Structural basis of the heterogeneity of asparagine-linked oligosaccharides of a Waldenstrom's macroglobulinemia immunoglobulin M

The extent of glycans heterogeneity in a pathological human immunoglobulin M ZAJ has been studied on oligosaccharides released by hydrazinolysis from the purified glycoprotein. After reduction with NaB3H4, asparagine-linked carbohydrate chains were separated by affinity chromatography on concanavalin A-Sepharose into oligomannosidic and N-acetyllactosaminic types. Glycans of the oligomannosidic type were further fractionated by HPLC and those of the N-acetyllactosamine type by preparative high-voltage electrophoresis. The primary structure of the main oligosaccharides was investigated on the basis of micro-methylation analysis, mass spectrometry and sequential exo-glycosidase digestion. Glycans of the oligomannosidic type varied in size from Man5GlcNAc2 to Man9GlcNAc2. N-Acetyllactosaminic glycans were found of the biantennary, bisected-biantennary and triantennary types. They presented a higher degree of heterogeneity due to the presence of a variable number of NeuAc and fucose residues. The new structures we report here were in addition to the major biantennary one we previously described on the basis of methylation analysis and 500 MHz 1H-NMR spectroscopy (Cahour, A., Debeire, P., Hartmann, L., Montreuil, J., Van Halbeek, H. and Vliegenthart, J.F.G. (1984) FEBS Lett. 170, 343-349): NeuAc(alpha 2-6)Gal(beta 1-4)GlcNAc(beta 1-2)Man(alpha 1-3)[Gal(beta 1-4)Glc-NAc(beta 1-2)Man(alpha 1-6)]Man(beta 1-4)]Glc-NAc(beta 1-4) [Fuc(alpha 1-6)]GlcNAc.     

Structural characterization of N-linked oligosaccharides on monoclonal antibody Nimotuzumab through process development

Nimotuzumab (TheraCIM, CIMAher, h-R3, humanized anti-EGF-R antibody), monoclonal antibody (mAb) manufactured at the Center of Molecular Immunology (Havana, Cuba) is currently being tested in several clinical trials. Nimotuzumab has a single N-glycosylation site in the Fc-CH2 fragment but no N-glycosylation site in the Fab region. The current study reports the full characterization of the mAb N-glycosylation and the consistency observed in several production batches from a perfusion mode culturing system that lasted between 68 and 150 days. It confirms that the N-glycan structures of Nimotuzumab expressed in the NS0 murine myeloma cell line are of the murine type. They consist mainly of fucosylated G0, G1 and G2 oligosaccharides, which are normally found in the CH2 region of IgG. Other minor species found were high mannose and sialylated structures. A small portion of the glycans were sialylated (～12%) and the only type of sialic acid detected was N-glycolyl-sialic acid, α2,6-linked to Gal. No Galα1-3Gal moieties were detected.     

Structural changes of immunoglobulin G oligosaccharides with age in healthy human serum

Age-related changes of IgG N-linked oligosaccharides isolated from normal human serum are reported for 403 individuals (male 227 and female 176), varying in age from 0 to 85 years. The IgG N-linked oligosaccharides were released from the protein by digestion with a glycoamidase and reductively aminated with the fluorescent reagent, 2-aminopyridine. The mixture of pyridylaminated oligosaccharides was separated at high resolution by HPLC using a reverse-phase column. From the results of neutral oligosaccharide analysis, agalactosyl glycoform and bisecting GlcNAc-containing glycoform were shown to increase with increasing age. Spearman's correlation coefficients were 0.503 and 0.473, respectively. Thus, in healthy people, an increase of both types of glycoforms correlates weakly with age. In addition, differences were demonstrated between male and female groups in their twenties. The quantity of agalactosyl glycoform was found to be lower in females than in males. No significant differences, however, were observed in the quantity of bisecting GlcNAc-containing glycoforms between males and females. Abbreviations: Gal, D-galactose: GlcNAc, N-acetyl-D-glucosamine; Man, D-mannose; Fc, C-terminal half of the heavy chain dimers of IgG; HPLC, high-performance liquid chromatography; IgG, immunoglobulin G; ODS, octadecylsilyl; PA, pyridylamino This revised version was published online in November 2006 with corrections to the Cover Date.     

Structural characterization of neutral oligosaccharides of human midcycle cervical mucin

It was previously shown that alkaline borohydride treatment of human midcycle cervical mucin releases a heterogeneous population of reduced neutral, sialylated, and sulfated oligosaccharides (Yurewicz, E. C., and Moghissi, K. S. (1981) J. Biol. Chem. 256, 11895-11905). Three major neutral oligosaccharides were isolated with approximate compositions of Fuc:Gal:GlcNAc:N-acetylgalactosaminitol (GalNAcol) = 0:2:1:1 (A1), 1:2:1:1 (A2), and 2:2:1:1 (A3). They comprised roughly 21%, 13%, and 8% of human cervical mucin oligosaccharide chains, respectively. In the present report, each was analyzed by periodate oxidation, methylation, and sequential degradation with glycosidases. A1 was shown to contain more than one component, but structural analyses clearly demonstrated the presence of one predominant (75%) tetrasaccharide. The proposed structure, Gal beta 1-4GlcNAc beta 1-6(Gal beta 1-3)GalNAcol, has previously been found in human gastric, submaxillary, and ovarian cyst mucins in their carbohydrate-to-protein linkage regions. beta-Galactosidase from Aspergillus niger selectively cleaved the Gal beta 1-4GlcNAc linkage in the intact tetrasaccharide. Enzymatic hydrolysis of the Gal beta 1-3GalNAcol linkage required prior removal of the Gal beta 1-4GlcNAc beta 1-unit attached to 0-6 of GalNAcol. The data for A2 indicated a mixture of two oligosaccharides, Gal beta 1-4,3(Fuc alpha 1-3,4)GlcNAc beta 1-6(Gal beta 1-3)GalNacol and Fuc alpha 1-2Gal beta 1-4GlcNac beta 1-6(Gal beta 1-3)-GalNacol, in an approximate molar ratio of 3 to 4:1, respectively. Two structures are consistent with the data obtained for A3: Fuc alpha 1-2Gal beta 1-4,3(Fuc alpha 1-3,4)GlcNAc beta 1-6(Gal beta 1-3)GalNAcol and/or Gal beta 1-4,3(Fuc alpha 1-3,4)GlcNac beta 1-6(Fuc alpha 1-2Gal beta 1-3)GalNacol. The results indicate that A1 represents the "core" tetrasaccharide of the larger human cervical mucin oligosaccharides A2 and A3.     

Conformation changes in the mechanism of cryoprecipitation of human monoclonal immunoglobulin M

The role of conformational changes in the mechanism of cryoprecipitation of human monoclonal immunoglobulin M (IgM) was studied. It was demonstrated that the variable moiety of the Fab-region of cryo-IgM has a site which comprises 5 to 6 charged amino acid residues. This site is responsible for intermolecular electrostatic interactions which lead to the formation of a precipitate with a decrease in temperature. This interaction is cooperative and stabilized by dipole molecules of H2O. The chain growth during aggregation is nuclear. The primary nucleus contains three IgM macromolecules. stability of the three-molecule nucleus is provided for by 16--17 intermolecular links. Using circular dichroism and fluorescent methods, it was found that the formation of a cryoprecipitate is accompanied by ionic pair release and conformational changes.     

Site-specific characterization of the N-linked oligosaccharides of a murine immunoglobulin M by high-performance liquid chromatography/electrospray mass spectrometry

Immunoglobulin M is an especially important product of the immune system because it plays a critical role in early protection against infections. In this report, the glycosylation pattern of the protective murine monoclonal IgM 12A1 to Cryptococcus neoformans polysaccharide was analyzed by high-performance liquid chromatography coupled with electrospray ionization mass spectrometry. Peptide mapping studies covering 88% of the deduced amino acid sequence indicated that of the six potential N-glycosylation sites in this antibody only five were utilized, as the tryptic peptide derived from monoclonal IgM 12A1 containing Asn-260 was recovered without carbohydrates. The oligosaccharide side chains of monoclonal IgM 12A1 were characterized at each of the N-glycosylation sites. Asn-166 possessed 20 monosialylated and nonsialylated, and fucosylated and nonfucosylated complex- and hybrid-type oligosaccharides and one high-mannose-type oligosaccharide. Thirteen oligosaccharides were attached to the site at Asn-401, including six complex-type, four hybrid-type, and three high-mannose-type oligosaccharides. Twelve hybrid-type oligosaccharides were attached to Asn-378, three of which had terminal sialic acids. Eleven hybrid-type oligosaccharides were attached to Asn-331, seven of which had terminal sialic acids. Only two high-mannose type oligosaccharides were attached to Asn-363. These results indicated great complexity in the structure and composition of oligosaccharides attached to individual IgM glycosylation sites.     

Structural characterization of mouse immunoglobulin allotypic determinants (allotopes) defined by monoclonal antibodies

We have generated a new series of monoclonal antibodies recognizing allotypic determinants on mouse IgG₁, IgG_2a, and IgG_2b. In this communication we describe their reactivity at the molecular level. A number of genetic specificities (as defined by reactivity with sera from inbred strains) were divided into subspecificities (allotopes) by these analyses. With the exception of one allotope located in the hinge region of Igh-1 ^b, all other 23 allotopes examined were preserved upon reduction and alkylation of immunoglobulin antigens. To further analyze the role of immunoglobulin conformation in presenting the allotopes, we assayed their presence on mixed Igh-1^a/Igh-4^a heavy chain molecules. The Igh-1^a determinants were maintained, but the Igh-4^a determinants were lost. Taken together, our results indicate that genetic polymorphisms at the Igh loci generate an enormous antigenic complexity, much of which relies on tertiary and quaternary protein structure for expression.     

Structural studies of a human hybrid immunoglobulin

The structure of an hypothesized hybrid or “Lepore-type” IgG contained in the serum of donor 2904 was investigated. Previous immunological studies suggested that this serum contained IgG with aγ3-γ1 hybrid heavy chain consisting of aγ3 Fd portion and aγ1 Fc. Structural studies have now shown that the carboxyl terminal end of the 2904 hybrid IgG, including much of the Fc fragment, isγ1 in character. However, the fingerprints of the Fc tryptic peptides at both pH 3.6 and pH 6.4 included a peptide, possibly from the hinge region, in peptide maps of Fc from aγG3 Gm (5) myeloma protein, but absent from maps of Fc peptides fromγG1 proteins. Gel filtration of the CNBr fragments of Fc from 2904 suggested that the hinge region isγ3-like. Papain cleavage experiments indicated an elevated level of resistant IgG, which agrees with immunological findings of an increasedγG2 subclass level. Our data confirm previous reports that theγ chain C-terminal octadecapeptides fromγG3 proteins have a subclass specific residue of arginine and indicate that within this subclass there is an allotypic variation related to the Gm type of the protein.     

Structural characterization of neutral oligosaccharides of human H+Leb+ gastric mucin

The structure of carbohydrate chains of human gastric mucin was investigated. The mucin, purified from pronase-degraded gastric aspirates of the secretors with blood group H+Leb, was subjected to alkaline borohydride treatment and a heterogeneous population of neutral (72.1%) and acidic (27.9%) oligosaccharide alditols was obtained. Ten oligosaccharides (I-X), representing 80.3% of the neutral oligosaccharide fraction, have been purified to homogeneity and their structures determined. These oligosaccharides ranged in length from 4 to 12 sugar units and contained mono-, bi-, and triantennary structures. Based on hemagglutination inhibition data in H-anti-H, Leb-anti-Leb, and I-anti-I systems, and the results of structural analyses, we proposed the following structures for these oligosaccharides: (formula, see text)     

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 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 human melanoma-associated antigen p97 with monoclonal antibodies

Monoclonal antibodies were used to study p97, a human melanoma-associated antigen (MAA). Four hybridomas, designated 4.1, 96.5, 118.1, and 8.2, were obtained by fusing mouse myeloma cells with spleen cells from mice immunized with human melanoma cells. Antibodies 4.1 and 8.2 were IgG1; antibodies 96.5 and 118.1 were IgG2a. Sequential immunoprecipitation (IP) and sodium-dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that all 4 antibodies recognized the same 97 kilodalton (kD) protein. Binding studies with 125I-labeled antibody showed that antibodies 4.1 and 96.5 bound the same epitope, p97a. Antibodies 118.1 and 8.2 defined epitopes p97b and p97c, respectively. Six monoclonal antibodies (M17, L1, L10, R10, I12, and K5) specific for gp95, a kD melanoma cell surface glycoprotein were also tested. Sequential IP showed that these antibodies bound p97; p97 and gp95 are thus identical. Binding studies showed that antibody m17 bound epitope p971, and antibodies L1, L10, and R19 bound epitope p97c. Antibodies I12 and K5 defined 2 other epitopes, p97d and p97e, respectively. SDS-PAGE under nonreducing conditions indicated that p97 is monomeric, probably with intrachain disulfide bonds. Cell-surface labeling of sialic acid residues and neuraminidase digestion showed that p97 is a sialoglycoprotein. Digestion of p97 with papain or trypsin produced a stable 40 kD fragment, which expressed epitopes p971, p97b, and p97c, but not p97d or p97e.     

Structural characterization of oligosaccharides isolated from the pectic polysaccharide rhamnogalacturonan II

Rhamnogalacturonan II (RG-II) is a structurally complex pectic (d-galactosyl-uronic acid-rich) polysaccharide that is present in the primary (growing) cell-walls of higher plants. RG-II is composed of ∼60 glycosyl residues. The isolation and structural characterization of 23 oligosaccharide fragments of the residue of RG-II that remained after removal of hepta- and di-saccharides by partial hydrolysis with acid are reported. In order to obtain the oligosaccharide fragments characterized herein, the carboxyl groups of RG-II were dideuterio-reduced, and the carboxyl-reduced polysaccharide was per-O-methylated. The per-O-methylated polysaccharide was fragmented by partial hydrolysis with acid, producing partially O-methylated oligosaccharides. These derivatized oligosaccharides were reduced, to afford a mixture of partially O-methylated oligoglycosyl-alditols, which was then per-O-methylated. The structures of the resulting per-O-methylated oligoglycosylalditols were determined by chemical-ionization mass spectrometry, electron-impact mass spectrometry, fast-atom-bombardment mass spectrometry, ¹H-n.m.r. spectroscopy, and analysis of corresponding, partially O-acetylated, partially O-methylated alditols. Seventeen of the oligosaccharides isolated from RG-II were parts of a single heptasaccharide, namely.     

Structure and location of asparagine-linked oligosaccharides in the Fc region of a human immunoglobulin D

Seven kinds of asparagine-linked oligosaccharides were bound to the Fc region of a human immunoglobulin D(NIG-65). The oligosaccharides quantitatively released from four species of glycopeptides by digestion with almond glycopeptidase, were separated by Bio-Gel p-4 column chromatography and were purified further by thin-layer chromatography. The sugars were identified with GC-MS following the permethylation of respective oligosaccharide. To Asn-68(NIG-65 Fc numbering (1)), two kinds of high-mannose-type oligosaccharides were bonded. To Asn-159, a kind of hybride-type and two kinds of bisected complex-type oligosaccharides were attached. From Asn-210, four kinds of bisected complex-type oligosaccharides were isolated.     

Polymerization of human immunoglobulin M.

The repolymerization of human IgM following mild reductive cleavage was studied as a model for intracellular polymer assembly. Repolymerization was found to require the presence of J chain and a disulfide exchanging system which could be furnished either intrinsically by the use of the monofunctional thiol mercaptoethylamine or extrinsically by the inclusion of a protein-mercaptan mixed disulfide, and/or a disulfide exchanging enzyme. The degree of repolymerization was dependent on the extent of monomer reduction and the product covalently incorporated one J chain per five monomer units. Disulfide exchanging enzyme probably served as a source of mixed disulfides rather than as an enzymatic catalyst of the reaction. These results are discussed in terms of a tentative mechanism for IgM polymerization.