Structural characterization of the N-linked oligosaccharides from tomato fruit.

The primary structures of the N-linked oligosaccharides from tomato fruit (Lycopersicon esculentum) have been elucidated. For the isolation of the protein fraction, two procedures were employed alternatively: a low temperature acetone powder method and ammonium sulfate precipitation of the tomato extract. After peptic digestion, the glycopeptides were purified by cation-exchange chromatography; the oligosaccharides were released by N-glycosidase A and fluorescently labelled with 2-aminopyridine. Structural characterization was accomplished by means of two-dimensional HPLC in combination with exoglycosidase digestions and MALDI-TOF mass spectrometry. Two varieties as well as two stages of ripening were investigated. In all the samples, the same sixteen N-glycosidic structures were detected; the two most abundant glycans showed identical properties to those of the major N-linked oligosaccharides of horseradish peroxidase and pineapple stem bromelain, respectively and accounted for about 65-78% of the total glycan amount; oligomannosidic glycans occurred only in small quantities (3-9%). The majority of the N-glycans were beta 1,2-xylosylated and carried an alpha 1,3-fucose residue linked to the terminal N-acetylglucosamine. This structural element contributes to cross-reactions among non-related glycoproteins and has been shown to be an IgE-reactive determinant (Tretter, Altmann, Kubelka, M?rz, & Becker, 1993). The presented study gives a possible structural explanation for reported immunological cross-reactivities between tomato and grass pollen extracts due to carbohydrate IgE epitopes (Petersen, Vieths, Aulepp, Schlaak, & Becker, 1996), thereby demonstrating the importance of the structural characterization of plant N-glycans for a more reliable interpretation of immunological data.     

Structural characterization of N-linked oligosaccharides on monoclonal antibody cetuximab by the combination of orthogonal matrix-assisted laser desorption/ionization hybrid quadrupole-quadrupole time-of-flight tandem mass spectrometry and sequential enzymatic digestion

Cetuximab is a novel therapeutic monoclonal antibody with two N-glycosylation sites: a conserved site in the CH2 domain and a second site within the framework 3 of the variable portion of the heavy chain. The detailed structures of these oligosaccharides were successfully characterized using orthogonal matrix-assisted laser desorption/ionization hybrid quadrupole-quadrupole time-of-flight mass spectrometry (oMALDI Qq-TOF MS) and tandem mass spectrometry (MS/MS) in combination with exoglycosidase digestion. The N-linked oligosaccharides were released by treatment with N-glycanase F, reductively aminated with anthranilic acid, and fractionated by normal phase high-performance liquid chromatography (NP-HPLC). The fluorescent-labeled oligosaccharide pool and fractions were analyzed by oMALDI Qq-TOF MS and MS/MS in negative ion mode. Each fraction was further digested with an array of exoglycosidase mixtures, and subsequent MALDI TOF MS analysis of the resulting products yielded information about structural features of the oligosaccharide. The combined data revealed the presence of 21 distinct oligosaccharide structures in cetuximab. These oligosaccharides differ mainly in degree of sialylation with N-glycolyl neuraminic acid and extent of galactosylation (zero-, mono-, di-, and alpha(1-3)-galactosidase). The individual oligosaccharides were further assigned to the specific sites in the Fab and Fc regions of the antibody. This study represents a unique approach in that MS/MS data were used to identify and confirm the oligosaccharide structures of a protein.     

Structural characterization of the N-linked oligosaccharides derived from HIVgp120 expressed in lepidopteran cells

The oligosaccharides of recombinant HIV gp120 expressed in lepidopteran Sf9 cells were analysed after hydrazine release by gel permeation and high pH anion exchange chromatography. N-Linked glycans were exclusively of the oligomannose series and no evidence for charged complex or hybrid type glycans was found. However a glycosylation reaction similar to those found in vertebrates was evident. The major glycoform of gp120, that comprised 30% of all the species analysed, was structurally identified by exoglycosidase digestion and found to be a core fucosylated structure, Man1,6(Man1,3)Man1,4GlcNAc(Fuc1,6)GlcNAc. Further confirmation of the ability of lepidopteran cells to fucosylate N-linked glycans was provided by an in vitro analysis of this reaction using authentic oligosaccharide substrates.     

Structural analysis of the N-linked oligosaccharides from murine glycophorin

Glycophorins, isolated from BALB/c mouse erythrocytes, were degraded under mild and strong reductive alkaline conditions and the N-linked oligosaccharides were isolated as alditols. The oligosaccharide alditols were fractionated and purified using gel filtration, concanavalin A-Sepharose affinity chromatography, and high-performance ion-exchange chromatography. Structural analysis was carried out by chemical analyses, periodate oxidation in combination with fast atom bombardment mass spectrometry, and 500-MHz 1H NMR spectroscopy. The results revealed the presence of sialylated biantennary, triantennary, and tetraantennary complex type oligosaccharides, all fucosylated at the innermost N-acetylglucosamine residue. The tri- and tetraantennary oligosaccharide-containing fractions also contained species elongated by one and/or two N-acetyllactosamine (-3Gal beta 1-4GlcNAc beta 1-) sequences. The N-linked oligosaccharides were shown to be combined only with one (the low molecular weight) of the two mouse glycophorins.     

Structural analysis of sulfated N-linked oligosaccharides in erythropoietin.

We previously demonstrated that high-performance liquid chromatography with electrospray ionization mass spectrometry (LC/MS) equipped with a graphitized carbon column (GCC) is useful for the structural analysis of carbohydrates in glycoproteins. Using LC/MS with GCC, sulfated N-linked oligosaccharides were found in erythropoietin (EPO) expressed in baby hamster kidney cells. Sulfation occurs in a part of the N-linked oligosaccharides in the EPO. Sulfated monosaccharide residue in the sulfated N-linked oligosaccharide was determined by exoglycosidase digestion followed by sugar mapping by LC/MS. The linkage position and branch-location of the sulfate group in the tetraantennary oligosaccharide were analyzed by (1)H-nuclear magnetic resonance. It was suggested that sulfation occurs on the C-6 position of GlcNAc located in the GlcNAcbeta1-4Manalpha1-3 branch.     

Structural characterization of the N-linked oligosaccharides in bile salt-stimulated lipase originated from human breast milk

The detailed structures of N- glycans derived from bile salt-stimulated lipase (BSSL) found in human milk were determined by combining exoglycosidase digestion with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The N- glycan structures were conclusively determined in terms of complexity and degree of fucosylation. Ion-exchange chromatography with pulsed amperometric detection, together with mass-spectral analysis of the esterified N- glycans, indicated the presence of monosialylated structures. The molecular mass profile of esterified N- glycans present in BSSL further permitted the more detailed studies through collision-induced dissociation (CID) and sequential exoglycosidase cleavages. The N- glycan structures were elucidated to be complex/dibranched, fucosylated/complex/dibranched, monosialylated/complex/dibranched, and monosialylated/fucosylated/dibranched entities.     

Structural characterization of the oligosaccharides of a human monoclonal anti-lipopolysaccharide immunoglobulin M

The oligosaccharide side chains of a human anti-lipopolysaccharide IgM produced by a human-human-mouse heterohybridoma were analyzed at each of its five conserved N-glycosylation sites. This antibody also has a potential sixth N-glycosylation site in the variable region of its heavy chain which is not glycosylated. The oligosaccharides were released by digestion with various endo- and exoglycosidases and analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and fluorophore-assisted carbohydrate electrophoresis. The antibody has various complex- and hybrid-type oligosaccharide structures at Asn 171, various sialylated complex-type oligosaccharides at Asn 332 and 395, and high-mannose-type oligosaccharides at Asn 402 and 563. Of note is the presence in this human IgM of oligosaccharides containing N-glycolylneuraminic acid and N-acetylneuraminic acid in the ratio of 98:2 as determined using anion- exchange chromatography. Furthermore, we observed oligosaccharide structures containing Gal alpha (1,3)Gal that have not been reported as components of human glycoproteins.      

Changes in N-linked oligosaccharides during seed development of Ginkgo biloba

Structural changes in N-linked oligosaccharides of glycoproteins during seed development of Ginkgo biloba have been explored to discover possible endogenous substrate(s) for the Ginko endo-beta-N-acetylglucosaminidase (endo-GB; Kimura, Y., et al. (1998) Biosci. Biotechnol. Biochem., 62, 253-261), which should be involved in the production of high-mannose type free N-glycans. The structural analysis of the pyridylaminated oligosaccharides with a 2D sugar chain map, by ESI-MS/MS spectroscopy, showed that all N-glycans expressed on glycoproteins through the developmental stage of the Ginkgo seeds have the xylose-containing type (GlcNAc2 approximately 0Man3Xyl1Fuc1 approximately 0GlcNAc2) but no high-mannose type structure. Man3Xyl1Fuc1GlcNAc2, a typical plant complex type structure especially found in vacuolar glycoproteins, was a dominant structure through the seed development, while the amount of expression of GlcNAc2Man3Xyl1Fuc1GlcNAc2 and GlcNAc1Man3Xyl1Fuc1GlcNAc2 decreased as the seeds developed. The dominantly occurrence of xylose-containing type structures and the absence of the high-mannose type structures on Ginkgo glycoproteins were also shown by lectin-blotting and immunoblotting of SDS-soluble glycoproteins extracted from the developing seeds at various developmental stages. Concerning the endogenous substrates for plant endo-beta-N-acetylglucosaminidase, these results suggested that the endogenous substrates might be the dolicol-oligosaccharide intermediates or some glycopeptides with the high-mannose type N-glycan(s) derived from misfolded glycoproteins in the quality control system for newly synthesized glycoproteins.     

Recovery and purification process development for monoclonal antibody production

Hundreds of therapeutic monoclonal antibodies (mAbs) are currently in development, and many companies have multiple antibodies in their pipelines. Current methodology used in recovery processes for these molecules are reviewed here. Basic unit operations such as harvest, Protein A affinity chromatography, and additional polishing steps are surveyed. Alternative processes such as flocculation, precipitation, and membrane chromatography are discussed. We also cover platform approaches to purification methods development, use of high throughput screening methods, and offer a view on future developments in purification methodology as applied to mAbs.     

Recovery and purification process development for monoclonal antibody production

Hundreds of therapeutic monoclonal antibodies (mAbs) are currently in development, and many companies have multiple antibodies in their pipelines. Current methodology used in recovery processes for these molecules are reviewed here. Basic unit operations such as harvest, Protein A affinity chromatography and additional polishing steps are surveyed. Alternative processes such as flocculation, precipitation and membrane chromatography are discussed. We also cover platform approaches to purification methods development, use of high throughput screening methods, and offer a view on future developments in purification methodology as applied to mAbs.Key words: monoclonal antibody, recovery, purification, chromatography, membrane, filtration, platform process     

Eliminating antibody polyreactivity through addition of N-linked glycosylation

Antibody polyreactivity can be an obstacle to translating a candidate antibody into a clinical product. Standard tests such as antibody binding to cardiolipin, HEp-2 cells, or nuclear antigens provide measures of polyreactivity, but its causes and the means to resolve are often unclear. Here we present a method for eliminating antibody polyreactivity through the computational design and genetic addition of N-linked glycosylation near known sites of polyreactivity. We used the HIV-1-neutralizing antibody, VRC07, as a test case, since efforts to increase VRC07 potency at three spatially distinct sites resulted in enhanced polyreactivity. The addition of N-linked glycans proximal to the polyreactivity-enhancing mutations at each of the spatially distinct sites resulted in reduced antibody polyreactivity as measured by (i) anti-cardiolipin ELISA, (ii) Luminex AtheNA Multi-Lyte ANA binding, and (iii) HEp-2 cell staining. The reduced polyreactivity trended with increased antibody concentration over time in mice, but not with improved overall protein stability as measured by differential scanning calorimetry. Moreover, glycan proximity to the site of polyreactivity appeared to be a critical factor. The results provide evidence that antibody polyreactivity can result from local, rather than global, features of an antibody and that addition of N-linked glycosylation can be an effective approach to reducing antibody polyreactivity.     

Structural and functional characterization of an agonistic anti-human EphA2 monoclonal antibody

We report here the three-dimensional structure of human ephrin type A receptor 2 (EphA2) bound to the Fab (fragment antigen binding) of an agonistic human antibody (1C1; IgG1/κ). The structure of the corresponding complex was solved at a resolution of 2.5 Å using molecular replacement and constitutes the first reported structure of a human ephrin receptor bound to an antibody. We have also defined the corresponding functional epitope using a mutagenesis-based approach. This study revealed discrete structural features that determine the fine specificity of 1C1 to EphA2. Our data also provided a molecular basis for 1C1 mechanism of action. More precisely, we propose that its agonistic, internalizing properties are the result of ligand mimicry by the third heavy-chain complementarity-determining region of 1C1. Because EphA2 is an important contributor to cancer formation and progression, these findings may have implications for designing the next generation of anti-tumor therapies.     

Preparation and characterization of monoclonal antibodies to an N-linked oligosaccharide

Two monoclonal antibodies to an N-linked oligosaccharide, MT-5 and MT-9, have been prepared by immunization with a pyridylaminated, asialylated, galactosylated, fucosylated, bisected biantennary sugar. The reactivity of these antibodies was monitored by their reaction with human asialoglycophorin in a solid-phase enzyme-linked immunosorbent assay. Both antibodies reacted with the sugar chains of various human glycoproteins such as immunoglobulin G, transferrin, gamma-glutamyl transpeptidase, alpha 1-acid glycoprotein, and alpha-fetoprotein. Treatment of asialoglycophorin with beta-N-acetylhexosaminidase or alpha-mannosidase resulted in reduction of the binding to these antibodies. The reactivity of MT-5 to asialoglycophorin was slightly inhibited by D-mannose and N-acetylglucosamine, whereas that of MT-9 was inhibited by D-mannose, N-acetyl-D-glucosamine, chitobiose, and L-fucose. The epitope specificity of MT-5 appears to be a sugar chain containing biantennary N-acetyl-D-glucosamine residues, the bisected N-acetyl-D-glucosamine residue, and a trimannosyl core. The epitope to which MT-9 is directed may be a complex made up of beta-mannose, chitobiose, and L-fucose. These studies indicate that immunization with pyridylaminated sugars can produce antibodies that recognize N-linked oligosaccharides. Monoclonal/polyclonal antibodies to the N-linked sugar chains of glycopeptides would be useful in such studies of proteins.     

An analytical system for the characterization of highly heterogeneous mixtures of N-linked oligosaccharides

A novel system for characterizing complex N-linked oligosaccharide mixtures that uses a combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), capillary electrophoresis (CE), and high-performance liquid chromatography (HPLC) has been developed. In this study, oligosaccharides released from recombinant TNK-tPA (tissue plasminogen activator) were derivatized with 5-amino-2-naphthalenesulfonic acid (ANSA). The negative charge imparted by the ANSA label facilitated the analysis of the oligosaccharides by MALDI-TOF MS by allowing the observation of both neutral and sialylated oligosaccharides in a single negative ion mode spectrum. Labeling with ANSA was also determined to be advantageous in the characterization of oligosaccharides by both HPLC and CE. The ANSA label was demonstrated to provide superior resolution over the commonly used label 8-aminopyrene-1,3,6-trisulfonic acid (APTS) in both the CE and HPLC analysis of oligosaccharides. To date, no other labels that enable the analysis of complex oligosaccharide mixtures in a single mass spectral mode, while also enabling high-resolution chromatographic and electrophoretic separation of the oligosaccharides, have been reported. By integrating the structural information obtained by MALDI-TOF MS analysis with the ability of CE and HPLC to discriminate between structural isomers, the complete characterization of complex oligosaccharide mixtures is possible.     

Sequence determination for N-linked oligosaccharides through the use of the nuclear Overhauser effect

A nuclear magnetic resonance technique, the nuclear Overhauser effect, has been used to confirm the presence of Man alpha 1-2, GlcNAc beta 1-2, and Man alpha 1-3 residues and to establish the branching pattern and sequences in four classes of N-linked oligosaccharides. This method offers a rapid and nondestructive approach to the elucidation of sequences in carbohydrate chains, in contrast to enzymatic and chemical methods.     

Glycosylated major urinary protein of the house mouse: characterization of its N-linked oligosaccharides

A minor component of the major urinary protein complex of the house mouse was chromatographically isolated and ascertained to be a previously suspected glycoprotein. Using highly sensitive mass-spectrometric techniques for sequencing and linkage analysis, the N-linked oligosaccharides of this glycoprotein were characterized. They were determined to be of the complex type with a wide heterogeneity. The heterogeneity was due to both the degree of sialylation and the presence of galactose residues in either beta(1-3) or beta(1-4) linkages. The biantennary structures were the most pronounced glycans, while tri- and tetraantennary entities were minor.     

Structural characterization of N-linked oligosaccharides of laminin from rat kidney: changes during diabetes and modulation by dietary fiber and butyric acid

Carbohydrates of laminin, a family of large multidomain glycoproteins, have been implicated in various cellular activities including maintaining the protein structure, its function and also basement membrane integrity. During the course of our investigation, we observed that purified laminin from kidneys of control, diabetic, and dietary fiber- and butyric acid-treated diabetic rats showed differences in binding to extracellular matrix components. This prompted us to determine whether there are structural changes in laminin oligosaccharides. In this study, we have characterized a few major N-linked oligosaccharides isolated from purified laminin in various experimental groups, viz. normal, diabetic and diabetic rats fed with dietary fiber and butyric acid. Sugar composition, as identified by GLC, revealed the presence of mannose, galactose and N-acetylglucosamine. In order to study fine structures of the oligosaccharides, N-linked oligosaccharides of laminin were released by Peptide-N-glycosidase F digestion, end-labeled with 2-anthranilic acid and fractionated by lectin affinity chromatography. Furthermore, structural elucidation carried out by MALDI-TOF MS/MS analysis showed variations in the oligosaccharide sequence of laminin during diabetes which were altered by the feeding of dietary fiber and butyric acid.     

Structural study of N-linked oligosaccharides of human intercellular adhesion molecule-3 (CD50).

The N-linked oligosaccharides were released from purified human intercellular adhesion molecule (ICAM)-3 by hydrazinolysis. Approximately 6 mol of oligosaccharides were released from 1 mol of ICAM-3. The oligosaccharides reduced with NaB[3H]4 were separated into neutral and acidic fractions by paper electrophoresis. Most of the acidic oligosaccharides were converted to neutral ones by digestion with sialidase, indicating that they are sialyl derivatives. The neutral and sialidase-treated acidic oligosaccharides were fractionated by serial lectin column chromatography followed by Bio-Gel P-4 column chromatography. Structural studies of each oligosaccharide by sequential exo- and endo-glycosidase digestion and by methylation analysis revealed that N-linked oligosaccharides of ICAM-3 are mainly of tri- and tetra-antennary complex-type, about 60% of which contain two to three poly N-acetyllactosamine chains terminated with the type 1 structure and those without the type 1 structure per oligosaccharide. In addition, a small amount of the high mannose-type oligosaccharide with six alpha-mannose residues, which could act as a ligand for the dendritic cell-specific ICAM-3 grabbing nonintegrin, was detected.