Nonreducing Terminal Galactose Research Articles

Raffinose degradation-related gene GhAGAL3 was screened out responding to salinity stress through expression patterns of GhAGALs family genes.

A-galactosidases (AGALs), the oligosaccharide (RFO) catabolic genes of the raffinose family, play crucial roles in plant growth and development and in adversity stress. They can break down the non-reducing terminal galactose residues of glycolipids and sugar chains. In this study, the whole genome of AGALs was analyzed. Bioinformatics analysis was conducted to analyze members of the AGAL family in Gossypium hirsutum, Gossypium arboreum, Gossypium barbadense, and Gossypium raimondii. Meanwhile, RT-qPCR was carried out to analyze the expression patterns of AGAL family members in different tissues of terrestrial cotton. It was found that a series of environmental factors stimulated the expression of the GhAGAL3 gene. The function of GhAGAL3 was verified through virus-induced gene silencing (VIGS). As a result, GhAGAL3 gene silencing resulted in milder wilting of seedlings than the controls, and a significant increase in the raffinose content in cotton, indicating that GhAGAL3 responded to NaCl stress. The increase in raffinose content improved the tolerance of cotton. Findings in this study lay an important foundation for further research on the role of the GhAGAL3 gene family in the molecular mechanism of abiotic stress resistance in cotton.

Practical Use of Analytical Tools for Glycan Structures Related to Functionality and Safety of Biopharmaceuticals

It is known that glycosylation of biopharmaceuticals relates to drug potency and half-life in blood. Since glycan modification is essential for the effector effect in antibody drugs, biopharmaceuticals are produced by using biosynthesis of mammalian cells but not microorganisms like as Escherichia coli. In particular, a biantennary N-glycan attached on Fc region of antibody is an important factor for interaction with its receptor FcRIIIa, and lack of a core-fucose residue attached to innermost GlcNAc at reducing end of N-glycan dramatically enhances interaction between FcRIIIa and IgG. Recently, it is reported that a particular galactose residue at non-reducing end of biantennary N-glycan also promotes the interaction with FcRIIIa receptor. On the other hand, glycan structure of post-translational modification by the use of mammalian-derived cells for production of biopharmaceuticals is different from that of human in some cases, and non-human type glycans is observed sometimes. For instance, high-mannose N-glycan is observed in unpreferable process step of manufacturing production and non-human type glycans are found depend on host cell type. Antigenicity of α1,3galactose (α1,3Gal) antigen and N-glycolylneuraminic acid (NeuGc) are observed at non-human type glycans well-known as xenoantigen. Because human lacks the enzyme activities of α1,3Gal and NeuGc synthesis for during the process of evolution with acquired gene mutations, immune response may act as a exogeneous antigen when they are injected to human patients. Therefore, contamination of antigenic glycosylation is considered to affect the safety of biopharmaceuticals. Currently, general standards have not been established regarding methods for glycan analysis from the viewpoint of the functionality and safety of biopharmaceuticals. Furthermore, it seems that the comprehensive supply system of glycan standards and recognition probes is still partial. We will report our developed tools regarding glycan standards and recognition probes. The glycan standards are available for quantitative analysis of reducing terminal core fucose and non-reducing terminal galactose (G2 and 6-G1 glycans) to evaluate the effector function of antibody drugs. The recognition probes derived from chicken are expected to be applied for qualitative analysis of an allergy risk factor caused by xenoantigen (α1,3Gal and NeuGc epitopes). The tools developed in this study would contribute to the analysis of glycan structures that affect the quality of biopharmaceuticals.

NMR assignments of the N-glycans of the Fc fragment of mouse immunoglobulin G2b glycoprotein.

The Fc portion of immunoglobulin G (IgG) promotes defensive effector functions in the immune system by interacting with Fcγ receptors and complement component C1q. These interactions critically depend on N-glycosylation at Asn297 of each CH2 domain, where biantennary complex-type oligosaccharides contain microheterogeneities resulting primarily from the presence or absence of non-reducing terminal galactose residues. Crystal structures of Fc have shown that a pair of N-glycans is located between the two CH2 domains. Here we applied our metabolic isotope labeling technique using mammalian cells for in-solution structural characterization of mouse IgG2b-Fc glycoforms with a molecular mass of 54kDa. Based on spectral assignments of the N-glycans as well as polypeptide backbones of Fc, we probed conformational perturbations of Fc induced by N-glycan trimming, especially enzymatic degalactosylation. The results indicated that degalactosylation structurally perturbed the Fc region through rearrangement of glycan-protein interactions. The spectral assignments of IgG2b-Fc glycoprotein will provide the basis for NMR investigation of its dynamic conformations and interactions with effector molecules in solution.

Remodeling of the Oligosaccharide Conformational Space in the Prebound State To Improve Lectin-Binding Affinity.

We developed an approach to improve the lectin-binding affinity of an oligosaccharide by remodeling its conformational space in the precomplexed state. To develop this approach, we used a Lewis X-containing oligosaccharide interacting with RSL as a model system. Using an experimentally validated molecular dynamics simulation, we designed a Lewis X analogue with an increased population of conformational species that were originally very minor but exclusively accessible to the target lectin without steric hindrance by modifying the nonreducing terminal galactose, which does not directly contact the lectin in the complex. This Lewis X mimetic showed 17 times higher affinity for the lectin than the native counterpart. Our approach, complementing the lectin-bound-state optimizations, offers an alternative strategy to create high-affinity oligosaccharides by increasing populations of on-pathway metastable conformers.

Effects of amino acid substitutions on the biological activity of anti-CD20 monoclonal antibody produced by transgenic silkworms (Bombyx mori)

Recombinant monoclonal antibodies (mAbs) have been used in various therapeutic applications including cancer therapy. Fc-mediated effector functions play a pivotal role in the tumor-killing activities of some tumor-targeting mAbs, and Fc-engineering technologies with glyco-engineering or amino acid substitutions at the antibody Fc region have been used to enhance cytotoxic activities including antibody-dependent cellular cytotoxicity (ADCC). We previously reported that the mAbs produced using transgenic silkworms showed stronger ADCC activity and lower complement-dependent cytotoxicity (CDC) activity than mAbs derived from Chinese hamster ovary (CHO) cells due to their unique N-glycan structure (lack of core-fucose and non-reducing terminal galactose). In this study, we generated anti-CD20 mAbs with amino acid substitutions using transgenic silkworms and analyzed their biological activities to assess the effect of the combination of glyco-engineering and amino acid substitutions on the Fc-mediated function of mAbs. Three types of amino acid substitutions at the Fc region (G236A/S239D/I332E, L234A/L235A, and K326W/E333S) modified the Fc-mediated biological activities of silkworm-derived mAbs as in the case of CHO-derived mAbs, resulting in the generation of Fc-engineered mAbs with characteristic Fc-mediated functions. The combination of amino acid substitutions at the Fc region and glyco-engineering using transgenic silkworm made it possible to generate Fc-engineered mAbs with suitable Fc-mediated biological functions depending on the pharmacological mechanism of their actions. Transgenic silkworms were shown to be a promising system for the production of Fc-engineered mAbs.

Structural characterization and anticoagulant activity of a sulfated polysaccharide from the green alga Codium divaricatum

A sulfated polysaccharide, designated CP2-1, was isolated from the green alga Codium divaricatum by water extraction and purified by anion-exchange and size-exclusion chromatography. CP2-1 is a galactan which is highly sulfated and substituted with pyruvic acid ketals. On the basis of chemical and spectroscopic analyses, the backbone of CP2-1 was mainly composed of (1→3)-β-d-galactopyranose residues, branched by single (1→)-β-d-galactopyranose units attached to the main chain at C-4 positions. The degree of branching was estimated to be about 12.2%. Sulfate groups were at C-4 of (1→3)-β-d-galactopyranose and C-6 of non-reducing terminal galactose residues. In addition, the ketals of pyruvic acid were found at 3,4- of non-reducing terminal galactose residues forming a five-membered ring. CP2-1 possessed a high anticoagulant activity as assessed by the activated partial thromboplastin time and thrombin time assays. The investigation demonstrated that CP2-1 was an anticoagulant-active sulfated polysaccharide distinguishing from other sulfated polysaccharides from marine green algae.

Human Lipooligosaccharide IGG That Prevents Endemic Meningococcal Disease Recognizes an Internal Lacto-N-neotetraose Structure

Antibodies that initiate complement-mediated killing of Neisseria meningitidis as they enter the bloodstream from the oropharynx protect against disseminated disease. Human IgGs that bind the neisserial L7 lipooligosaccharide (LOS) are bactericidal for L3,7 and L2,4 meningococci in the presence of human complement. These strains share a lacto-N-neotetraose (nLc4) LOS α chain. We used a set of mutants that have successive saccharide deletions from the nLc4 α chain to characterize further the binding and bactericidal activity of nLc4 LOS IgG. We found that the nLc4 α chain conforms at least four different antigens. We separately purified IgG that required the nLc4 (non-reducing) terminal galactose (Gal) for binding and IgG that bound the truncated nLc3 α chain that lacks this Gal residue. IgG that bound the internal nLc3 α chain killed both L3,7 and L2,4 strains, whereas IgG that required the nLc4 terminal Gal residue for binding killed L2,4 stains but not L3,7 strains. These results show that the diversity of LOS antibodies in human serum is as much a function of the conformation of multiple antigens by a single glycoform as of the production of multiple glycoforms. Differences in sensitivity to killing by human nLc4 LOS IgG may account for the fact that fully two-thirds of endemic group B meningococcal disease in infants and children is caused by L3,7 strains, but only 20% is caused by L2,4 stains.

The DC2.3 Gene in Caenorhabditis elegans Encodes a Galectin That Recognizes the Galactose.BETA.1.RAR.4Fucose Disaccharide Unit

Galectins comprise a large family of β-galactoside-binding proteins in animals and fungi. We previously isolated cDNAs of 10 galectin and galectin-like genes (lec-1 to lec-6 and lec-8 to lec-11) from Caenorhabditis elegans and characterized the carbohydrate-binding properties of their recombinant proteins. In the present study, we isolated cDNA corresponding to an open reading frame of the DC2.3a gene from C. elegans total RNA; this cDNA encodes another potential galectin. A recombinant DC2.3a protein was expressed in Escherichia coli and used for analysis. The protein displayed hemagglutinating activity against rabbit erythrocytes, bound to an asialofetuin-Sepharose column, and was eluted with lactose. Furthermore, frontal affinity chromatography (FAC) analysis confirmed that DC2.3a recognized oligosaccharides with a non-reducing terminal galactose. According to these results, we designated DC2.3 as lec-12. The carbohydrate-binding property of the recombinant DC2.3a/LEC-12a was essentially similar to that of LEC-6. Additionally, DC2.3a/LEC-12a and LEC-6 showed higher affinities for the galactoseβ1→4fucose (Galβ1→4Fuc) disaccharide than for N-acetyllactosamine. This suggests that the principal recognition unit is the Galβ1→4Fuc disaccharide as in the case of the C. elegans galectins. However, the recombinant DC2.3a/LEC-12a showed weak affinity for N-glycan E3, which was previously shown to be a preferential endogenous ligand for LEC-6. The DC2.3a/LEC-12a endogenous ligand structures appear to be somewhat different but contain the same galactose-fucose recognition motif.

Structural Basis for Langerin Recognition of Diverse Pathogen and Mammalian Glycans through a Single Binding Site

Langerin mediates the carbohydrate-dependent uptake of pathogens by Langerhans cells in the first step of antigen presentation to the adaptive immune system. Langerin binds to an unusually diverse number of endogenous and pathogenic cell surface carbohydrates, including mannose-containing O-specific polysaccharides derived from bacterial lipopolysaccharides identified here by probing a microarray of bacterial polysaccharides. Crystal structures of the carbohydrate-recognition domain from human langerin bound to a series of oligomannose compounds, the blood group B antigen, and a fragment of β-glucan reveal binding to mannose, fucose, and glucose residues by Ca2+ coordination of vicinal hydroxyl groups with similar stereochemistry. Oligomannose compounds bind through a single mannose residue, with no other mannose residues contacting the protein directly. There is no evidence for a second Ca2+-independent binding site. Likewise, a β-glucan fragment, Glcβ1–3Glcβ1–3Glc, binds to langerin through the interaction of a single glucose residue with the Ca2+ site. The fucose moiety of the blood group B trisaccharide Galα1–3(Fucα1–2)Gal also binds to the Ca2+ site, and selective binding to this glycan compared to other fucose-containing oligosaccharides results from additional favorable interactions of the nonreducing terminal galactose, as well as of the fucose residue. Surprisingly, the equatorial 3-OH group and the axial 4-OH group of the galactose residue in 6SO4–Galβ1–4GlcNAc also coordinate Ca2+, a heretofore unobserved mode of galactose binding in a C-type carbohydrate-recognition domain bearing the Glu-Pro-Asn signature motif characteristic of mannose binding sites. Salt bridges between the sulfate group and two lysine residues appear to compensate for the nonoptimal binding of galactose at this site.

Structure of a SusD homologue, BT1043, involved in mucin O-glycan utilization in a prominent human gut symbiont.

Mammalian distal gut bacteria have an expanded capacity to utilize glycans. In the absence of dietary sources, some species rely on host-derived mucosal glycans. The ability of Bacteroides thetaiotaomicron, a prominent human gut symbiont, to forage host glycans contributes to both its ability to persist within an individual host and its ability to be transmitted naturally to new hosts at birth. The molecular basis of host glycan recognition by this species is still unknown but likely occurs through an expanded suite of outermembrane glycan-binding proteins that are the primary interface between B. thetaiotaomicron and its environment. Presented here is the atomic structure of the B. thetaiotaomicron protein BT1043, an outer membrane lipoprotein involved in host glycan metabolism. Despite a lack of detectable amino acid sequence similarity, BT1043 is a structural homologue of the B. thetaiotaomicron starch-binding protein SusD. Both structures are dominated by tetratrico peptide repeats that may facilitate association with outer membrane beta-barrel transporters required for glycan uptake. The structure of BT1043 complexed with N-acetyllactosamine reveals that recognition is mediated via hydrogen bonding interactions with the reducing end of beta-N-acetylglucosamine, suggesting a role in binding glycans liberated from the mucin polypeptide. This is in contrast to CBM 32 family members that target the terminal nonreducing galactose residue of mucin glycans. The highly articulated glycan-binding pocket of BT1043 suggests that binding of ligands to BT1043 relies more upon interactions with the composite sugar residues than upon overall ligand conformation as previously observed for SusD. The diversity in amino acid sequence level likely reflects early divergence from a common ancestor, while the unique and conserved alpha-helical fold the SusD family suggests a similar function in glycan uptake.

Isolation of Sulfated Galactan from Codium fragile and Its Antiviral Effect

A sulfated galactan (FG) was isolated from the green alga, Codium fragile. Chemical analysis revealed that FG mainly consisted of D-galactose with pyruvic acid (12.3%) and sulfate (11.0%). Methylation and NMR analyses showed that FG was composed of -->3)-beta-D-Galp-(1-->, beta-D-Galp-(1--> and -->3,6)-beta-D-Galp-(1--> residue. In addition, pyruvic acid was suggested to be present as (1'-carboxy)-ethylidene cyclic ketal at O-3 and O-4 of non-reducing terminal galactose residues, whereas sulfate was substituted at O-4 of other galactose residues. When the antiviral potency of FG was evaluated, it inhibited the replication of herpes simplex virus type 2 (HSV-2), and the mechanism of action was suggested to be interference in the early steps such as virus adsorption to and penetration into host cells. Furthermore, it was shown that FG directly reduced virus infection rates. In a genital infection model using HSV-2-infected mice, FG improved mortality and lesion scores and reduced virus yields by intravaginal administration. These results suggest that FG might be a potent candidate as a prophylactic agent for HSV-2 infection.

Diverse Sugar-Binding Specificities of Marine Invertebrate C-Type Lectins

The sugar-binding specificities of C-type lectins isolated from marine invertebrates were investigated by frontal affinity chromatography (FAC) using 100 oligosaccharides. The lectins included BRA-2 and BRA-3, multiple lectins from the hemolymph of the acorn barnacle, Megabalanus rosa, and BRL from the acorn barnacle, Balanus rostatus. The diverse sugar-binding specificities of the C-type lectins were determined by FAC analysis. BRA-2 recognized alpha2-6 sialylation but not alpha2-3 sialylation on glycans. On the other hand, BRA-3 showed high affinity for oligosaccharides with alpha-linked non-reducing terminal galactose, but not for sialylated forms, and BRL showed enhanced recognition activity towards Lewis(x) and Lewis(a) epitopes.

Structure of a highly pyruvylated galactan sulfate from the Pacific green alga Codium yezoense (Bryopsidales, Chlorophyta)

A polysaccharide fraction consisting of d-galactose, sulfate, and pyruvate in a molar proportion of 4:2:1 was isolated from the green seaweed Codium yezoense by water extraction followed by ion-exchange chromatography. To elucidate its structure, modified polysaccharides were prepared by desulfation, depyruvylation, and by total removal of non-carbohydrate substituents. Structures of the native polysaccharide and of the products of its chemical modifications were investigated by methylation analysis as well as by 1D and 2D 1H and 13C NMR spectroscopy. The polysaccharide devoid of sulfate and pyruvate was subjected to two subsequent Smith degradations to afford a rather low-molecular and essentially linear (1→3)-β- d-galactan. A highly ramified structure was suggested for the native polysaccharide, which contains linear backbone segments of 3-linked β- d-galactopyranose residues connected by (1→6) linkages, about 40% of 3-linked residues being additionally substituted at C-6, probably by short oligosaccharide residues also containing (1→3) and (1→6) linkages. Sulfate groups were found mainly at C-4 and in minor amounts at C-6. Pyruvate was found to form mainly five-membered cyclic ketals with O-3 and O-4 of the non-reducing terminal galactose residues. The minor part of pyruvate forms six-membered cyclic ketals with O-4 and O-6. The absolute configurations of ketals ( R for six-membered ketals and S for five-membered ones) were established using NMR spectral data.

Discrimination of Isomeric Fragment Ions Observed in Tandem Mass Spectra of Biantennary Oligosaccharides by Use of Selective Isotope Labeling

We report herein the first mass spectrometric analyses of isotopomers of a pyridylamino (PA)-derivatized biantennary oligosaccharide, in which one of two non-reducing terminal galactose residues is selectively enriched in 13C by an enzymatic reaction. By use of these isotopomeric PA-oligosaccharides, individual fragment ions observed in tandem mass spectra of MALDI-QIT-TOF-MS were unambiguously assigned. It was shown that the oligosaccharide ion that lacks the N-acetyllactosamine previously linked to the Manα1 →6 antenna dominates its isomeric counterpart ion. We propose that tandem mass spectrometric analyses of oligosaccharides isotopically labeled at a selected antenna would provide us with the basis of mechanisms of formation of glycosidic linkage fragment ions.

Intact cell adhesion to glycan microarrays

A rapid and reproducible method was developed to detect and quantify carbohydrate-mediated cell adhesion to glycans arrayed on glass slides. Monosaccharides and oligosaccharides were covalently attached to glass slides in 1.7-mm-diameter spots (200 spots/slide) separated by a Teflon gasket. Primary chicken hepatocytes, which constitutively express a C-type lectin that binds to nonreducing terminal N-acetylglucosamine residues, were labeled with a fluorescent dye and incubated in 1.3-microL aliquots on the glycosylated spots. After incubating to allow cell adhesion, nonadherent cells were removed by immersing the slide in phosphate buffered saline, inverting, and centrifuging in a sealed custom acrylic chamber so that cells on the derivatized spots were subjected to a uniform and controlled centrifugal detachment force while avoiding an air-liquid interface. After centrifugation, adherent cells were fixed in place and detected by fluorescent imaging. Chicken hepatocytes bound to nonreducing terminal GlcNAc residues in different linkages and orientations but not to nonreducing terminal galactose or N-acetylgalactosamine residues. Addition of soluble GlcNAc (but not Gal) prior to incubation reduced cell adhesion to background levels. Extension of the method to CD4+ human T-cells on a 45-glycan diversity array revealed specific adhesion to the sialyl Lewis x structure. The described method is a robust approach to quantify selective cell adhesion using a wide variety of glycans and may contribute to the repertoire of tools for the study of glycomics.

Crystal Structure of the Carbohydrate Recognition Domain of the H1 Subunit of the Asialoglycoprotein Receptor

The human asialoglycoprotein receptor (ASGPR), also called hepatic lectin, is an integral membrane protein and is responsible for the clearance of desialylated, galactose-terminal glycoproteins from the circulation by receptor-mediated endocytosis. It can be subdivided into four functional domains: the cytosolic domain, the transmembrane domain, the stalk and the carbohydrate recognition domain (CRD). The galactose-binding domains belong to the superfamily of C-type (calcium-dependent) lectins, in particular to the long-form subfamily with three conserved intramolecular disulphide bonds. It is able to bind terminal non-reducing galactose residues and N-acetyl-galactosamine residues of desialated tri or tetra-antennary N-linked glycans. The ASGPR is a potential liver-specific receptor for hepatitis B virus and Marburg virus and has been used to target exogenous molecules specifically to hepatocytes for diagnostic and therapeutic purposes. Here, we present the X-ray crystal structure of the carbohydrate recognition domain of the major subunit H1 at 2.3 Å resolution. While the overall fold of this and other known C-type lectin structures are well conserved, the positions of the bound calcium ions are not, indicating that the fold is stabilised by alternative mechanisms in different branches of the C-type lectin family. It is the first CRD structure where three calcium ions form an intergral part of the structure. In addition, the structure provides direct confirmation for the conversion of the ligand-binding site of the mannose-binding protein to an asialoglycoprotein receptor-like specificity suggested by Drickamer and colleagues. In agreement with the prediction that the coiled-coil domain of the ASGPR is separated from the CRD and its N-terminal disulphide bridge by several residues, these residues are indeed not α-helical, while in tetranectin they form an α-helical coiled-coil.

Naturally occurring anti-alpha-galactosyl antibodies: relationship to xenoreactive anti-alpha-galactosyl antibodies.

Antibodies produced by an individual without a known history of sensitization to the relevant antigen are called "natural" antibodies. Some natural antibodies, called xenoreactive antibodies, react with the cells of foreign species. Most xenoreactive antibodies in humans and higher primates bind to a nonreducing terminal galactose expressed by pigs and other lower mammals. Although human natural antibodies which bind to one or more of a variety of terminal alpha-galactosyl structures have been identified previously, the antigen recognized by anti-alpha-galactosyl antibodies on the cells of foreign species is thought to be exclusively Galalpha1-3Gal. Thus, anti-alpha-galactosyl antibodies which do not react with Galalpha1-3Gal are thought to be nonxenoreactive. Here, we identify natural antibodies in human serum which bind to Galalpha1-6Hexosepyrranosides but not Galalpha1-3Gal, indicating that these antibodies are not xenoreactive. Various lower mammals were found to have natural anti-Galalpha1-2Gal antibodies in their sera, suggesting that at least some anti-Galalpha1-2Gal antibodies might not be xenoreactive and indicating, surprisingly, that anti-alpha-galactosyl antibodies are much more phylogenetically disperse than previously known. Also surprising was the finding that some natural antibodies which bind to Galalpha1-3Gal in vitro do not bind to porcine xenografts. These studies show that naturally occurring anti-alpha-galactosyl antibodies in mammalian serum include antibodies with a greater variety of reactivities than previously thought, only some of which would bind to a porcine xenograft. Further, these studies show that the methods used to detect anti-alpha-galactosyl antibodies of relevance in xenotransplantation must be carefully evaluated to avoid detection of anti-alpha-galactosyl antibodies which would not bind to a porcine organ and which therefore are not involved in xenograft rejection.

Glycosidase-catalysed synthesis of α-galactosyl epitopes important in xenotransplantation and toxin binding using the α-galactosidase from Penicillium multicolor

The α-galactosidase from Penicillium multicolor catalyses highly regioselective galactosyl transfer on to mono- and di-saccharide acceptors that have a non-reducing terminal galactose unit to give products containing the α-D-Galp-(1→3)-D-Galp epitope found on pig tissue and which is responsible for the hyperacute rejection response in xenotransplantation of pig organs into man.

Structural study of N-linked sugar chains of sheep erythrocyte membrane glycoproteins.

Our previous study showed that non-reducing terminal galactose residues of N-linked sugar chains present in sheep erythrocyte membrane glycoproteins are important for rosette formation with T lymphoblastic cells [Ogasawara et al. (1995) Immunol Lett 48: 35-38]. As a first step to elucidate the significant structures of sugar chains involved in rosette formation, we analysed N-linked sugar chains released from the membrane glycoproteins by hydrazinolysis. The oligosaccharides were labeled with NaB3H4 and fractionated using columns of Aleuria aurantia lectin-Sepharose, MonoQ and Bio-Gel P-4. Structural analyses of oligosaccharides by sequential exoglycosidase digestion in combination with methylation analysis revealed that the membrane glycoproteins contain bi- (19%), tri- (33%), and tetraantennary (44%) complex-type oligosaccharides and that the oligosaccharides having exposed galactose residues amount to 40% of the total.

Characterization of Monoclonal Antibody Glycosylation: Comparison of Expression Systems and Identification of Terminal α-Linked Galactose

CAMPATH-1H is a recombinant humanized murine monoclonal immunoglobulin (IgG1) which recognizes the CDw52 antigen of human lymphocytes, and has been the subject of clinical trials for the treatment of non-Hodgkin's lymphoma and rheumatoid arthritis. Peptide mapping by liquid chromatography–mass spectrometry was used to confirm the predicted amino acid sequences and profile glycosylation for two CAMPATH isotypes expressed in a murine myeloma cell line (NS0) and a single isotype expressed in both Chinese hamster ovary (CHO) and NS0 lines. The three major glycoforms identified in CAMPATH are fucosylated biantennary structures, containing zero, one, or two galactose residues. Glycosylation of the IgG1form of CAMPATH expressed in CHO cells is consistent with normal human IgG. However, IgG1and IgG4expressed in NS0 cells include two potentially immunogenic glycoforms which contain either one or two nonreducing terminal α-linked galactose residues. Oligosaccharide structures were characterized by a combination of tandem mass spectrometry, methylation analysis, and exoglycosidase digestion. The strategy used here is designed to be widely applicable to recombinant glycoproteins.