A Panel of Recombinant Mucins Carrying a Repertoire of Sialylated O-Glycans Based on Different Core Chains for Studies of Glycan Binding Proteins

Sialylated glycans serve as key elements of receptors for many viruses, bacteria, and bacterial toxins. The microbial recognition and their binding specificity can be affected by the linkage of the terminal sugar residue, types of underlying sugar chains, and the nature of the entire glycoconjugate. Owing to the pathobiological significance of sialylated glycans, we have engineered Chinese hamster ovary (CHO) cells to secrete mucin-type immunoglobulin-fused proteins carrying terminal α2,3- or α2,6-linked sialic acid on defined O-glycan core saccharide chains. Besides stably expressing P-selectin glycoprotein ligand-1/mouse immunoglobulin G2b cDNA (PSGL-1/mIgG2b), CHO cells were stably transfected with plasmids encoding glycosyltransferases to synthesize core 2 (GCNT1), core 3 (B3GNT6), core 4 (GCNT1 and B3GNT6), or extended core 1 (B3GNT3) chains with or without the type 1 chain-encoding enzyme B3GALT5 and ST6GAL1. Western blot and liquid chromatography-mass spectrometry analysis confirmed the presence of core 1, 2, 3, 4, and extended core 1 chains carrying either type 1 (Galβ3GlcNAc) or type 2 (Galβ4GlcNAc) outer chains with or without α2,6-linked sialic acids. This panel of recombinant mucins carrying a repertoire of sialylated O-glycans will be important tools in studies aiming at determining the fine O-glycan binding specificity of sialic acid-specific microbial adhesins and mammalian lectins.

[1]  M. Hollingsworth,et al.  A family of human beta3-galactosyltransferases. Characterization of four members of a UDP-galactose:beta-N-acetyl-glucosamine/beta-nacetyl-galactosamine beta-1,3-galactosyltransferase family. , 1998, The Journal of biological chemistry.

[2]  Carolyn R Bertozzi,et al.  The chemistry and biology of mucin-type O-linked glycosylation. , 2005, Bioorganic & medicinal chemistry.

[3]  E. Tian,et al.  Recent insights into the biological roles of mucin-type O-glycosylation , 2009, Glycoconjugate Journal.

[4]  Niclas G Karlsson,et al.  Small-scale analysis of O-linked oligosaccharides from glycoproteins and mucins separated by gel electrophoresis. , 2002, Analytical chemistry.

[5]  Jining Liu,et al.  Anti-pig antibody adsorption efficacy of {alpha}-Gal carrying recombinant P-selectin glycoprotein ligand-1/immunoglobulin chimeras increases with core 2 {beta}1, 6-N-acetylglucosaminyltransferase expression. , 2005, Glycobiology.

[6]  H. Gabius,et al.  Detection and functions of mammalian lectins--with emphasis on membrane lectins. , 1991, Biochimica et biophysica acta.

[7]  H. Narimatsu.  Human glycogene cloning: focus on β3-glycosyltransferase and β4-glycosyltransferase families , 2006 .

[8]  J. Holgersson,et al.  Glycosyltransferases involved in type 1 chain and Lewis antigen biosynthesis exhibit glycan and core chain specificity. , 2006, Glycobiology.

[9]  A. Guzman-Aranguez,et al.  Structure and biological roles of mucin-type O-glycans at the ocular surface. , 2010, The ocular surface.

[10]  S. Pohl,et al.  Genetic engineering of recombinant glycoproteins and glycosylation pathway in mammalian host cells , 1999, Glycoconjugate Journal.

[11]  S. Kornfeld,et al.  Purification and characterization of UDP-N-acetylgalactosamine: polypeptide N-acetylgalactosaminyltransferase from bovine colostrum and murine lymphoma BW5147 cells. , 1986, The Journal of biological chemistry.

[12]  A. Srinivasan,et al.  Glycan topology determines human adaptation of avian H5N1 virus hemagglutinin , 2008, Nature Biotechnology.

[13]  K. Dimock,et al.  Enterovirus 70 Binds to Different Glycoconjugates Containing α2,3-Linked Sialic Acid on Different Cell Lines , 2005, Journal of Virology.

[14]  References , 1971 .

[15]  H. Clausen,et al.  Identification and characterization of large galactosyltransferase gene families: galactosyltransferases for all functions. , 1999, Biochimica et biophysica acta.

[16]  E. Hounsell,et al.  O-linked protein glycosylation structure and function , 1996, Glycoconjugate Journal.

[17]  Nicolai Bovin,et al.  Receptor specificity of influenza viruses from birds and mammals: new data on involvement of the inner fragments of the carbohydrate chain. , 2005, Virology.

[18]  J. Paulson,et al.  Purification of a Gal beta 1 to 4GlcNAc alpha 2 to 6 sialyltransferase and a Gal beta 1 to 3(4)GlcNAc alpha 2 to 3 sialyltransferase to homogeneity from rat liver. , 1982, The Journal of biological chemistry.

[19]  R. Dwek,et al.  Concepts and principles of O-linked glycosylation. , 1998, Critical reviews in biochemistry and molecular biology.

[20]  J. Holgersson,et al.  Removal of xenoreactive human anti-pig antibodies by absorption on recombinant mucin-containing glycoproteins carrying the Gal alpha1,3Gal epitope. , 1997, Transplantation.

[21]  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.

[22]  N. Karlsson,et al.  O-glycan repertoires on a mucin-type reporter protein expressed in CHO cell pools transiently transfected with O-glycan core enzyme cDNAs. , 2015, Journal of biotechnology.

[23]  A. Varki,et al.  Human-specific Regulation of α2–6-linked Sialic Acids* , 2003, Journal of Biological Chemistry.

[24]  B. Domon,et al.  A systematic nomenclature for carbohydrate fragmentations in FAB-MS/MS spectra of glycoconjugates , 1988, Glycoconjugate Journal.

[25]  James C Paulson,et al.  Glycan microarray analysis of the hemagglutinins from modern and pandemic influenza viruses reveals different receptor specificities. , 2006, Journal of molecular biology.

[26]  J. Ko,et al.  Experimental Section , 1960 .

[27]  Dong-Yup Lee,et al.  Global insights into the Chinese hamster and CHO cell transcriptomes , 2015, Biotechnology and bioengineering.

[28]  J. Holgersson,et al.  Core saccharide dependence of sialyl Lewis X biosynthesis , 2008, Glycoconjugate Journal.

[29]  Aravind Srinivasan,et al.  Glycans as receptors for influenza pathogenesis , 2010, Glycoconjugate Journal.

[30]  J. Marth,et al.  Novel Sulfated Lymphocyte Homing Receptors and Their Control by a Core1 Extension β1,3-N-Acetylglucosaminyltransferase , 2001, Cell.

[31]  Kelvin H. Lee,et al.  The genomic sequence of the Chinese hamster ovary (CHO)-K1 cell line , 2011, Nature Biotechnology.

[32]  B. Haab,et al.  Global Comparisons of Lectin–Glycan Interactions Using a Database of Analyzed Glycan Array Data* , 2013, Molecular & Cellular Proteomics.

[33]  A. Varki.  Sialic acids in human health and disease. , 2008, Trends in molecular medicine.

[34]  M. Hollingsworth,et al.  A Family of Human β3-Galactosyltransferases , 1998, The Journal of Biological Chemistry.

[35]  N. Karlsson,et al.  Shiga-like toxin binds with high avidity to multivalent O-linked blood group P1 determinants on mucin-type fusion proteins. , 2014, Glycobiology.

[36]  B. Furie,et al.  Expression cloning of a functional glycoprotein ligand for P-selectin , 1993, Cell.

[37]  M. Johansson,et al.  Detailed O-glycomics of the Muc2 mucin from colon of wild-type, core 1- and core 3-transferase-deficient mice highlights differences compared with human MUC2. , 2012, Glycobiology.

[38]  M. Breimer,et al.  Studies of Lewis antigens and H. pylori adhesion in CHO cell lines engineered to express Lewis b determinants. , 2008, Glycobiology.

[39]  J. Dennis,et al.  Regulation of UDP-GlcNAc:Gal beta 1-3GalNAc-R beta 1-6-N-acetylglucosaminyltransferase (GlcNAc to GalNAc) in Chinese hamster ovary cells. , 1993, The Journal of biological chemistry.

[40]  R. Cummings.  Structure and function of the selectin ligand PSGL-1. , 1999, Brazilian journal of medical and biological research = Revista brasileira de pesquisas medicas e biologicas.

[41]  J. Paulson,et al.  Glycan terminator , 2009, Nature Structural &Molecular Biology.

[42]  P. Stanley,et al.  Regulatory mutations in CHO cells induce expression of the mouse embryonic antigen SSEA-1 , 1983, Cell.

[43]  U. Rova,et al.  Pichia pastoris-produced mucin-type fusion proteins with multivalent O-glycan substitution as targeting molecules for mannose-specific receptors of the immune system. , 2011, Glycobiology.