Jacques U. Baenziger

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.

A hepatic reticuloendothelial cell receptor specific for SO4-4GalNAcβ1, 4GlcNAcβ1,2Manα that mediates rapid clearance of lutropin

Abstract We have identified a receptor in hepatic endothelial and Kupffer cells that binds oligosaccharides terminating with the sequence SO 4 -4GalNAcβ1,4GlcNAcβ1,2-Manα (S4GGnM). This receptor can account for the rapid removal of the glycoprotein hormone lutropin, which bears unique Asn-linked oligosaccharides terminating in S4GGnM, from the circulation. Hepatic endothelial cells express 579,000 S4GGnM receptors at their surface and bind lutropin with an apparent K d of 1.63 × 10 −7 M. Bound ligand is rapidly internalized. Binding does not require divalent cations, is reversed by incubation at pH 5.0 or below, and is inhibited by fucoidin but not by hyaluronate, heparin, chondroitin sulfate, or dextran sulfate. We propose that the S4GGnM-specific receptor represents a major mechanism for clearance of certain sulfated glycoproteins from the blood, including members of the glycoprotein hormone family.

Galactose and N-acetylgalactosamine-specific endocytosis of glycopeptides by isolated rat hepatocytes

We have examined the kinetics of binding and uptake of iodinated glycoproteins and glycopeptides bearing terminal Gal or GalNAc moieties in an isolated rat hepatocyte system. Asparagine-linked, triantennary complex oligosaccharides with three terminal Gal residues are endocytosed with the same kinetics as asialo-orosomucoid, whereas biantennary, complex oligosaccharides with one or two terminal Gal residues are not endocytosed Glycopeptides bearing as few as four O-glycosidically-linked Gal beta 1, 3GalNAc or GalNAc moieties are also rapidly endocytosed, while glycopeptides with one or two more closely spaced moieties are not endocytosed. All the endocytosable glycoproteins and glycopeptides have similar apparent dissociation constants and a similar number of binding sites on the surface of the intact hepatocyte. The ligand-binding properties of the receptor in the plasma membrane of intact cells differ from those of the solubilized receptor, suggesting that interaction with other as yet undefined cellular components confers the ability to discriminate among closely related oligosaccharide structures. This is consistent with a model in which only glycopeptides bearing terminal Gal or GalNAc residues that fall within a restricted spatial relationship can induce a conformational alteration in the receptor which is required for uptake to occur. The endocytosis of a number of glycoproteins such as human asialo-ceruloplasmin can be accounted for by the presence of a single, complex oligosaccharide with the appropriate structure.

Expression Cloning of a New Member of the ABO Blood Group Glycosyltransferases, iGb3 Synthase, That Directs the Synthesis of Isoglobo-glycosphingolipids

The large array of different glycolipids described in mammalian tissues is a reflection, in part, of diverse glycosyltransferase expression. Herein, we describe the cloning of a UDP-galactose: β-d-galactosyl-1,4-glucosylceramide α-1,3-galactosyltransferase (iGb3 synthase) from a rat placental cDNA expression library. iGb3 synthase acts on lactosylceramide, LacCer (Galβ1,4Glcβ1Cer) to form iGb3 (Galα1,3Galβ1,4Glcβ1Cer) initiating the synthesis of the isoglobo-series of glycosphingolipids. The isolated cDNA encoded a predicted protein of 339 amino acids, which shows extensive homology (40–50% identity) to members of the ABO gene family that includes: murine α1,3-galactosyltransferase, Forssman (Gb5) synthase, and the ABO glycosyltransferases. In contrast to the murine α1,3-galactosyltransferase, iGb3synthase preferentially modifies glycolipids over glycoprotein substrates. Reverse transcriptase-polymerase chain reaction revealed a widespread tissue distribution of iGb3 synthase RNA expression, with high levels observed in spleen, thymus, and skeletal muscle. As an indirect consequence of the expression cloning strategy used, we have been able to identify several potential glycolipid biosynthetic pathways where iGb3 functions, including the globo- and isoglobo-series of glycolipids.

Separation of neutral oligosaccharides by high-performance liquid chromatography☆

Abstract We have developed a method for separating reduced, neutral oligosaccharides by high-performance liquid chromatography on columns of MicroPak AX-5 (Varian Associates) with a mobile phase consisting of acetonitrile:H 2 O. Individual glucose oligomers containing from 1 to 20 glucose moieties can be separated in a single 1-h analysis with a solvent program of decreasing acetonitrile concentration. We have applied this method to both the analysis and preparative isolation of glycoprotein-derived oligosaccharides obtained by enzymatic release with endoglycosidases or chemical release by hydrazinolysis. Introduction of 3 H by reduction with NaB 3 H 4 permits the detection of subnanomole quantities of oligosaccharides. This method offers previously unattainable rapidity and resolution for the analysis of oligosaccharides.

Oligosaccharides containing β1,4-linked N-acetylgalactosamine, a paradigm for protein-specific glycosylation

The carbohydrate moieties found on glycoproteins have long been recognized as having great potential to bear biologically important information. However, actual examples of systems in which oligosaccharides play defined physiological roles have remained limited. These oligosaccharides with known biologic functions typically have distinctive structural features and are generally confined to specific glycoproteins. Synthesis of structurally unique oligosaccharides on specific glycoproteins at defined times is essential if these structures are to fulfill their biologic purpose. Since cells produce many distinct oligosaccharides as newly synthesized glycoproteins pass through the endoplasmic reticulum and the Golgi, mechanisms are required to assure that the correct structures are added to the numerous glycoproteins being synthesized. Determining how synthesis of the vast array of oligosaccharides produced by each cell is regulated is essential for understanding the biologic importance of these complex structures. Asn-linked oligosaccharides arise by processing of a common precursor structure, which is transferred en bloc from dolichol to the nascent peptide chain in the endoplasmic reticulum (1). As a result Asn-linked oligosaccharides have a common core region and differ primarily in the number and location of their peripheral branches and terminal modifications. Since all newly synthesized glycoproteins pass through the same subcellular compartments and are exposed to the same transferases, structural differences in oligosaccharides on individual glycoproteins and/or at individual glycosylation sites must in some fashion reflect the influence of the protein moiety on one or more glycosyltransferases. This suggests that key glycosyltransferases recognize features encoded within the peptide as well as the oligosaccharide of the target glycoprotein. Among the three glycosyltransferases thus far demonstrated to display peptide as well as oligosaccharide recognition, UDP-glucose:glycoprotein glucosyltransferase, UDP-N-acetylglucosamine: lysosomal enzyme N-acetylglucosamine-1-phosphotransferase, and UDP-GalNAc:glycoprotein hormone b1,4-N-acetylgalactosaminyltransferase (b1,4-GalNAcT, reviewed in Ref. 2), one of the most extensively characterized is the b1,4-GalNAcT, which produces the terminal sequence GalNAcb1,4GlcNAcb1-R on glycoproteins that contain a specific peptide recognition determinant in addition to an appropriate oligosaccharide acceptor. The product of the b1,4-GalNAcTmay be further modified by the addition of sulfate, sialic acid, or fucose, thus producing a range of unique oligosaccharide structures defined by the presence of b1,4-linked GalNAc as illustrated in Fig. 1. Each of these structures has the potential to be recognized by a specific receptor or binding protein and thus mediate a distinct biological function. As will become apparent below, the b1,4-GalNAcT is a key component of a well characterized system, which includes unique oligosaccharide structures, highly specific glycosyltransferases, and oligosaccharide-specific receptors. This is therefore an excellent model system for understanding proteinspecific glycosylation.

Cloning of Gb3 Synthase, the Key Enzyme in Globo-series Glycosphingolipid Synthesis, Predicts a Family of α1,4-Glycosyltransferases Conserved in Plants, Insects, and Mammals

We have cloned Gb3synthase, the key α1,4-galactosyltransferase in globo-series glycosphingolipid (GSL) synthesis, via a phenotypic screen, which previously yielded iGb3 synthase, the α1,3-galactosyltransferase required in isoglobo-series GSL (Keusch, J. J., Manzella, S. M., Nyame, K. A., Cummings, R. D., and Baenziger, J. U. (2000) J. Biol. Chem.33). Both transferases act on lactosylceramide, Galβ1,4Glcβ1Cer (LacCer), to produce Gb3(Galα1,4LacCer) or iGb3 (Galα1,3LacCer), respectively. GalNAc can be added sequentially to either Gb3 or iGb3 yielding globoside and Forssman from Gb3, and isogloboside and isoForssman from iGb3. Gb3synthase is not homologous to iGb3 synthase but shows 43% identity to a human α1,4GlcNAc transferase that transfers a UDP-sugar in an α1,4-linkage to a β-linked Gal found in mucin. Extensive homology (35% identity) is also present between Gb3synthase and genes in Drosophila melanogaster andArabidopsis thaliana, supporting conserved expression of an α1,4-glycosyltransferase, possibly Gb3 synthase, throughout evolution. The isolated Gb3 synthase cDNA encodes a type II transmembrane glycosyltransferase of 360 amino acids. The highest tissue expression of Gb3 synthase RNA is found in the kidney, mesenteric lymph node, spleen, and brain. Gb3 glycolipid, also called Pk antigen or CD77, is a known receptor for verotoxins. CHO cells that do not express Gb3 and are resistant to verotoxin become susceptible to the toxin following transfection with Gb3 synthase cDNA.

Loss of Dermatan-4-Sulfotransferase 1 Function Results in Adducted Thumb-Clubfoot Syndrome

Adducted thumb-clubfoot syndrome is an autosomal-recessive disorder characterized by typical facial appearance, wasted build, thin and translucent skin, congenital contractures of thumbs and feet, joint instability, facial clefting, and coagulopathy, as well as heart, kidney, or intestinal defects. We elucidated the molecular basis of the disease by using a SNP array-based genome-wide linkage approach that identified distinct homozygous nonsense and missense mutations in CHST14 in each of four consanguineous families with this disease. The CHST14 gene encodes N-acetylgalactosamine 4-O-sulfotransferase 1 (D4ST1), which catalyzes 4-O sulfation of N-acetylgalactosamine in the repeating iduronic acid-alpha1,3-N-acetylgalactosamine disaccharide sequence to form dermatan sulfate. Mass spectrometry of glycosaminoglycans from a patients fibroblasts revealed absence of dermatan sulfate and excess of chondroitin sulfate, showing that 4-O sulfation by CHST14 is essential for dermatan sulfate formation in vivo. Our results indicate that adducted thumb-clubfoot syndrome is a disorder resulting from a defect specific to dermatan sulfate biosynthesis and emphasize roles for dermatan sulfate in human development and extracellular-matrix maintenance.

A pituitary N-acetylgalactosamine transferase that specifically recognizes glycoprotein hormones

The glycoprotein hormones lutropin (LH) and follitropin (FSH), which have common alpha-subunits but hormone-specific beta-subunits, are both synthesized in the gonadotroph. However, they bear Asn-linked oligosaccharides that differ in structure. Those on LH terminate with the sequence SO4-4GalNAc beta 1----4GlcNAc beta 1----2Man alpha, whereas those on FSH terminate with the sequence sialic acid alpha-Gal beta 1----4GlcNAc beta 1----2Man alpha. A GalNAc-transferase was identified in bovine pituitary membranes that recognizes features of the alpha-subunit peptide and adds GalNAc to its oligosaccharides with an apparent Michaelis constant of 25 micromolar. The different patterns of glycosylation for LH and FSH indicate that access to the protein recognition marker on the alpha-subunit is modulated by the associated beta-subunit. The tightly regulated synthesis of sulfated and sialylated oligosaccharides on the pituitary glycoprotein hormones suggests these oligosaccharides have an important biological role.

From legumes to leukocytes: biological roles for sulfated carbohydrates.

Carbohydrates attached to proteins and lipids characteristically display complex and heterogeneous structures. However, it is becoming increasingly clear that carbohydrates with definite biological functions also exhibit unique structural features. A number of glycoproteins and glycolipids have been shown to bear oligosaccharides containing sulfate. Often, addition of a sulfate moiety turns a relatively common structural motif into a unique carbohydrate with the potential to be recognized by a specific receptor or lectin. This is clearly the case in three systems in which sulfated oligosaccharides have been shown to play a well‐defined biological role: 1) control of the circulatory half‐life of luteinizing hormone, 2) symbiotic interactions between leguminous plants and nitrogen‐fixing bacteria, and 3) homing of lymphocytes to lymph nodes. The rapidly growing list of glycoproteins and glycolipids identified as bearing sulfated oligosaccharides suggests that sulfated carbohydrates play important biological roles in numerous other systems as well.—Hooper, L. V., Manzells, S. M., Baenziger, J. U. From legumes to leukocytes: biological roles for sulfated carbohydrates. FASEB J. 10, 1137‐1146 (1996)