-Galp as the major sugar component. The results of methylation linkage analysis, and 1H and 13C NMR studies on the native and modified polysaccharides, indicated that WSTP-IA is an L-arabino-D-galactan with a beta-(1-->6)-D-galactan main chain in which at least one in every two D-Galp residues carries single residues of either L-Araf (alpha-/beta-) or beta-D-Galp end-group as substituents at O-3. WSTP-IB is a linear beta-(1-->6)-D-galactan. This is the first report of polysaccharides containing a beta-(1-->6)-D-galactan main chain isolated from plant sources.

Chemical structure of the core region of Campylobacter jejuni serotype O:2 lipopolysaccharide.

The complete structure for the core region of Campylobacter jejuni serotype O:2 lipopolysaccharide (LPS) was assigned through studies on derivatives of the liberated oligosaccharide (OS 2) and the intact LPS. Structure determinations were performed using 1H-NMR spectroscopy, methylation studies supported by fast-atom-bombardment mass spectrometry and linkage analysis by gas chromatography/mass spectrometry, Smith degradation, and oxidation with chromium trioxide. It was concluded that complete oligosaccharide chains had the following structure: [formula: see text]

Chemical structures of the core regions of Campylobacter jejuni serotypes O:1, O:4, O:23, and O:36 lipopolysaccharides.

Complete structures, including the location of N-acetylneuraminic acid (Neu5Ac) residues, were assigned for the core regions of Campylobacter jejuni serotypes O:1, O:4, and O:23 and O:36 lipopolysaccharides (LPS). In continuation of earlier studies, structure determinations of liberated oligosaccharides and, where necessary, of intact LPS, were by 1H-NMR spectroscopy, Smith degradation, chromium trioxide and enzymic degradations, in conjunction with methylation studies supported by fast-atom-bombardment mass spectrometry and linkage analyses by gas chromatography/mass spectrometry. It was concluded on the basis of the following structures, in which each was linked 1-->5 to a terminal 3-deoxy-D-manno-octulosonic acid residue, that the core regions with qualitatively similar sugar compositions showed serotypic differences in one or more of their sequences, linkage types, and anomeric configurations: [formula: see text] [corrected]. The outer regions of each structure carry Neu5Ac residues linked 2-->3 to available beta-D-Galp residues and show striking similarities with various glycosphingolipids of the ganglioside family. However, Neu5Ac epitopes are not apparently involved in determining serospecificity.