Glycobiology Advance Access originally published online on December 29, 2004
Glycobiology 2005 15(6):571-583; doi:10.1093/glycob/cwi037
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Glycobiology vol. 15 no. 6 © Oxford University Press 2004; all rights reserved.
Anti-pig antibody adsorption efficacy of 
-Gal carrying recombinant P-selectin glycoprotein ligand-1/immunoglobulin chimeras increases with core 2 ß1, 6-N-acetylglucosaminyltransferase expression
3 Division of Clinical Immunology, Karolinska Institutet, Karolinska University Hospital, S-141 86 Stockholm, Sweden; 4 Department of Clinical Chemistry, Sahlgrenska University Hospital, S-413 45 Göteborg, Sweden; 5 Department of Surgery, Sahlgrenska University Hospital, S-413 45 Göteborg, Sweden; 6 Clinical Research Centre, Karolinska Institutet, Karolinska University Hospital, S-141 86 Stockholm, Sweden; and 7 University College of South Stockholm, S-141 89 Stockholm, Sweden
1 These authors contributed equally to this work.
2 To whom correspondence should be addressed; e-mail: jan.holgersson{at}labmed.ki.se
Received on July 2, 2003; revised on September 13, 2004; accepted on December 23, 2004
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 
1,3 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 1,3 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 ß1,6 N-acetylglucosaminyltransferase in CHO-K1 cells led to increased -Gal epitope density and improved anti-pig antibody adsorption efficacy. ESI-MS/MS of O-glycans released from PSGL-1/mIgG2b produced in an 1,3 galactosyl- and core 2 ß1,6 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 1,3 galactosyltransferase in CHO-K1 cells. In conclusion, the density of -Gal epitopes on PSGL-1/mIgG2b 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.
Key words: glycoconjugate / mass spectrometry / mucin / natural antibodies / xenotransplantation
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