Structural features of free and covalently bound glycans

Abstract

Carbohydrates act in many cellular functions and biological processes such as cell-cell recognition and adhesion, inflammation, fertilization, signal transduction, and development. In this context, structural information is required to understand molecular mechanisms involving carbohydrates. The placental glycoprotein hormone human chorionic gonadotropin (hCG) indirectly stimulates the corpus luteum to produce progesterone during the first trimester of pregnancy, until the placenta itself acquires the ability to produce this pregnancy-sustaining steroid. Nowadays, the therapeutic glycoprotein is produced in several ways, including isolation from the urine of pregnant woman and recombinant production protocols. In view of the importance of the glycosylation pattern for biological activity, it is essential to analyze the glycan chains connected to the protein backbone of recombinant materials. In the present work the “high-mannosylation” glycosylation pattern of the N-glycans of hCG expressed in Pichia pastoris GS115 is reported. Detailed neutral oligosaccharide structures in the range Man8GlcNAc2 - Man15GlcNAc2 could be established. Phosphate-containing oligosaccharides ranged from Man9PGlcNAc2 to Man13PGlcNAc2. As glycosylations are site-specific, also attention was paid to the N-glycosylation pattern per attachment site. Comparison of the N-glycosylation profiles of hCG produced in the P. pastoris GS115 and X-33 strains showed typical differences, i.e. high- versus hyper-mannosylation, a finding of high importance for the therapeutic glycoprotein production in P. pastoris cells. Additionally, an optimized culturing protocol for the production of hCG in GS115 cells was established. This new route made it possible to produce biologically active 15N-labeled hCG in good quantities against a realistic price, of importance for detailed conformational analysis by NMR spectroscopy. Another part of the thesis work focuses on the flexibility of the Asn-34 linked N-glycans of bovine RNase B (1-124) and BS (21-124; prepared from B via subtilisin digestion), in the context of the observation that the N-glycan releasing enzyme PNGase-F is active on native RNase BS but not on RNase B. Structural investigations by NMR spectroscopy showed that the glycan at Asn-34 was more mobile in RNase BS (now Asn-14) than in RNase B. MD simulations indicated that the region around Asn-34 in RNase B is relatively rigid, whereby the α-helix (residues 1-20) had a stabilizing effect. In RNase BS, the α-helix (residues 23-32) was significantly more flexible. A model of the RNase B(S) / PNGase-F complex could be built allowing to explain the difference in enzymatic activity. A third part of the thesis work focuses on conformational aspects of proteoglycans. NMR spectroscopy and molecular modeling studies were performed on five octasaccharides isolated from shark cartilage proteoglycan CS-D, which present different binding properties towards mAb 473HD, but also to mAb CS-56 and MO-225. It could be established that the sulfate group at position 2 of GlcA in disaccharide unit GlcA2S(α1-3)GalNAc6S, as wells as the presence of an exo-cyclic negative tail in disaccharide units GlcA(α1-3)GalNAc6S and Δ4,5HexA(α1-3)GalNAc6S are important for antibody recognition. These results will form the basis of further studies focused on the unravelling of the structures of the complexes between CS-D and recognizing antibodies.