Glycobiology Advance Access published online on December 29, 2004
Glycobiology, doi:10.1093/glycob/cwi037
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1 Division of Clinical Immunology, Karolinska Institutet, Karolinska University Hospital, S-141 86 Stockholm
* To whom correspondence should be addressed. We have previously described the construction of a P-selectin glycoprotein ligand-1 - mouse immunoglobulin Fc fusion protein, which when transiently co-expressed with the porcine
Received July 2, 2003
Revised September 13, 2004
Accepted December 23, 2004
Article
Anti-pig antibody adsorption efficacy of
-Gal carrying recombinant P-selectin glycoprotein ligand-1/immunoglobulin chimeras increases with core 2 beta 1,6-N- acetylglucosaminyltransferase expression
2 Division of Clinical Immunology and; Departments of Clinical Chemistry, Karolinska Institutet, Karolinska University Hospital, S-141 86 Stockholm
3 Departments of Surgery, Sahlgrenska University Hospital, S-413 45 Göteborg
4 Division of Clinical Research Centre, Karolinska Institutet, Karolinska University Hospital, S-141 86 Stockholm; Departments of University College of South Stockholm, S-141 89 Stockholm, Sweden
Jan Holgersson, E-mail: jan.holgersson{at}labmed.ki.se
Abstract 
1,3 galactosyltransferase in COS cells become a very efficient adsorber of xenoreactive, anti-pig antibodies. In order 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. Upon 1,3 galactosyltransferase coexpression, high affinity adsorbers were produced by both COS and 293T cells, while an adsorber of lower affinity was derived from CHO-K1 cells. Stable co-expression of a core 2 â1,6 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 â1,6 Nacetylglucosaminyltransferase 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.
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