NMR investigations of the N‐linked oligosaccharides at individual glycosylation sites of human lutropin

Abstract

Human lutropin or luteinizing hormone (hLH) is a heterodimeric glycoprotein, composed of two subunits, hLHa (N-glycosylated at Asn52 and Asn78) and hLHP (N-glycosylated at Asn30). The sugar chains were liberated by hydrazinolysis from intact hLHP and from glycopeptides obtained after tryptic digestion of hLHa, subsequently reduced and fractionated as alditols by anion-exchange and ion-suppression amine-adsorption HPLC and identified mainly by one-dimensional (1 D) and two-dimensional (2D) 'H-NMR spectroscopy. The results indicate predominantly diantennary, N-acetyllactosamine-type structures at all three glycosylation sites. The oligosaccharides attached to Am52 (hLHa) and Am30 (hLHB) show a remarkably similar pattern, with mainly chain-terminating 4-sulphated 2-deoxy-2-N-acetylamino-~-galactose (GalNAc) and a sulphatedisialylated structure as the major single component. However, virtually all N-glycans on the b subunit bear a fucose residue a1 -6-linked to the proximal GlcNAc, whereas those at Am52 (and Asn78) of the a subunit are predominantly nonfucosylated. The oligosaccharides at Asn78 (hLHa) are sialylated rather than sulphated and contain the unique sequence NeuAca2-6GalNAcpl-4GlcNAcpl-2Manal-3 as part of the majority of mono-and disialylated compounds. The major single constituent at Asn78 has the following structure:Man~l-4GlcNAc~1-4GlcNAc-ol. /3 NeuAca2-6GalNAc~1-4GlcNAc~1-2ManalhLH is a heterodimeric glycoprotein hormone that consists of two non-covalently associated subunits, both highly crosslinked by disulphide bonds: the a subunit (hLHa) bears Nglycans at positions Am52 and Asn78, the p subunit (hLHb) is N-glycosylated at Asn30. The hormone, which is a vital component of the reproductive process, is produced in the anterior pituitary gland and stimulates steroidogenesis in ovary and testis [l -31. The a subunit shares an identical amino acid sequence and carbohydrate attachment sites with the respective a subunits of human follitropin (folliclestimulating hormone) and thyrotropin (thyroid-stimulating hormone), produced in the same gland, and with that of human chorionic gonadotropin from the placenta; the four closely related hormones differ structurally from each other with regard to their hormone-specific fl subunits and the carbohydrate structures attached to both subunits [3 -71. NuCorrespondence to A. G. C. Renwick, Department of Biochemistry, University of Auckland, Private Bag, Auckland, New ZealandAbbreviations. COSY, scalar shift-correlated NMR spectroscopy; ID, one-dimensional; 2D, two-dimensional; FAB-MS, fast-atombombardment mass spectrometry; Fuc, L-fucose; GalNAc, 2-deoxy-2-N-acetylamino-~-galactose; GlcNAc-01, N-acetylglucosaminitol; hLH, human lutropin; hLHa,a subunit of hLH; hLH/l,D subunit of hLH.Enzyme. Trypsin (EC 3.4.21.4).merous investigations, mostly on human chorionic gonadotropin, suggest that the sugar chains are not required for hormone-receptor binding, but play a critical role in receptor activation via the adenylate-cyclase enzyme system [2, 3, 8 -10 (and the referenc...

Molecular structures of glycoprotein hormones and functions of their carbohydrate components

“…Although there are major differences between the structures of the peripheral carbohydrate chains of these hormones [188,190], the presence of chain-terminating negatively charged groups in both has led to a proposal that all glycoprotein hormones may function by interaction with structured water in the receptor cavity [223].…”

“…Detailed investigation of the N-linked oligosaccharides of human lutropin [188] revealed predominantly diantennary complex type structures at all three glycosylation sites (Fig. 5).…”

“…Weisshaar et al [187,188] isolated the N-linked oligosaccharides from tryptic digests of the a-subunits of ovine and human lutropin, and from each fl-subunit. These were individually subjected to hydrazinolysis, and the oligosaccharide products were reduced to their alditols with sodium borohydride [187,188].…”

“…These were individually subjected to hydrazinolysis, and the oligosaccharide products were reduced to their alditols with sodium borohydride [187,188]. Each was then fractionated by anion-exchange and ion-suppression amine-adsorption h.p.l.c.…”

“…The application of new, rigorous, analytical techniques for the structural determination of complex carbohydrates [147,226,227] has greatly facilitated the acquisition of detailed information concerning the N-linked oligosaccharides at individual glycosylation sites in ovine and human lutropin [187,188], human chorionic gonadotropin [190] and human thyrotropin [192]. Similarly, many investigations have probed salient features of the protein backbones of these hormones using circular dichroism [228,229], monoclonal antibodies [230][231][232][233][234][235] and synthetic peptides [236][237][238][239], especially domains that appear to be vital in hormonal receptor interactions.…”