title: 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.
abstract: We have previously described the construction of a P-selectin glycoprotein ligand-1-mouse immunoglobulin Fc fusion protein, which when transiently coexpressed with the porcine alpha1,3 galactosyltransferase in COS cells becomes a very efficient adsorber of xenoreactive, anti-pig antibodies. To relate the adsorption capacity with the glycan expression of individual fusion proteins produced in different cell lines, stable CHO-K1, COS, and 293T cells producing this fusion protein have been engineered. On alpha1,3 galactosyltransferase coexpression, high-affinity adsorbers were produced by both COS and 293T cells, whereas an adsorber of lower affinity was derived from CHO-K1 cells. Stable coexpression of a core 2 beta1,6 N-acetylglucosaminyltransferase in CHO-K1 cells led to increased alpha-Gal epitope density and improved anti-pig antibody adsorption efficacy. ESI-MS/MS of O-glycans released from PSGL-1/mIgG(2b) produced in an alpha1,3 galactosyl- and core 2 beta1,6 N-acetylglucosaminyltransferase expressing CHO-K1 cell clone revealed a number of structures with carbohydrate sequences consistent with terminal Gal-Gal. In contrast, no O-glycan structures with terminal Gal-Gal were identified on the fusion protein when expressed alone or in combination with the alpha1,3 galactosyltransferase in CHO-K1 cells. In conclusion, the density of alpha-Gal epitopes on PSGL-1/mIgG(2b) was dependent on the expression of O-linked glycans with core 2 structures and lactosamine extensions. The structural complexity of the terminal Gal-Gal expressing O-glycans with both neutral as well as sialic acid-containing structures is likely to contribute to the high adsorption efficacy.
authors.affiliation: Division of Clinical Immunology, Karolinska Institutet, Karolinska University Hospital, S-141 86 Stockholm, Sweden.
authors.forename: Jining
authors.initials: J
authors.lastname: Liu
authors.forename: Anki
authors.initials: A
authors.lastname: Gustafsson
authors.forename: Michael E
authors.initials: ME
authors.lastname: Breimer
authors.forename: Anders
authors.initials: A
authors.lastname: Kussak
authors.forename: Jan
authors.initials: J
authors.lastname: Holgersson
mesh_terms: D000042:Absorption; D000818:Animals; D000910:Antibodies, Heterophile / Q000276:immunology*; D000918:Antibody Specificity; D016466:CHO Cells; D019556:COS Cells; D002240:Carbohydrate Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003412:Cricetulus; D004187:Disaccharides / Q000276:immunology*; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology*; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000276:immunology* / Q000302:isolation & purification; D051379:Mice; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000096:biosynthesis* / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D013552:Swine
chemical_list: D000910:Antibodies, Heterophile; D004187:Disaccharides; D007074:Immunoglobulin G; D008562:Membrane Glycoproteins; C084882:P-selectin ligand protein; D011993:Recombinant Fusion Proteins; C055075:galactosyl-(1-3)galactose; D017351:N-Acetylglucosaminyltransferases; C053024:beta-1,3-galactosyl-O-glycosyl-glycoprotein beta-1,6-acetylglucosaminyl transferase