Biosynthesis of sulfated asparagine-linked oligosaccharides on bovine lutropin

The asparagine-linked oligosaccharides on bovine lutropin (bLH) are unusual, containing GalNAc and sulfate but no galactose or sialic acid. Oligosaccharides from metabolically radiolabeled or purified bLH consist of non- (neutral), mono- (S-1), and di- (S-2) sulfated structures. We have previously shown that S-2 is a complex type oligosaccharide bearing two peripheral branches with the sequence SO4----GalNAc----GlcNAc attached to a typical Man3GlcNAc2 core (Green, E.D., van Halbeek, H., Boime, I., and Baenziger, J.U. (1985) J. Biol. Chem. 260, 15623-15630). We have now characterized the S-1 oligosaccharides on bLH which, in contrast to S-2, consist of several different structures of both the hybrid and complex types. The sulfate on S-1 oligosaccharides is located exclusively within the peripheral sequence SO4----GalNAc----GlcNAc. The GalNAc bearing hybrid structures, either with or without sulfate, cannot be processed to mono- or disulfated complex oligosaccharides due to the inability of either alpha-mannosidase II or GlcNAc-transferase II to act on GalNAc containing oligosaccharides. Since both Gal and GalNAc are added to oligosaccharides on some pituitary hormones, for example bovine and ovine follitropin and human lutropin, the Gal- and GalNAc-transferases appear to be key elements in regulating the synthesis of sulfated oligosaccharides on bLH and the other pituitary glycoprotein hormones.     

Asparagine-linked oligosaccharides on lutropin, follitropin, and thyrotropin. II. Distributions of sulfated and sialylated oligosaccharides on bovine, ovine, and human pituitary glycoprotein hormones

The asparagine-linked oligosaccharides on the pituitary glycoprotein hormones lutropin (LH), follitropin (FSH), and thyrotropin (TSH) consist of a heterogeneous array of neutral, sulfated, sialylated, and sulfated/sialylated structures. In the accompanying paper (Green, E.D., and Baenziger, J.U. (1987) J. Biol. Chem. 262, 25-35), we elucidated the structures of the anionic asparagine-linked oligosaccharides found on the bovine, ovine, and human pituitary glycoprotein hormones. In this study, we determined the relative quantities of the various asparagine-linked oligosaccharides on LH, FSH, and TSH from these three animal species. The proportions of sulfated versus sialylated oligosaccharides varied markedly among the different hormones. Both hormone- and animal species-specific differences in the types and distributions of sulfated, sialylated, and sulfated/sialylated structures were evident. In particular, LH and FSH, which are synthesized in the same pituitary cell and bear alpha-subunits with the identical amino acid sequence, contained significantly different distributions of sulfated and sialylated oligosaccharides. For all three animal species, the ratio of sialylated to sulfated oligosaccharides differed by greater than 10-fold for LH and FSH, with sulfated structures dominating on LH and sialylated structures on FSH. Sialylated oligosaccharides were also heterogeneous with respect to sialic acid linkage (alpha 2,3 versus alpha 2,6). In addition to differences in the proportion of sulfated and sialylated structures on LH and FSH, there were site-specific variations in the amount of mono- and disulfated oligosaccharides at different glycosylation sites on LH alpha-beta dimers. The differences in oligosaccharide structures among the various pituitary glycoprotein hormones as well as among the various glycosylation sites within a single hormone support the hypothesis that glycosylation may serve important functional roles in the expression and/or regulation of hormone bioactivity.     

Asparagine-linked oligosaccharides on lutropin, follitropin, and thyrotropin. I. Structural elucidation of the sulfated and sialylated oligosaccharides on bovine, ovine, and human pituitary glycoprotein hormones

We have elucidated the structures of the anionic asparagine-linked oligosaccharides present on the glycoprotein hormones lutropin (luteinizing hormone), follitropin (follicle-stimulating hormone), and thyrotropin (thyroid-stimulating hormone). Purified hormones, isolated from bovine, ovine, and human pituitaries, were digested with N-glycanase, and the released oligosaccharides were reduced with NaB[3H]4. The 3H-labeled oligosaccharides from each hormone were then fractionated by anion-exchange high performance liquid chromatography (HPLC) into populations differing in the number of sulfate and/or sialic acid moieties. The anionic oligosaccharides were further purified as well as structurally characterized using a variety of preparative and analytical techniques, including HPLC, endo- and exoglycosidase digestions, and lectin affinity chromatography. The sulfated, sialylated, and sulfated/sialylated structures, which together comprised 67-90% of the asparagine-linked oligosaccharides on the pituitary glycoprotein hormones, were highly heterogeneous and displayed hormone- as well as animal species-specific features. The sulfated oligosaccharides consisted of hybrid and complex type oligosaccharides with one or two branches terminating in SO4-4GalNAc beta 1,4. In contrast, the sialylated oligosaccharides consisted of a wide array of differing structures containing two or three peripheral branches as well as one, two, or three sialic acid moieties. A previously uncharacterized dibranched oligosaccharide, bearing one residue each of sulfate and sialic acid, was found on all of the hormones except bovine lutropin. In this study, we describe the purification and detailed structural characterizations of the sulfated, sialylated, and sulfated/sialylated oligosaccharides found on lutropin, follitropin, and thyrotropin from several animal species. In the accompanying paper (Green, E.D., and Baenziger, J.U.(1987) J. Biol. Chem. 262, 36-44) we demonstrate the marked quantitative differences among the pituitary glycoprotein hormones in terms of sulfation, sialylation, and underlying oligosaccharide structures, as well as provide evidence for site-specific synthesis of oligosaccharides on individual hormones.     

Pituitary glycoprotein hormone oligosaccharides: structure, synthesis and function of the asparagine-linked oligosaccharides on lutropin, follitropin and thyrotropin

Luteinizing hormone (LH), follicle-stimulating hormone (FSH) and thyroid-stimulating hormone (TSH) from pituitary and chorionic gonadotropin (CG) from placenta are a family of closely related glycoproteins. Each hormone is a heterodimer, consisting of an alpha- and a beta-subunit. Within an animal species, the alpha-subunits of all four glyco-protein hormones have an identical amino acid sequence, whereas each beta-subunit is distinct and confers hormone-specific features to the heterodimer. LH and FSH are synthesized within the same cell, the gonadotroph of the anterior pituitary, but are predominantly stored in separate secretory granules. We have characterized the asparagine-linked oligosaccharides on bovine, ovine and human LH, FSH and TSH. The various pituitary hormones were found to contain unique sulfated oligosaccharides with the terminal sequence SO4-4GalNAc beta 1----4GlcNAc beta 1----2Man alpha, sialylated oligosaccharides with the terminal sequence SA alpha Gal beta GlcNAc beta Man alpha, or both sulfated and sialylated structures. Despite synthesis of LH and FSH in the same pituitary cell, sulfated oligosaccharides predominate on LH while sialylated oligosaccharides predominate on FSH for all three animal species. We have examined the reactions leading to synthesis of the sulfated oligosaccharides to determine which steps are hormone specific. The sulfotransferase is oligosaccharide specific, requiring only the sequence GalNAc beta 1----4GlcNAc beta 1----2Man alpha. In contrast, the GalNAc-transferase appears to be protein specific, accounting for the preferential addition of GalNAc to LH, TSH, and free (uncombined) alpha-subunits compared with FSH and other pituitary glycoproteins. The predominance of sulfated oligosaccharide structures on LH may account for sorting of LH and FSH into separate secretory granules. Differences in sulfation and sialylation of LH, FSH and TSH may also play a role in the regulation of hormone bioactivity.     

High-performance capillary electrophoresis of sialylated oligosaccharides of human milk

Oligosaccharides in human milk inhibit enteric pathogens in vitro and in vivo. Neutral milk oligosaccharides vary among individuals and over the course of lactation. To study such variation in the acidic milk oligosaccharides, a sensitive, convenient, quantitative method is needed. High-performance capillary electrophoresis of underivatized acidic oligosaccharides with detection by UV absorbance at 205 nm proved to be sensitive to the femtomole level. Eleven standard oligosaccharides ranging from tri- to nonasaccharide (3'-sialyllactose, 6'-sialyllactose, 3'-sialyllactosamine, 6'-sialyllactosamine, disialyltetraose, 3'-sialyl-3-fucosyllactose, sialyllacto-N-tetraose-a, sialyllacto-N-tetraose-b, sialyllacto-N-neotetraose-c, disialyllacto-N-tetraose, and disialomonofucosyllacto-N-neohexaose) were resolved; baseline resolutions of 3'-sialyllactose, 6'-sialyllactose, and other structural isomers were achieved. Peak areas were linear from 30 to 2000 pg and were reproducible with a coefficient of variation between 4 and 9%. There was no evidence of quantitative interference of one oligosaccharide with another. In studies using pooled human milk, addition of increasing amounts of authentic standard oligosaccharides produced the expected positive increments in detected values, indicating quantitative recovery without interference by other milk components. The identities of the major sialylated acidic oligosaccharides of pooled human milk agreed with the results of previous studies employing other analytical methods. Comparison of oligosaccharide profiles of milk samples from different donors revealed extensive variation, especially in the structural isomers of sialyllacto-N-tetraose. This sensitive, highly reproducible method requires only simple sample workup and is useful in defining variations in human milk acidic oligosaccharides and investigating their possible relationship with diseases of infants.     

Annotation and structural analysis of sialylated human milk oligosaccharides

Sialylated human milk oligosaccharides (SHMOs) are important components of human milk oligosaccharides. Sialic acids are typically found on the nonreducing end and are known binding sites for pathogens and aid in neonates' brain development. Due to their negative charge and hydrophilic nature, they also help modulate cell-cell interactions. It has also been shown that sialic acids are involved in regulating the immune response and aid in brain development. In this study, the enriched SHMOs from pooled milk sample were analyzed by HPLC-Chip/QTOF MS. The instrument employs a microchip-based nano-LC column packed with porous graphitized carbon (PGC) to provide excellent isomer separation for SHMOs with highly reproducible retention time. The precursor ions were further examined with collision-induced dissociation (CID). By applying the proper collision energy, isomers can be readily differentiated by diagnostic peaks and characteristic fragmentation patterns. A set of 30 SHMO structures with retention times, accurate masses, and MS/MS spectra was deduced and incorporated into an HMO library. When combined with previously determined neutral components, a library with over 70 structures is obtained allowing high-throughput oligosaccharide structure identification.     

Structural studies of sialylated oligosaccharides of human midcycle cervical mucin

It was previously shown that reductive alkali treatment of purified human cervical mucin releases a heterogeneous population of reduced neutral, sialylated, and sulfated oligosaccharides (Yurewicz, E. C., and Moghissi, K. S. (1981) J. Biol. Chem. 256, 11895-11904). Four major sialylated oligosaccharide fractions were isolated with approximate compositions of Fuc:GlcNac:Gal:NeuAc:N-acetylgalactosaminitol (GalNAcol) = 0:0:0:1:1 (B1a), 0:0:1:1:1 (B2b), 0:1:2:1:1 (B3a), and 1:1:2:1:1 (B4a), where Fuc is fucose. They comprised roughly 3, 11, 7, and 6% of recovered oligosaccharide chains, respectively. On the basis of periodate oxidations, methylation analyses, and sequential degradations with glycosidases, the following structures were determined. (Formula: see text) Oligosaccharides 1 and 2 are characterized by the presence of N-acetylneuraminic acid in alpha 2,6-linkage to N-acetylgalactosaminitol. The remaining oligosaccharides contain N-acetylneuraminic acid in alpha 2,3-linkage to galactose residues. Oligosaccharides 3 and 4 and oligosaccharides 5 and 6 were isolated as unresolved isomeric mixtures in fractions B3a and B4a, respectively. Oligosaccharides 3 and 4 were distinguished on the basis of susceptibility to digestion with Aspergillus niger beta-galactosidase whereas oligosaccharides 5 and 6 were distinguished on the basis of differential rates of digestion with beef kidney alpha-fucosidase. The structural data indicate the presence of at least two sialyltransferases in human cervical epithelium and further suggest a potential physiologically significant competition between sialyltransferase and beta-N-acetylglucosaminyltransferase for C-6 of the N-acetylgalactosamine residue O-glycosidically linked to serine/threonine of the polypeptide core.     

Large-scale production of CMP-NeuAc and sialylated oligosaccharides through bacterial coupling

A large-scale production system of cytidine 5′-monophospho-N-acetylneuraminic acid (CMP-NeuAc) and sialyloligosaccharides was established by a whole-cell reaction through the combination of recombinant Escherichia coli strains and Corynebacterium ammoniagenes. For the production of CMP-NeuAc, two recombinant E. coli strains were generated that overexpressed the genes of CMP-NeuAc synthetase and CTP synthetase, respectively. C. ammoniagenes contributed to the formation of UTP from orotic acid. CMP-NeuAc was accumulated at 27 mM (17 g/l) after a 27-h reaction starting with orotic acid and N-acetylneuraminic acid. When E. coli cells that overexpressed the α-(2 → 3)-sialyltransferase gene of Neisseria gonorrhoeae were put into the CMP-NeuAc production system, 3′-sialyllactose was accumulated at 52 mM (33 g/l) after an 11-h reaction starting with orotic acid, N-acetylneuraminic acid, and lactose. Almost no oligosaccharide byproducts other than 3′-sialyllactose were observed after the reaction. The production of 3′-sialyllactose at a 5-l jar fermenter scale was almost the same as that at a beaker scale, which indicated the high potential of the 3′-sialyllactose production on an industrial scale. Received: 9 July 1999 / Received revision: 17 September 1999 / Accepted: 10 October 1999     

The broad diversity of neutral and sialylated oligosaccharides derived from human salivary mucins.

Mucin glycopeptides were prepared from the salivary mucins of 20 healthy donors with blood group O. The carbohydrate chains of the high-molecular-weight mucins were released by alkaline borohydride treatment. Neutral and monosialylated oligosaccharide-alditols were purified by ion-exchange chromatography, gel filtration, and HPLC. The structures of the oligosaccharide-alditols were determined by high-resolution 1H-NMR spectroscopy in combination with fast atom bombardment mass spectrometry and methylation analysis. Thirty-seven oligosaccharide-alditols were characterized and illustrate the extreme diversity of the salivary mucins carbohydrate chains. This diversity might represent a mosaic of bacterial adhesion sites and be involved in the early events of the nonimmune defense of the oral cavity. Among these 37 oligosaccharide-alditols, 31 have not been previously described in human saliva and five of these are novel structures: [formula: see text]     

Structures of novel sialylated O-linked oligosaccharides isolated from human erythrocyte glycophorins

The O-linked oligosaccharides attached to human erythrocyte glycophorins were extensively characterized. In addition to the previously described disialylated tetrasaccharide, NeuNAc alpha 2----3Gal beta 1----3 (Neu-NAc alpha 2----6)GalNAcOH and monosialylated trisaccharide, NeuNAc alpha 2----3Gal beta 1----3GalNAcOH, novel trisialylated oligosaccharides were isolated. Methylation analysis, fast atom bombardment-mass spectrometry, and enzymatic degradation were used to elucidate the following novel structures: formula; see text: These results suggest that O-linked oligosaccharides with a disialosyl group, NeuNAc alpha 2----8NeuNAc alpha 2----, may be present in various tissues.     

Carbohydrate recognition by pentadecapeptide ligands for a series of sialylated oligosaccharides

Sialyloligosaccharides of glycoproteins and glycosphingolipids play important roles in biological events on cell membranes. GT1b is a ganglioside having a trisialyloligosaccharide and is a receptor for tetanus toxin. In the present study, pentadecapeptide ligands for GT1b were obtained by phage display selection from a random peptide library with the use of a GT1b monolayer. The artificial pentadecapeptides had high affinity for GT1b which tended to increase depending on the number of sialic acids in sialyloligosaccharides. Arg, Ser, and hydrophobic amino acids were found in a consensus motif and may contribute to carbohydrate recognition. The consensus motif of the GT1b-binding peptides was different from that of GM1-, GM2-, GM3-, or GD1a-binding peptides. Peptide ligands for GT1b should be investigated for trisialyloligosaccharide functions and the development of therapeutic agents against trisialyloligosaccharide-related diseases.     

Cell-free sulfation of human and bovine pituitary hormones. Comparison of the sulfated oligosaccharides of lutropin, follitropin, and thyrotropin

Lutropin (LH), follitropin (FSH), and thyrotropin (TSH) from pituitary and human chorionic gonadotropin (hCG) from placenta are a family of glycoprotein hormones, each with an alpha and beta subunit. The alpha subunits of all four hormones have the same amino acid sequence, whereas biological specificity is determined by their unique beta subunits. The carbohydrate compositions of these hormones indicate the structures of their Asn-linked oligosaccharides are not identical. Sulfate is present on most, but not all, of these hormones, and for bovine LH is attached to GalNAc (Green, E.D., van Halbeek, H., Boime, I., and Baenziger, J.U. (1985) J. Biol. Chem. 260, 15623-15630). We used a reconstituted cell-free system to study sulfation of bovine (b) and human (h) glycoprotein hormones and its relationship to glycosylation. Exogenously added bLH, bTSH, bFSH, hLH, and hTSH are sulfated exclusively on the oligosaccharides of both alpha and beta subunits. The distribution of sulfated oligosaccharide structures varies among the hormones and appears to result from differences in the extent and/or pathway of oligosaccharide processing. Significant amounts of disulfated, dibranched complex oligosaccharides are present on all the sulfated hormones. Human FSH is not susceptible to sulfation unless first treated with neuraminidase. The sulfated oligosaccharides obtained from bovine FSH and desialylated human FSH are unlike those of the other hormones. Therefore, there is differential processing of the oligosaccharides on pituitary hormones. For FSH and LH, which are believed to be synthesized in the same cell, we would suggest that the unique beta subunits may regulate processing of all oligosaccharides present on the alpha-beta dimers.     

Expression and bioactivity of a single chain recombinant equine luteinizing hormone (reLH)

To study structure-activity relationships and the role of equine gonadotropins in the normal and pathophysiology of equine reproduction, the availability of purified hormones is essential. Previous expression studies in transfected CHO cells showed inefficient assembly of the human and bovine alpha and beta subunits, resulting in low levels of recombinant LH. The ability to express a single chain bearing genetically linked alpha and beta subunits bypasses this rate-limiting assembly step. A chimera was constructed by overlap PCR in which the carboxy terminal end of the eLHbeta subunit was genetically fused to the amino end of the alpha subunit. This gene was transfected into CHO cells and the recombinant product was purified through multiple steps, including a Fractogel resin separation. Serial dilutions of pituitary derived native eLH and the single chain reLH were compared in an eLH radioimmunoassay (RIA); the concentration curves between the single chain recombinant eLH and the native eLH standard were parallel. The biological activity of the analog was determined in vitro and in vivo using homologous equine models. Testicular tissue from five colts was processed for Leydig cell cultures. Increasing doses of reLH were incubated with equine Leydig cells for 24h in vitro and testosterone production was determined by RIA. Recombinant eLH stimulated a greater than 15-fold increase in testosterone production in a dose-dependent manner. Quarter Horse breeding stallions were treated with either reLH (n=5) or saline (n=3) and plasma testosterone concentrations were measured by RIA. Recombinant eLH stimulated a four-fold increase in circulating testosterone concentrations compared to the saline control. Therefore, the single chain recombinant will be effective for a variety of structure-function analyses and for breeding management in the horse.     

Genetic engineering of Escherichia coli for the economical production of sialylated oligosaccharides

We have previously described a microbiological process for the conversion of lactose into 3'sialyllactose and other ganglioside sugars by living Escherichia coli cells expressing the appropriate recombinant glycosyltransferase genes. In this system the activated sialic acid donor (CMP-Neu5Ac) was generated from exogenous sialic acid, which was transported into the cells by the permease NanT. Since sialic acid is an expensive compound, a more economical process has now been developed by genetically engineering E. coli K12 to be capable of generating CMP-Neu5Ac using its own internal metabolism. Mutant strains devoid of Neu5Ac aldolase and of ManNAc kinase were shown to efficiently produce 3'sialyllactose by coexpressing the alpha-2,3-sialyltransferase gene from Neisseria meningitidis with the neuC, neuB and neuACampylobacter jejuni genes encoding N-acetylglucosamine-6-phosphate-epimerase, sialic acid synthase and CMP-Neu5Ac synthetase, respectively. A sialyllactose concentration of 25 g l(-1) was obtained in long-term high cell density culture with a continuous lactose feed. This high concentration and low cost of fermentation medium should make possible to use sialylated oligosaccharides in new fields such as the food industry.     

Inhibition of nonopsonic Helicobacter pylori-induced activation of human neutrophils by sialylated oligosaccharides

Certain strains of Helicobacter pylori have nonopsonic neutrophil-activating capacity. Some H. pylori strains and the neutrophil-activating protein of H.pylori (HPNAP) bind selectively to gangliosides of human neutrophils. To determine if there is a relationship between the neutrophil-activating capacity and the ganglioside-binding ability, a number of H. pylori strains, and HPNAP, were incubated with oligosaccharides, and the effects on the oxidative burst of subsequently challenged neutrophils was measured by chemiluminescence and flow cytometry. Both by chemiluminescence and flow cytometry a reduced response was obtained by incubation of H.pylori with sialic acid-terminated oligosaccharides, whereas lactose had no effect. The reductions obtained with different sialylated oligosaccharides varied to some extent between the H. pylori strains, but in general 3'-sialyllactosamine was the most efficient inhibitor. Challenge of neutrophils with HPNAP gave no response in the chemiluminescence assay, and a delayed moderate response with flow cytometry. Preincubation of the protein with 3'-sialyllactosamine gave a slight reduction of the response, while 3'-sialyllactose had no effect. The current results suggest that the nonopsonic H. pylori-induced activation of neutrophils occurs by lectinophagocytosis, the recognition of sialylated glycoconjugates on the neutrophil cell surface by a bacterial adhesin leads to phagocytosis and an oxidative burst with the production of reactive oxygen metabolites.     

Structural elucidation of the disulfated oligosaccharide from bovine lutropin

The Asn-linked oligosaccharides of the pituitary hormone lutropin (LH) contain both sulfate and GalNAc. Bovine pituitary explants incorporate [3H]glucosamine, [3H]mannose, [3H]fucose, and [35S]sulfate into the Asn-linked oligosaccharides of LH. Endoglycosidase F or N-glycanase releases the [3H]glucosamine- and [3H]mannose-labeled oligosaccharides from the protein, which resolve on anion-exchange high pressure liquid chromatography as neutral (S-0), mono- (S-1), and disulfated (S-2) species. Based on sequential enzyme digestion, methylation, periodate oxidation, and nuclear magnetic resonance studies, the proposed structure for S-2 is as follows: formula see text. Sulfate is confined to position 3 or 4 of GalNAc based on periodate and methylation data and can be removed by methanolysis. The presence of beta-linked GalNAc at a position typically occupied by Gal has not previously been observed.     

Microheterogeneity of the oligosaccharides carried by the recombinant bovine lactoferrin expressed in Mamestra brassicae cells

The development of therapeutic glycoprotein production usingthe baculovirus expression system depends on the ability ofinsect cell lines to reproduce site specific mammalian-likeN-glycans. A combination of ¹H-NMR and mass spectrometry techniques(MALD-MS, ES-MS, and CID-MS-MS) allowed us to elucidate theN-linked oligosaccharides microheterogeneity on three differentN-glycosylation sites, Asn₂₃₃, Asn₄₇₆, and Asn₅₄₅, of a baculovirus-expressedrecombinant bovine lactoferrin produced in Mamestra brassicae.Two families of N-glycan structures have been found: first,oligomannosidic glycans (Man₉₅GlcNAC₂) and secondly, short truncatedpartially fucosylated glycans (Man_3–2[Fuc_0–1]GlcNAc₂).These results indicate that Mamestra brassicae cell line isnot able to synthesize complex N-glycans, even if an     

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

Human lutropin or luteinizing hormone (hLH) is a heterodimeric glycoprotein, composed of two subunits. hLH alpha (N-glycosylated at Asn52 and Asn78) and hLH beta (N-glycosylated at Asn30). The sugar chains were liberated by hydrazinolysis from intact hLH beta and from glycopeptides obtained after tryptic digestion of hLH alpha, subsequently reduced and fractionated as alditols by anion-exchange and ion-suppression amine-adsorption HPLC and identified mainly by one-dimensional (1D) and two-dimensional (2D) 1H-NMR spectroscopy. The results indicate predominantly diantennary. N-acetyllactosamine-type structures at all three glycosylation sites. The oligosaccharides attached to Asn52 (hLH alpha) and Asn30 (hLH beta) show a remarkably similar pattern, with mainly chain-terminating 4-sulphated 2-deoxy-2-N-acetylamino-D-galactose (GalNAc) and a sulphated/sialylated structure as the major single component. However, virtually all N-glycans on the beta subunit bear a fucose residue alpha 1-6-linked to the proximal GlcNAc, whereas those at Asn52 (and Asn78) of the alpha subunit are predominantly non-fucosylated. The oligosaccharides at Asn78 (hLH alpha) are sialylated rather than sulphated and contain the unique sequence NeuAc alpha 2-6 GalNAc beta 1-4GlcNAc beta 1-2 Man alpha 1-3 as part of the majority of mono- and disialylated compounds. The major single constituent at Asn78 has the following structure: [formula, see text]