Glycan mapping of glycotherapeutics.

Glycosylation of proteins may, or may not, affect biological activity, either directly or indirectly. In addition, variability in glycosylation arises within the manufacturing procedure. Therefore, glycosylation would be an important parameter when assessing product quality. Regulatory authorities require biotechnological and biological products to be characterised, including determination of their biological activity, physicochemical and immunochemical properties, their purity and their impurity profiles. Here we outline how we can decide if and which types of glycan analysis and methodologies to use for glycoprotein therapeutics to answer the scientific questions as well as meeting regulatory requirements that already exist or are going to be developed/implemented.

ADP-ribosylation activity in pertussis vaccines and its relationship to the in vivo histamine-sensitisation test.

Pertussis toxin (PTx) is a major virulence factor produced by Bordetella pertussis. In its detoxified form (PTd), it is an important component of acellular pertussis vaccines although some residual PTx activity may likely be present because of the limitations of the detoxification processes used. Furthermore, different detoxification procedures have been shown to result in different amino acid side-chain modifications for the resulting PTd. The histamine-sensitisation test (HIST) in mice is currently used for the safety testing of these vaccines. However, an alternative test is needed because of large assay variability and ethical concerns. The ADP-ribosylation enzyme activity of PTx is thought to be the major factor responsible for the histamine-sensitising activity detected in vivo. In the present study, the ADP-ribosylation activity in different acellular pertussis-based combination vaccine formulations was measured and compared with reactivity in the HIST. The results indicated that different products showed differences in ADP-ribosylation activity and a level which would be significant in relation to the reactivity seen in the HIST could not be defined, except for vaccines that contain genetically detoxified PTx, which do not have enzymatic activity nor in vivo toxicity. Different detoxification procedures as well as formulation factors could contribute to this variation. Relying solely on the residual enzyme activity of PTx in vaccines containing chemically detoxified PTd may not fully reflect the in vivo reactivity observed by the HIST. Refinement of the in vitro test to include a step which monitors the B-subunit activity of PTx may provide a better correlation with the in vivo HIST.

Comparative analysis of HIV-1 recombinant envelope glycoproteins from different culture systems.

The productivity of stable Chinese hamster ovary cell lines secreting HIV-1 monomeric (IIIB gp120) and oligomeric (UG21 gp140) recombinant envelope glycoproteins was compared in serum-containing (S+), serum-free (S-) and protein-free (P-) culture media. UG21 gp140 expression was greatest in S+ medium, while IIIBgp120 production was lower than gp140 in all three media but highest in S-. UG21 gp140 production was highest in standard 850-cm2 roller bottle cultures in S+ media, peaking after 14 days of incubation, while expression levels in the three media were 0.5 (S+), 0.4 (S-) and 0.2 (P-) mg/l, from which 90, 80 and 12% of gp140, respectively, could be purified by immunoaffinity chromatography. Purified UG21 gp140 from S+ and S- media possessed biological functionality as evidenced by CD4 and monoclonal antibody (Mab) binding. In contrast, UG21 gp140 from P- medium appears to be misfolded and non-functional. Despite the possession of a different N-linked glycan profile, UG21 gp140 from S- media shows very similar CD4 and Mab binding characteristics to S+ UG21 gp140. The relevance of these findings to HIV vaccine development is discussed.

High-performance liquid chromatographic profiling of fluorescent labelled N-glycans on glycoproteins.

Monitoring protein glycosylation is becoming increasingly important as novel recombinant glycoprotein therapeutics, such as glycoprotein hormones, cytokines and clotting factors, are introduced into clinical use. In this report, we describe an HPLC strategy and an improved and simplified pre-column derivatization procedure to profile N-linked glycans obtained from a variety of commercially available glycoproteins as examples. N-Glycans were first released by peptide:N-glycosidase F and labelled with the fluorescent label, 4-aminobenzoic acid by reductive amination. The labelled N-Glycans were then resolved by normal-phase HPLC and the N-glycan profile could be further improved by separating the N-glycans first according to charge by anion-exchange HPLC prior to the normal-phase HPLC. If required, identification of the fractionated derivatized oligosaccharides can be determined by mass spectrometry. The whole profiling process is simple and can be implemented in most laboratories. Because of the high sensitivity, batch glycan-analysis of low-yield recombinant glycoproteins such as samples in ampoules or obtained in the early stage of production development is possible.

Developments in reduction and replacement of in vivo toxicity and potency tests for pertussis vaccines.

The regulatory control of pertussis vaccines, as for other biological products, requires that they conform to specified standards of safety and efficacy. The current potency test for whole cell vaccines, the intracerebral mouse protection test (AMPT) is still the only such assay that has shown a correlation with protection in children. An alternative in vivo assay based on non-lethal aerosol challenge of mice has been assessed as a replacement for the current AMPT. An in vitro assay based on determination of reactive nitrogen/oxygen intermediates produced as a result of macrophage activation has also been investigated as a potential replacement for the in vivo challenge test. On the other hand, for safety testing, an enzymatic-HPLC coupled assay using a fluorescein-labelled G alpha(i3)C20 peptide to measure the enzymatic ribosylation activity of active pertussis toxin was evaluated for its suitability as a replacement for the current histamine sensitisation test (HIST). An assay for adenylate cyclase toxin (ACT)-related toxicity, based on measuring the ACT-induced oxidative burst in macrophage-like cell cultures has also been investigated. Although some questions still need to be answered in relation to the development of suitable replacements for in vivo tests of pertussis vaccines, the prospects for further improvements are promising.

Expression of glycoconjugates bearing the Lewis X epitope during neural differentiation of P19 EC cells.

The Lewis X (Le(x)) bearing glycolipids were noticeably increased in amounts during the course of neural differentiation of P19 EC cells induced by retinoic acid (RA, all-trans form). Applying neoglycolipid technology and in situ TLC-LSIMS, the oligosaccharide chains of these scarce Le(x) bearing glycolipids were partially characterized after released by endoglycoceramidase and subsequent conversion into neoglycolipids. In order to understand the enzymatic basis for the expression of Le(x) bearing glycolipids, we measured glycolipid, glycoprotein and oligosaccharide fucosyltransferase (Fuc-T) activities using appropriate substrates in P19 EC cells with or without RA treatment. All three Fuc-Ts were increased after RA treatment and the highest activity was in the differentiated neural cells. We then investigated the two possible Fuc-T genes that might be responsible for these changes using RT-PCR analysis. Mouse Fuc-TIX (mFuc-TIX) transcript was detected in all cell types but it was only strongly expressed in RA-induced aggregates and neural cells. In the case of mouse Fuc-TIV (mFuc-TIV) gene, its transcript was only detectable in RA-induced aggregates and not found in either undifferentiated or RA-induced neural cells. These results strongly support that RA induces only a transient expression of the mFuc-TIV gene in cell aggregates but a more persistent expression of the mFuc-TIX gene at the transcription level throughout neural cell differentiation. The mFuc-TIX gene is probably the main cause for the increased expression of Le(x) glycoconjugates during neural differentiation of P19 EC cells.

Role of aromatic residues at the lipid-water interface in micelle-bound bacteriophage M13 major coat protein.

Analyses of transmembrane domains of proteins have revealed that aromatic residues tend to cluster at or near the lipid-water interface of the membrane. To assess protein-membrane interactions of such residues, a viable mutant library was generated of the major coat protein of bacteriophage M13 (a model single membrane-spanning protein) in which one or the other of its interfacial tyrosine residues (Tyr-21 and Tyr-24) is mutated. Using the interfacial tryptophan (Trp-26) as an intrinsic probe, blue shifts in fluorescence emission spectra and quenching constants indicated that mutants with a polar amino acid substitution (such as Y24D or Y24N) are less buried in a deoxycholate micelle environment than in the wild type protein. These polar mutants also exhibited alpha-helix to beta-structure transition temperatures in incremental-heating circular dichroism studies relatively lower than those of wild type and nonpolar mutants (such as Y21V, Y21I, and Y24A), indicating that specific side chains in the lipid-water interface influence local protein-micelle interactions. Mutant Y21F exhibited the highest transition temperature, suggesting that phenylalanine is ostensibly the most effective interfacial anchoring residue. Using phage viability as the assay in a combination of site-directed and saturation mutagenesis experiments, it was further observed that both Tyr residues could not simultaneously be "knocked out". The overall results support the notion that an interfacial Tyr is a primary recognition element for precise strand positioning in vivo, a function that apparently cannot be performed optimally by residues with simple aliphatic character.

The cysteine-rich domain of the macrophage mannose receptor is a multispecific lectin that recognizes chondroitin sulfates A and B and sulfated oligosaccharides of blood group Lewis(a) and Lewis(x) types in addition to the sulfated N-glycans of lutropin.

The mannose receptor (MR) is an endocytic protein on macrophages and dendritic cells, as well as on hepatic endothelial, kidney mesangial, tracheal smooth muscle, and retinal pigment epithelial cells. The extracellular portion contains two types of carbohydrate-recognition domain (CRD): eight membrane-proximal C-type CRDs and a membrane-distal cysteine-rich domain (Cys-MR). The former bind mannose-, N-acetylglucosamine-, and fucose-terminating oligosaccharides, and may be important in innate immunity towards microbial pathogens, and in antigen trapping for processing and presentation in adaptive immunity. Cys-MR binds to the sulfated carbohydrate chains of pituitary hormones and may have a role in hormonal clearance. A second feature of Cys-MR is binding to macrophages in marginal zones of the spleen, and to B cell areas in germinal centers which may help direct MR-bearing cells toward germinal centers during the immune response. Here we describe two novel classes of carbohydrate ligand for Cys-MR: chondroitin-4 sulfate chains of the type found on proteoglycans produced by cells of the immune system, and sulfated blood group chains. We further demonstrate that Cys-MR interacts with cells in the spleen via the binding site for sulfated carbohydrates. Our data suggest that the three classes of sulfated carbohydrate ligands may variously regulate the trafficking and function of MR-bearing cells.

Fluorescent neoglycolipids. Improved probes for oligosaccharide ligand discovery.

A second generation of lipid-linked oligosaccharide probes, fluorescent neoglycolipids, has been designed and synthesized for ligand discovery within highly complex mixtures of oligosaccharides. The aminolipid 1,2-dihexadecyl-sn-glycero-3-phosphoethanolamine (DHPE), which has been used extensively to generate neoglycolipids for biological and structural studies, has been modified to incorporate a fluorescent label, anthracene. This new lipid reagent, N-aminoacetyl-N-(9-anthracenylmethyl)-1, 2-dihexadecyl-sn-glycero-3-phosphoethanolamine (ADHP), synthesized from anthracenaldehyde and DHPE gives an intense fluorescence under UV light. Fluorescent neoglycolipids derived from a variety of neutral and acidic oligosaccharides by conjugation to ADHP, by reductive amination, can be detected and quantified by spectrophotometry and scanning densitometry, and resolved by TLC and HPLC with subpicomole detection. Antigenicities of the ADHP-neoglycolipids are well retained, and picomole levels can be detected using monoclonal carbohydrate sequence-specific antibodies. Among O-glycans from an ovarian cystadenoma mucin, isomeric oligosaccharide sequences, sialyl-Lea- and sialyl-Lex-active, could be resolved by HPLC as fluorescent neoglycolipids, and sequenced by liquid secondary-ion mass spectrometry. Thus the neoglycolipid technology now uniquely combines high sensitivity of immuno-detection with a comparable sensitivity of chemical detection. Principles are thus established for a streamlined technology whereby an oligosaccharide population is carried through ligand detection and ligand isolation steps, and sequence determination by mass spectrometry, enzymatic sequencing and other state-of-the-art technologies for carbohydrate analysis.

Synthesis and enzymology of modified N-benzyloxycarbonyl-L-cysteinylglycyl-3,3-dimethylaminopropylamide++ + disulphides as alternative substrates for trypanothione reductase from Trypanosoma cruzi: Part 3.

Kinetic data for alternative substrates of recombinant trypanothione reductase from Trypanosoma cruzi were measured for a series of N-substituted-L-cysteinylglycyl-3-dimethylaminopropylamides, in which the cysteine N-substituent was either a variant of the benzyloxycarbonyl group or was L-phenylalanine or L-tryptophan. Replacing the benzylic ether oxygen atom by CH2 or NH had relatively minor effects on kcat, but raised the value of K(m) 4.5- and 10-fold, respectively. Similarly, relative to the carbobenzoxy group, an N-L-phenylalanyl or N-L-tryptophanyl replacement on the cysteine hardly altered kcat, but increased K(m) values by 16.6 and 7.4 fold, respectively. These observations were consistent with the K(m) values referring primarily to binding for this series of nonspecific substrates.

High prevalence of 2-mono- and 2,6-di-substituted manol-terminating sequences among O-glycans released from brain glycopeptides by reductive alkaline hydrolysis.

Di- to heptasaccharides isolated from total nondialyzable brain glycopeptides after release by alkaline borohydride treatment have been subjected to mass spectrometric and nuclear magnetic resonance spectroscopic analyses supplemented by TLC-MS analyses of derived neoglycolipids. A family of Manol-terminating oligosaccharides has been revealed which includes novel sequences with a 2, 6-disubstituted Manol: In contrast to the Manol-terminating HNK-1 antigen-positive chains described previously that occur as a minor population [Yuen, C.-T., Chai, W., Loveless, R.W., Lawson, A.M., Margolis, R.U. & Feizi, T. (1997) J. Biol. Chem. 272, 8924-8931], the above oligosaccharides are abundant. The ratio of these compounds to the classical N-acetylgalactosaminitol-terminating oligosaccharides is about 1 : 3. Thus, there appears to be in higher eukaryotes a major alternative pathway related to the yeast-type protein O-mannosylation, the enzymatic basis and functional importance of which now require investigation.

Core-branching pattern and sequence analysis of mannitol-terminating oligosaccharides by neoglycolipid technology.

The occurrence of mannitol-terminating oligosaccharides (2-substituted or 2,6-disubstituted) among the O-glycans released by alkaline borohydride treatment from glycoproteins of the nervous system has prompted the development of a microscale method to analyze the core-branching pattern and sequence by the neoglycolipid (NGL) technology, analogous to a method previously described for GalNAcol-terminating oligosaccharides (M. S. Stoll, E. F. Hounsell, A. M. Lawson, W. Chai, and T. Feizi, Eur. J. Biochem. 189, 499-507, 1990). The approach involves the selective cleavage at the core mannitol by mild periodate treatment and analysis of the reaction products as NGLs by in situ TLC/liquid secondary ion mass spectrometry. Oxidation conditions have been optimized using as reference compounds 2-, 3-, 4-, or 6-monosubstituted mannobi-itols, 3,6-disubstituted mannitol-terminating pentasaccharides, and 2-mono- and 2,6-disubstituted mannitol-terminating neutral and sialylated oligosaccharides isolated from brain glycopeptides. When a 2:1 molar ratio of periodate to alditol is used, the core mannitol is cleaved at the C3-C4 threo-diol bond and in the absence of a threo-diol cleavage occurs to a lesser extent at erythro-diols. Saccharide ring diols are not cleaved under these conditions, and it is also shown that the side chain of sialic acid on the oligosaccharide is largely unaffected. Substituents at 2- and 6-positions of the core mannitol can be identified, and the method is applicable to neutral and sialylated oligosaccharide alditols. Typically, the starting material is 5 nmol of oligosaccharide and 0.5-1 nmol of derivatives is applied for analysis. By this strategy, the core-branching pattern and position of sialic acid of two branched monosialylated mannitol-terminating oligosaccharide isomers have been determined.

Recombinant GM2-activator protein stimulates in vivo degradation of GA2 in GM2 gangliosidosis AB variant fibroblasts but exhibits no detectable binding of GA2 in an in vitro assay.

The interaction between glycosphingolipids and recombinant human GM2-activator was studied in a microwell binding assay. A-series gangliosides like GM3, GM2 and GM1 were strongly bound by the recombinant human GM2 activator. A weak binding was observed to GD1b and sulfatide, while neutral glycolipids were not bound. Optimal binding occurred at pH 4.2 and was inhibited by increasing concentrations of citrate buffer and NaCl. In contrast with these in vitro results the recombinant human GM2-activator is able to restore the degradation of GA2 in fibroblasts from patients with the AB variant of GM2 gangliosidosis in vivo.

Monoclonal antibody 91.9H raised against sulfated mucins is specific for the 3'-sulfated Lewisa tetrasaccharide sequence.

The IgG1hybridoma antibody, 91.9H, was originally raised against sulfated mucins isolated from normal human colonic mucosa. Previous studies have shown that the 91.9H antigen is expressed on normal colonic epithelial cells and the sulfomucins that they produce, but not in the normal small intestine and stomach. Tissue-specific changes occur in 91.9H antigen expression in disease: the antigen diminishes in colonic carcinomas, whereas in regions of gastric mucosa showing intestinal metaplasia and in gastric carcinomas, the antigen is expressed as a "neo-antigen." This report is concerned with elucidation, by the neoglycolipid technology, of the determinant recognized by antibody 91.9H using sulfated and sialyl oligosaccharides of Lewisa(Lea) and Lextypes, and analogs that lack sulfate, sialic acid, or fucose. Binding experiments with the lipid-linked oligosaccharides immobilized on chromatograms or on microwells, and inhibition of binding experiments with free oligosaccharides based on di-, tri- and tetrasaccharide backbones, show that the 91.9H antigenic determinant is based on a trisaccharide backbone, and consists of the 3'-sulfated Leatetrasaccharide sequence, which is a potent ligand for the E- and L-selectins. The antibody gives a relatively low signal with the 3'-sulfated non-fucosylated backbone, and has no detectable cross-reaction with the 3'-sulfated Lexisomer, nor with sialyl-Leaand -Lexanalogues. Antibody 91.9H is a valuable addition, therefore, to the repertoire of reagents for mapping details of the distribution, and determining the relative importance of sulfated and sialyl oligosaccharides as ligands for the selectins, in normal and pathological epithelia and endothelia.

Nonreductive release of O-linked oligosaccharides from mucin glycoproteins for structure/function assignments as neoglycolipids: application in the detection of novel ligands for E-selectin.

The neoglycolipid technology comprises several microprocedures involving the generation of lipid-linked oligosaccharide probes for carbohydrate recognition studies in conjunction with oligosaccharide sequence determination by mass spectrometry. Although applicable to any desired oligosaccharides, procedures are greatly facilitated if the oligosaccharides are nonreduced, as conjugation is by reductive amination of a reducing end aldehyde to a phosphatidylethanolamine. Using bovine submaxillary mucin as a model for release of O-glycans in the reducing state, and based on yields of neoglycolipids and side-products from "peeling" reactions and degradation, aqueous ethylamine 70% w/v at 22 degrees C for 48 h has been selected in preference to other conditions, triethylamine, sodium hydroxide, and hydrazine. The integrity of the main acidic and neutral oligosaccharides released under these conditions, di- to octasaccharides, was established by analyses of free oligosaccharides by liquid secondary ion mass spectrometry (LSIMS) and of the derived neoglycolipids by TLC-LSIMS; the repertoire compared favorably with that of the oligosaccharide alditols generated by conventional reductive alkaline borohydride treatment. More forcing conditions of ethylamine 70% w/v at 65 degrees C for 6 h were required to release oligosaccharides from porcine gastric mucin; di- to nonasaccharides were obtained of which about one-third had an intact core GalNAc. Relative to yields after reductive alkaline hydrolysis, the overall yields for these two glycoproteins were 20% and 40-50% for acidic and neutral oligosaccharides, respectively. Among O-glycans released from an ovarian cystadenoma glycoprotein using ethylamine, three variants of the sulfated Le(a/x) sequences were identified as ligands for the endothelial adhesion molecule E-selectin, one of which is based on the unusual backbone Gal-3/4GlcNAc-3Gal-3Gal.

Valency dependent patterns of binding of human L-selectin toward sialyl and sulfated oligosaccharides of Le(a) and Le(x) types: relevance to anti-adhesion therapeutics.

The human L-selectin is known to bind to immobilized 3'-sialyl-Le(x) and -Le(a) oligosaccharides both under static and physiological flow conditions. Here the reactivities toward 3'-sulfated and 3'-sialyl-Le(a) and -Le(x) pentasaccharides are compared by in-vitro binding and inhibition assays using preparations of human L-selectin-IgG-Fc chimera in which the selectin is predominantly in di- and tetrameric form (paucivalent) or in the form of a complex with anti-IgG (multivalent). Affinity for the sulfated ligands is marginally greater than for the sialyl ligands, as judged by concentrations required to give 50% inhibition of the multivalent selectin binding to the immobilized sulfated and sialyl ligands. There is a striking difference, however, in the avidities of binding of the two L-selectin forms toward the sulfated and sialyl ligands when these are immobilized in the clustered state: the paucivalent selectin gives detectable binding only to the sulfated ligands when these are immobilized as neoglycolipids on plastic microwells (up to 100 pmol immobilized per well) whereas the multivalent L-selectin binds well to both classes of ligand. Moreover, binding of the paucivalent selectin form is effectively inhibited only by the sulfated ligand, although binding of the multivalent selectin is inhibitable by both the sulfated and sialyl ligands. Such striking valency-dependent differences in ligand binding avidity and inhibitability may be manifest in vivo with the membrane-bound L-selectin, as marked variations occur in its density of expression on leukocytes. Thus, for the purpose of selecting inhibitors for development of therapeutic anti-inflammatory compounds, experimental designs based on the paucivalent L-selectin would more clearly single out compounds with broad spectrum anti-adhesive activities toward the both the high- and low-avidity interactions of the cell adhesion protein.

Brain contains HNK-1 immunoreactive O-glycans of the sulfoglucuronyl lactosamine series that terminate in 2-linked or 2,6-linked hexose (mannose).

The monoclonal antibody HNK-1 originally raised to an antigenic marker of natural killer cells also binds to selected regions in nervous tissue. The antigen is a carbohydrate that has attracted much interest as its expression is developmentally regulated in nervous tissue, and it is found, and proposed to be a ligand, on several of the adhesive glycoproteins of the nervous system. It is also expressed on glycolipids and proteoglycans, and is the target of monoclonal auto-antibodies that give rise to a demyelinating disease. The epitope, as characterized on glycolipids isolated from the nervous system, is expressed on 3-sulfated glucuronic acid joined by beta1-3-linkage to a neolacto backbone. Here we exploit the neoglycolipid technology, in conjunction with immunodetection and in situ liquid secondary ion mass spectrometry, to characterize HNK-1-positive oligosaccharide chains derived by reductive alkaline release from total brain glycopeptides. The immunoreactive oligosaccharides detected are tetra- to octasaccharides that are very minor components among a heterogeneous population, each representing less than 0.1% of the starting material. Their peripheral and backbone sequences resemble those of the HNK-1-positive glycolipids. An unexpected finding is that they terminate not with N-acetylgalactosaminitol but with hexitol (2-substituted and 2,6-disubstituted). In a tetrasaccharide investigated in the greatest detail, the hexitol is identified as 2-substituted mannitol.

The Le(x) carbohydrate sequence is recognized by antibody to L5, a functional antigen in early neural development.

The L5 antigenic determinant was previously suggested to be a carbohydrate epitope present on murine cell recognition molecules in the developing brain and to be an early neural marker in the chick embryo. Here, we show that L5 immunoreactivity is associated with complex-type N-glycosidic oligosaccharides. To identify the carbohydrate structure recognized by the L5 antibody, we investigate its binding to N-linked oligosaccharides derived from L5 glycoproteins and to known glycans. Results of mass spectrometric analyses of L5-positive neoglycolipids prepared from L5 glycoproteins are consistent with those for N-glycans containing a 3-fucosyl N-acetyllactosamine sequence. We also investigate L5 binding to structurally defined, lipid-linked oligosaccharides based on the blood group type I and II backbones. Chromatogram binding assays, ELISA, and inhibition studies show that the antibody reacts strongly with carbohydrate chains presenting the 3-fucosyl N-acetyllactosamine sequence [Lewisx (Le(x)) or X-hapten] also recognized by anti-SSEA-1 and anti-CD15. Histochemical studies with different antibodies recognizing the Lex sequence show partially overlapping patterns of immunoreactivity during early neural development in the chick embryo. Therefore, we suggest that the epitope recognized by L5 antibody is closely related to those for anti-SSEA-1 and anti-CD15.