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Wedepohl S.,Central Institute of Laboratory Medicine and Pathobiochemistry | Wedepohl S.,Free University of Berlin | Kaup M.,Central Institute of Laboratory Medicine and Pathobiochemistry | Riese S.B.,Central Institute of Laboratory Medicine and Pathobiochemistry | And 4 more authors.
Journal of Proteome Research | Year: 2010

The leukocytic adhesion receptor L-selectin plays a crucial role in the first step of the adhesion cascade, enabling leukocytes to migrate into surrounding tissues during inflammation and immune surveillance. We analyzed the site-specific N-glycosylation of the lectin and EGF-like domain of L-selectin using recombinant variants ('LEHis'). The three glycosylation sites of LEHis were mutated to obtain singly glycosylated variants that were expressed in HEK293F cells. aα1-Acid glycoprotein (AGP), expressed in the same system, was used to distinguish between cell type- and protein-specific glycosylation. Using mass spectrometry and exoglycosidase digestions, we established that LEHis was mostly bearing multifucosylated diantennary N-glycans with a major fraction terminating with GalNAc residues replacing the more common Gal. We could also show that parts of the GalNAc residues were sulfated. Furthermore, we identified novel diantennary glycan structures terminating with the motif GalNAc-GalNAc or SO4-GalNAc-GalNAc, which have not been described for N-glycans yet. Interestingly, none of these specific features were found in the N-glycan profile of AGP. This indicates that protein intrinsic information of L-selectin leads to decoration with specific N-glycans, which in turn may be related to L-selectin function. © 2010 American Chemical Society. Source

Berger M.,Central Institute of Laboratory Medicine and Pathobiochemistry | Kaup M.,Central Institute of Laboratory Medicine and Pathobiochemistry | Blanchard V.,Central Institute of Laboratory Medicine and Pathobiochemistry
Advances in Biochemical Engineering/Biotechnology | Year: 2012

Glycosylation is a post-translational modification that is of paramount importance in the production of recombinant pharmaceuticals as most recombinantly produced therapeutics are N- and/or O-glycosylated. Being a cell-systemdependent process, it also varies with expression systems and growth conditions, which result in glycan microheterogeneity and macroheterogeneity. Glycans have an effect on drug stability, serum half-life, and immunogenicity; it is therefore important to analyze and optimize the glycan decoration of pharmaceuticals. This review summarizes the aspects of protein glycosylation that are of interest to biotechnologists, namely, biosynthesis and biological relevance, as well as the tools to optimize and to analyze protein glycosylation. © Springer-Verlag Berlin Heidelberg 2011. Source

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