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

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

[1]  P. Stanley,et al.  A dominant mutation to ricin resistance in Chinese hamster ovary cells induces UDP-GlcNAc:glycopeptide beta-4-N-acetylglucosaminyltransferase III activity. , 1984, The Journal of biological chemistry.

[2]  R. Dwek,et al.  Association of rheumatoid arthritis and primary osteoarthritis with changes in the glycosylation pattern of total serum IgG , 1985, Nature.

[3]  R. Parekh,et al.  Release of oligosaccharides from glycoproteins by hydrazinolysis. , 1994, Methods in enzymology.

[4]  K. Titani,et al.  Comparative studies of asparagine-linked sugar chains of immunoglobulin G from eleven mammalian species. , 1993, Comparative biochemistry and physiology. B, Comparative biochemistry.

[5]  Shigeru FujiiS,et al.  Structural Heterogeneity of Sugar Chains in Immunoglobulin G , 1990 .

[6]  S L Morrison,et al.  Effect of C2-associated carbohydrate structure on Ig effector function: studies with chimeric mouse-human IgG1 antibodies in glycosylation mutants of Chinese hamster ovary cells. , 1998, Journal of immunology.

[7]  K. Anumula.  Rapid quantitative determination of sialic acids in glycoproteins by high-performance liquid chromatography with a sensitive fluorescence detection. , 1995, Analytical biochemistry.

[8]  E. Nickerson,et al.  A mutation in human CMP-sialic acid hydroxylase occurred after the Homo-Pan divergence. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[9]  J. Pevsner,et al.  The expanding β4-galactosyltransferase gene family: messages from the databanks , 1998 .

[10]  Ajit Varki,et al.  Oligosaccharides in vertebrate development , 1995 .

[11]  R. Dwek,et al.  Rheumatoid arthritis as a glycosylation disorder. , 1988, British journal of rheumatology.

[12]  R. Schnaar,et al.  The Chicken Genome Contains Two Functional Nonallelic β1,4-Galactosyltransferase Genes , 1997, The Journal of Biological Chemistry.

[13]  R. Dwek,et al.  Glycosylation changes of IgG associated with rheumatooid arthritis can activate complement via the mannose-binding protein , 1995, Nature Medicine.

[14]  H. Schachter.  Biosynthetic controls that determine the branching and microheterogeneity of protein-bound oligosaccharides. , 1986, Advances in experimental medicine and biology.

[15]  A. Varki,et al.  Diversity in the sialic acids , 1992, Glycobiology.

[16]  P. Stanley.  Glycosylation mutants of animal cells. , 1984, Annual review of genetics.

[17]  A. Varki,et al.  Factors controlling the glycosylation potential of the Golgi apparatus. , 1998, Trends in cell biology.

[18]  M. Naiki,et al.  Immunogenicity of N-glycolylneuraminic acid-containing carbohydrate chains of recombinant human erythropoietin expressed in Chinese hamster ovary cells. , 1995, Journal of biochemistry.

[19]  S L Morrison,et al.  Studies of aglycosylated chimeric mouse-human IgG. Role of carbohydrate in the structure and effector functions mediated by the human IgG constant region. , 1989, Journal of immunology.

[20]  B. Kumpel,et al.  Functional interactions of aglycosylated monoclonal anti-D with Fc gamma RI+ and Fc gamma RIII+ cells. , 1991, Immunology.

[21]  R. Jefferis,et al.  A comparative study of the N-linked oligosaccharide structures of human IgG subclass proteins. , 1990, The Biochemical journal.

[22]  D. Phillips,et al.  The three-dimensional structure of the carbohydrate within the Fc fragment of immunoglobulin G. , 1983, Biochemical Society transactions.

[23]  M. Moscarello,et al.  Branch specificity of purified rat liver Golgi UDP-galactose: N-acetylglucosamine beta-1,4-galactosyltransferase. Preferential transfer of of galactose on the GlcNAc beta 1,2-Man alpha 1,3-branch of a complex biantennary Asn-linked oligosaccharide. , 1984, The Journal of biological chemistry.

[24]  T. Rademacher,et al.  Galactosylation of human IgG monoclonal anti-D produced by EBV-transformed B-lymphoblastoid cell lines is dependent on culture method and affects Fc receptor-mediated functional activity. , 1994, Human antibodies and hybridomas.

[25]  A. Varki,et al.  Selectin ligands. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[26]  R. Jefferis,et al.  Aglycosylation of human IgG1 and IgG3 monoclonal antibodies can eliminate recognition by human cells expressing Fc gamma RI and/or Fc gamma RII receptors. , 1989, The Biochemical journal.

[27]  A. Varki,et al.  Sialic acids as ligands in recognition phenomena , 1997, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[28]  J. Briggs,et al.  A high-throughput microscale method to release N-linked oligosaccharides from glycoproteins for matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis. , 1998, Glycobiology.

[29]  D R Burton,et al.  Effector functions of a monoclonal aglycosylated mouse IgG2a: binding and activation of complement component C1 and interaction with human monocyte Fc receptor. , 1985, Molecular immunology.

[30]  R. Parekh,et al.  Different culture methods lead to differences in glycosylation of a murine IgG monoclonal antibody. , 1992, The Biochemical journal.

[31]  F. Smith,et al.  COLORIMETRIC METHOD FOR DETER-MINATION OF SUGAR AND RELATED SUBSTANCE , 1956 .

[32]  A. Shimizu,et al.  Structural and numerical variations of the carbohydrate moiety of immunoglobulin G. , 1982, Journal of immunology.

[33]  A. Varki,et al.  Biological roles of oligosaccharides: all of the theories are correct , 1993, Glycobiology.

[34]  S. Kornfeld,et al.  Assembly of asparagine-linked oligosaccharides. , 1985, Annual review of biochemistry.

[35]  A. Varki,et al.  A structural difference between the cell surfaces of humans and the great apes. , 1998, American journal of physical anthropology.

[36]  P. Stanley,et al.  CHO cells provide access to novel N-glycans and developmentally regulated glycosyltransferases. , 1996, Glycobiology.

[37]  P. Lerouge,et al.  N-Glycosylation of a mouse IgG expressed in transgenic tobacco plants. , 1999, Glycobiology.

[38]  P. Stanley,et al.  The Chinese hamster ovary glycosylation mutants LEC11 and LEC12 express two novel GDP-fucose:N-acetylglucosaminide 3-alpha-L-fucosyltransferase enzymes. , 1984, The Journal of biological chemistry.

[39]  P Jackson,et al.  The use of polyacrylamide-gel electrophoresis for the high-resolution separation of reducing saccharides labelled with the fluorophore 8-aminonaphthalene-1,3,6-trisulphonic acid. Detection of picomolar quantities by an imaging system based on a cooled charge-coupled device. , 1990, The Biochemical journal.

[40]  H. Wigzell,et al.  Biological significance of carbohydrate chains on monoclonal antibodies. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[41]  R. Cummings,et al.  Transcriptional Regulation of 1,3-Galactosyltransferase in Embryonal Carcinoma Cells by Retinoic Acid , 1996, The Journal of Biological Chemistry.

[42]  R. Dwek,et al.  Agalactosyl glycoforms of IgG autoantibodies are pathogenic. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[43]  A. Jones,et al.  Analysis of acidic oligosaccharides and glycopeptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. , 1996, Analytical chemistry.