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 (English)In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 15, no 6, 571-583 p.Article in journal (Refereed) Published
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 alpha 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 alpha 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 beta 1,6 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 alpha 1,3 galactosyl- and core 2 beta 1,6 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 alpha 1,3 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.
Place, publisher, year, edition, pages
2005. Vol. 15, no 6, 571-583 p.
Biochemistry and Molecular Biology
IdentifiersURN: urn:nbn:se:sh:diva-14457DOI: 10.1093/glycob/cwi037ISI: 000229286000004OAI: oai:DiVA.org:sh-14457DiVA: diva2:469394