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Effects of the N-linked glycans on the 3D structure of the free alpha-subunit of human chorionic gonadotropin.

Erbel PJ, Karimi-Nejad Y, van Kuik JA, Boelens R, Kamerling JP, Vliegenthart JF
Biochemistry 39 6012-21 (2000)
Cited: 25 times
EuropePMC logo PMID: 10821673

Abstract

To gain insight into intramolecular carbohydrate-protein interactions at the molecular level, the solution structure of differently deglycosylated variants of the alpha-subunit of human chorionic gonadotropin have been studied by NMR spectroscopy. Significant differences in chemical shifts and NOE intensities were observed for amino acid residues close to the carbohydrate chain at Asn78 upon deglycosylation beyond Asn78-bound GlcNAc. As no straightforward strategy is available for the calculation of the NMR structure of intact glycoproteins, a suitable computational protocol had to be developed. To this end, the X-PLOR carbohydrate force field designed for structure refinement was extended and modified. Furthermore, a computational strategy was devised to facilitate successful protein folding in the presence of extended glycans during the simulation. The values for phi and psi dihedral angles of the glycosidic linkages of the oligosaccharide core fragments GlcNAc2(beta1-4)GlcNAc1 and Man3(beta1-4)GlcNAc2 are restricted to a limited range of the broad conformational energy minima accessible for free glycans. This demonstrates that the protein core affects the dynamic behavior of the glycan at Asn78 by steric hindrance. Reciprocally, the NMR structures indicate that the glycan at Asn78 affects the stability of the protein core. The backbone angular order parameters and displacement data of the generated conformers display especially for the beta-turn 20-23 a decreased structural order upon splitting off the glycan beyond the Asn78-bound GlcNAc. In particular, the Asn-bound GlcNAc shields the protein surface from the hydrophilic environment through interaction with predominantly hydrophobic amino acid residues located in both twisted beta-hairpins consisting of residues 10-28 and 59-84.

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  9. Homogeneous human complex-type oligosaccharides in correctly folded intact glycoproteins: evaluation of oligosaccharide influence on protein folding, stability, and conformational properties. Kajihara Y, Tanabe Y, Sasaoka S, Okamoto R. Chemistry 18 5944-5953 (2012)
  10. Studies on the relevance of the glycan at Asn-52 of the alpha-subunit of human chorionic gonadotropin in the alphabeta dimer. Erbel PJ, Haseley SR, Kamerling JP, Vliegenthart JF. Biochem. J. 364 485-495 (2002)
  11. Deglycosylation to obtain stable and homogeneous Pichia pastoris-expressed N-A1 domains of carcinoembryonic antigen. Sainz-Pastor N, Tolner B, Huhalov A, Kogelberg H, Lee YC, Zhu D, Begent RH, Chester KA. Int. J. Biol. Macromol. 39 141-150 (2006)
  12. Network analysis of dynamically important residues in protein structures mediating ligand-binding conformational changes. Saldaño TE, Tosatto SCE, Parisi G, Fernandez-Alberti S. Eur Biophys J 48 559-568 (2019)
  13. Expression and purification of neurolin immunoglobulin domain 2 from Carrassius auratus (goldfish) in Escherichia coli. Drees C, Stürmer CA, Möller HM, Fritz G. Protein Expr. Purif. 59 47-54 (2008)
  14. Enzymatic removal of asparagine-linked carbohydrate chains from heterodimer human chorionic gonadotrophin and effect on bioactivity. Richard CA, Creinin MD, Kubik CJ, DeLoia JA. Reprod. Fertil. Dev. 19 933-946 (2007)
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  16. Glycosylation and Crowded Membrane Effects on Influenza Neuraminidase Stability and Dynamics. Seitz C, Deveci İ, McCammon JA. J Phys Chem Lett 14 9926-9934 (2023)


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