Mobilities of the Inner Three Core Residues and the Man(R1f6) Branch of the Glycan at Asn78 of the R-Subunit of Human Chorionic Gonadotropin Are Restricted by the Protein † Carol W. E. M. Thijssen-van Zuylen, ‡,§ Tonny de Beer, ‡,| Bas R. Leeflang, ‡,⊥ Rolf Boelens, ⊥ Robert Kaptein, ⊥ Johannis P. Kamerling, ‡ and Johannes F. G. Vliegenthart* ,‡ Department of Bio-Organic Chemistry and Department of NMR Spectroscopy, BijVoet Center, Utrecht UniVersity, Padualaan 8, 3584 CH, Utrecht, The Netherlands ReceiVed July 30, 1997; ReVised Manuscript ReceiVed NoVember 10, 1997 ABSTRACT: Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein hormone involved in the maintenance of the corpus luteum in early pregnancy. The free R-subunit of hCG has a biological activity of its own, namely, stimulation of prolactin secretion from term pregnancy decidual cells [Blithe, D. L., et al. (1991) Endocrinology 129, 2257-2259]. Glycosylation at Asn78 of the R-subunit is required for the stability of the protein, but the exact nature of the stabilizing effect is not known. In our previous study, it was indicated that GlcNAc-1 at Asn78 has a reduced mobility, whereas the glycan at Asn52 is highly mobile [De Beer, T., et al. (1996) Eur. J. Biochem. 241, 229-242]. In the present investigation, it is shown that the PNGase F susceptibility of the Asn52-linked glycan in the free R-subunit is absent in the heterodimer. Thus, the high mobility of the glycan at Asn52 may be characteristic for the free R-subunit. For accurate modeling of RhCG, knowledge of the behavior of each of the glycans is essential. In this context, the mobility of the glycans and their interactions with the protein are explored by NMR spectroscopy using desialylated, partially deglycosylated free R-subunit (as-pdR) carrying glycans at Asn78 only. NOEs between GlcNAc-2 and several amino acid residues indicate that GlcNAc-2 is involved in stabilizing RhCG. From the values of 13 C relaxation parameters T 2 and T 1F of the constituting monosaccharide residues, it was concluded that the inner three residues have a severely restricted mobility. The Man-4 and Man-4′ residues of the diantennary oligosaccharide exhibit a similar relaxation behavior, suggesting that the Man-4′ branch occurs in a single conformation of the C5-C6 linkage of Man-3 instead of in rapidly interconverting conformations that are known to exist for this linkage for the free oligosaccharide. Human chorionic gonadotropin (hCG 1 ) is the placental member of the glycoprotein hormone family that further includes the pituitary hormones follitropin, lutropin, and thyrotropin. hCG exerts its activity through binding to a G-protein-coupled receptor in luteal cells resulting in in- creased adenylate cyclase activity. This is essential for the maintenance of the corpus luteum in early pregnancy (1- 3). The hormone is a heterodimer consisting of two noncovalently associated glycosylated subunits, R and . The R-subunit consists of 92 amino acids that are identical for all members of the hormone family. It is N-glycosylated at Asn52 and Asn78. Site-directed mutagenesis revealed that the glycan at Asn52 is essential for biological activity (4), whereas the glycan at Asn78 is involved in stabilizing the protein (5). The carbohydrate structures attached to Asn78 are mono- and diantennary N-acetyllactosamine type glycans in a ratio of 4:6 [(6), Figure 1]. Only a few studies have been carried out to determine the structure and conformation of the N-linked oligosaccharide part of intact glycoproteins in solution. Hen phosvitin has been subjected to NMR spectroscopy, and a high degree of similarity of chemical shifts and coupling patterns between the glycan part in the intact protein and model oligosaccha- rides was found, suggesting similar conformations (7). An extensive study on CD2 revealed that the Asn-linked GlcNAc residue stabilizes the protein structure by counterbalancing an unfavorable clustering of five positive charges (8, 9) and that only the resonances corresponding to GlcNAc-1 and -2 differ significantly from those of comparable free glycans. Also for CD58, only contacts between the Asn-linked † This investigation was supported by The Netherlands Foundation for Chemical Research (SON) with financial aid from The Netherlands Organization for Scientific Research (NWO). * Corresponding author. ‡ Department of Bio-Organic Chemistry. § Present address: Department of Analytical Chemistry for Research, N.V. Organon, P.O. Box 20, 5340 BH Oss, The Netherlands. | Present address: Department of Pharmacology, University of Colorado HSC, Box C236, 4200 East Ninth Avenue, Denver, CO 80262. ⊥ Department of NMR Spectroscopy. 1 Abbreviations: (R)hCG, (R-subunit of) human chorionic gonadot- ropin; PNGase F, peptide-N 4 -(N-acetyl--glucosaminyl)asparagine ami- dase F; sialidase, acylneuraminyl hydrolase; as-pdR, RhCG desialylated and site-specifically deglycosylated at Asn52, sialidase, and PNGase F-treated native RhCG; MLEV, composite spin-lock pulse devised by M. Levitt; ROE, rotating-frame nuclear Overhauser enhancement; TPPI, time-proportional phase increment; INEPT, insensitive nuclei enhanced by polarization transfer; HSQC, 1 H-detected heteronuclear single- quantum coherence spectroscopy. 1933 Biochemistry 1998, 37, 1933-1940 S0006-2960(97)01854-0 CCC: $15.00 © 1998 American Chemical Society Published on Web 01/30/1998