Effects of the N-Linked Glycans on the 3D Structure of the Free α-Subunit of Human Chorionic Gonadotropin

Abstract

To gain insight into intramolecular carbohydrate-protein interactions at the molecular level, the solution structure of differently deglycosylated variants of the R-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 φ and ψ dihedral angles of the glycosidic linkages of the oligosaccharide core fragments GlcNAc2( 1-4)GlcNAc1 and Man3( 1-4)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 -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 -hairpins consisting of residues 10-28 and 59-84.Human chorionic gonadotropin (hCG) 1 is a glycoprotein hormone involved in the maintenance of the corpus luteum during early pregnancy. It is a heterodimer consisting of noncovalently associated R-and -subunits. Both subunits are glycosylated and contain a cystine knot motif formed by three disulfide bridges as a central structural element (1,2). Besides the role of R-subunit (RhCG or RhCG[glycan 52,78 ]) in the R -dimer, it has been discovered that the noncombined R-subunit of hCG (hCG-R f ), which is produced in significant amounts by the placenta during pregnancy, has an independent biological activity in being able to stimulate prolactin secretion from human decidual cells (3, 4).Glycosylation of proteins has been shown to have many functions, including stabilization of the protein structure (5). In the R-subunit of hCG, the two glycosylation sites at Asn52 and Asn78, respectively, have remarkably different properties with respect to the stability of the subunit as shown by sitedirected mutagenesis (6). The absence of glycosylation at Asn52 does not affect folding and stability of the R-subunit. In contrast, mutant R-subunits lacking glycosylation at Asn78 are poorly secreted and rapidly degraded, although the precise molecular origin of thi...

Cell signaling, post-translational protein modifications and NMR spectroscopy

“…This, in turn, renders them amenable to high-resolution NMR studies, as exemplified by recent work on the glycosylated, 55 kDa Fc fragment of immunoglobulin G (Barb et al 2011). Thus, NMR constitutes the preferred tool for characterizing the structural and dynamic properties of sugar moieties in glyco-proteins (Fletcher et al 1994; Wyss et al 1995; Metzler et al 1997; Erbel et al 2000; Slynko et al 2009; Barb and Prestegard 2011). On the protein side, NMR has also been used to investigate the conformational properties of glycosylated-, and neighboring protein residues.…”

“…Once established, N-glycosylation itself is rather long lived, whereas individual glycan structures may experience dynamic compositional changes during a protein’s lifetime (Stanley et al 2009; Schwarz and Aebi 2011). With regard to glycosylation-induced changes in local protein backbone conformations, N-glycosylation has been reported to promote β -turn conformations (Meyer and Moller 2007), stabilize folded protein domains (Wyss et al 1995) and increase the degree of order in human chorionic gonadotropin (Erbel et al 2000). Immunoglobulin G N-glycans exhibit conformational exchange between two states, one giving rise to contacts between the glycan chain and the immunoglobulin, the other one preventing such contacts (Barb and Prestegard 2011; Barb et al 2012).…”

Experimental and computational study of inter- and intra- species specificity of gonadotropins for various gonadotropin receptors

“…The PDB database contains several hGTH structures: hCGa (PDB entries 1E9J, 1HD4, 1DZ7 and 1HCV (Spinelli et al, 1996;Erbel et al, 1999;Erbel et al, 2000)), hCG (PDB entries 1QFW, 1HRP, 1HCN (Lapthorn et al, 1994;Wu et al, 1994;Tegoni et al, 1999)), hFSH (PDB 1FL7 (Fox et al, 2001)), hFSH bound to hFSHR-ECD (PDB 1XWD (Fan and Hendrickson, 2005)) and the TSH binding domain of hTSHR (PDB 3GO4 (Sanders et al, 2007)). To compare these available structures we structurally superimposed them using the Discovery Studio 2.5 superposition by sequence alignment protocol.…”

Thyroid-Stimulating Hormone and Thyroid-Stimulating Hormone Receptor Structure-Function Relationships

“…It is known that the region ␣33-57 is largely involved in signal transduction and significantly altered by subunit dissociation. According to Erbel et al (47), comparison of X-ray structures of intact deglycosylated hCG and NMR structure of ␣-subunit in solution revealed that the dissociation of the hormone dimer induces large structural changes in the ␣-subunit, resulting in increased mobility of the glycan at Asn-52. Recently also, the structure of intact and deglycosylated hCG in complex with two Fv fragments of high-affinity anti-␣ and anti-␤ monoclonal antibodies has been solved.…”