Closely related mammals have distinct asialoglycoprotein receptor carbohydrate specificities

We recently reported that the rat asialoglycoprotein receptor binds oligosaccharides terminating with sialic acid (Sia) alpha2,6GalNAc. Despite a high percentage of identical amino acids in their sequences, orthologues of the asialoglycoprotein receptor (ASGP-R) in different mammals differ in their specificity for terminal Siaalpha2,6GalNAc. The recombinant subunit 1 of the ASGP-R from the rat (RHL-1 or rat hepatic lectin) and the mouse (MHL-1 or mouse hepatic lectin), which differ at only 12 positions in the amino acid sequence of their carbohydrate recognition domains, binds Siaalpha2,6GalNAcbeta1,4GlcNAcbeta1,2Man-bovine serum albumin and GalNAcbeta1,4GlcNAcbeta1,2Man-bovine serum albumin in ratios of 16:1.0 and 1.0:1.0, respectively. Mutagenesis was used to show that amino acids both in the immediate vicinity of the proposed binding site for terminal GalNAc and on the alpha2 helix that is distant from the binding site contribute to the specificity for terminal Siaalpha2,6GalNAc. Thus, multiple amino acid sequence alterations in two key locations contribute to the difference in specificity observed for the rat and mouse ASGP-Rs. We hypothesize that the altered specificity of ASPG-R orthologues in such evolutionarily closely related species reflects rapidly changing requirements for recognition of endogenous or exogenous oligosaccharides in vivo.     

The Asialoglycoprotein Receptor Regulates Levels of Plasma Glycoproteins Terminating with Sialic Acid ��2,6-Galactose

The asialoglycoprotein receptor (ASGP-R) is an abundant, carbohydrate-specific, endocytic receptor expressed by parenchymal cells of the liver. We recently demonstrated that the ASGP-R mediates the clearance of glycoproteins bearing Siaα2,6GalNAc as well as those bearing terminal Gal or GalNAc. We now report that glycoproteins such as haptoglobin, serum amyloid protein (SAP), and carboxylesterase that bear oligosaccharides with terminal Siaα2,6Gal are elevated in the plasma of ASGP-R-deficient mice. Because of their abundance in plasma, glycoproteins bearing terminal Siaα2,6Gal will saturate the ASGP-R and compete with each other on the basis of their relative affinities for the ASGP-R and their relative abundance. We propose that the ASGP-R mediates the clearance of glycoproteins that bear oligosaccharides terminating with Siaα2,6Gal and thereby helps maintain the relative concentrations of these glycoproteins in the blood.The asialoglycoprotein receptor (ASGP-R)³ was initially identified and characterized by Ashwell and co-workers (1, 2) on the basis of its ability to rapidly remove glycoproteins bearing oligosaccharides terminating with β1,4-linked Gal from the circulation. The ASGP-R has been extensively characterized since its initial discovery; however, its biologic function in vivo has remained unclear. This endocytic receptor is highly abundant with 500,000 receptors expressed at the plasma membrane of hepatocytes (3–5) and is rapidly internalized (3, 6). The abundance of the ASGP-R and its rapid rate of internalization in combination with the large number of hepatocytes that are present in the liver, 1.35 × 10⁸/g of liver (7, 8), results in an enormous potential capacity to remove glycoproteins from the circulation. Until recently, mice that have had either subunit of the ASGP-R ablated, subunit 1 ASGP-R1(-/-) or subunit 2 ASGP-R2(-/-), have not been reported to have altered levels of circulating glycoproteins in their blood or to have a physiologic phenotype (9, 10). However, Grewal et al. (11) have reported that the ASGP-R plays a role in von Willebrand factor homeostasis and promotes thrombocytopenia during Steptococcus pneumoniae sepsis by removing platelets that have had their surface sialic acid removed by the bacterial neuraminidase.We recently established that glycoproteins bearing Asn-linked oligosaccharides terminating with the sequence Siaα2,6GalNAcβ1,4GlcNAc are recognized by the ASGP-R and rapidly removed from the blood (12, 13). Glycoproteins bearing terminal Siaα2,6GalNAcβ1,4GlcNAc are the first examples of endogenous glycoproteins that can be recognized by the ASGP-R without further modification; i.e. removal of terminal Sia. Glycoproteins bearing these structures, for example the prolactin-like proteins (14), glycodelin (15), urokinase (16), and glycoprotein hormones (17), are not highly abundant, suggesting that the ASGP-R recognizes and clears additional more abundant glycoproteins. The most likely candidates are glycoproteins bearing Asn-linked oligosaccharides that terminate with the sequence Siaα2,6Galβ1, 4GlcNAc. We have reported that the ASGP-R recognizes these structures with an avidity that is in the micromolar range (13). The avidity of the ASGP-R for structures terminating with Siaα2,6Galβ1,4GlcNAc is predicted to be sufficient to mediate binding and clearance of glycoproteins bearing structures terminating with Siaα2,6Galβ1,4GlcNAc from the blood. This concept is supported by indications that neo-glycoproteins bearing structures terminating with Siaα2,6Galβ1,4GlcNAc are removed from the blood at a faster rate than those bearing Siaα2,3Galβ1,4GlcNAc (18). Slow clearance of glycoproteins bearing Siaα2,6Galβ1,4GlcNAc, however, hampers accurate measurement of their half-lives by injection of radiolabeled ligands.We now report that multiple glycoproteins bearing oligosaccharides that terminate with Siaα2,6Galβ1,4GlcNAc are elevated in the plasma of ASGP-R-deficient ASGP-R2(-/-) mice as compared with wild-type (Wt) mice. The elevation of multiple glycoproteins bearing terminal Siaα2,6Galβ1,4GlcNAc supports our proposal that the ASGP-R accounts for the clearance of these glycoproteins. This previously undiscerned role of the ASGP-R now allows us to develop a model of how this receptor may contribute to the regulation of the concentration of many different glycoproteins in the blood.     

N-Acetylglucosaminyltransferase IX acts on the GlcNAc beta 1,2-Man alpha 1-Ser/Thr moiety, forming a 2,6-branched structure in brain O-mannosyl glycan

Mammals contain O-linked mannose residues with 2-mono- and 2,6-di-substitutions by GlcNAc in brain glycoproteins. It has been demonstrated that the transfer of GlcNAc to the 2-OH position of the mannose residue is catalyzed by the enzyme, protein O-mannose beta1,2-N-acetylglucosaminyltransferase (POMGnT1), but the enzymatic basis of the transfer to the 6-OH position is unknown. We recently reported on a brain-specific beta1,6-N-acetylglucosaminyltransferase, GnT-IX, that catalyzes the transfer of GlcNAc to the 6-OH position of the mannose residue of GlcNAcbeta1,2-Manalpha on both the alpha1,3- and alpha1,6-linked mannose arms in the core structure of N-glycan (Inamori, K., Endo, T., Ide, Y., Fujii, S., Gu, J., Honke, K., and Taniguchi, N. (2003) J. Biol. Chem. 278, 43102-43109). Here we examined the issue of whether GnT-IX is able to act on the same sequence of the GlcNAcbeta1,2-Manalpha in O-mannosyl glycan. Using three synthetic Ser-linked mannose-containing saccharides, Manalpha1-Ser, GlcNAcbeta1,2-Manalpha1-Ser, and Galbeta1,4-GlcNAcbeta1,2-Manalpha1-Ser as acceptor substrates, the findings show that (14)C-labeled GlcNAc was incorporated only into GlcNAcbeta1,2-Manalpha1-Ser after separation by thin layer chromatography. To simplify the assay, high performance liquid chromatography was employed, using a fluorescence-labeled acceptor substrate GlcNAcbeta1,2-Manalpha1-Ser-pyridylaminoethylsuccinamyl (PAES). Consistent with the above data, GnT-IX generated a new product which was identified as GlcNAcbeta1,2-(GlcNAcbeta1,6-)Manalpha1-Ser-PAES by mass spectrometry and (1)H NMR. Furthermore, incorporation of an additional GlcNAc residue into a synthetic mannosyl peptide Ac-Ala-Ala-Pro-Thr(Man)-Pro-Val-Ala-Ala-Pro-NH(2) by GnT-IX was only observed in the presence of POMGnT1. Collectively, these results strongly suggest that GnT-IX may be a novel beta1,6-N-acetylglucosaminyltransferase that is responsible for the formation of the 2,6-branched structure in the brain O-mannosyl glycan.     

Structural analysis of murine zona pellucida glycans. Evidence for the expression of core 2-type O-glycans and the Sd(a) antigen

Murine sperm initiate fertilization by binding to specific oligosaccharides linked to the zona pellucida, the specialized matrix coating the egg. Biophysical analyses have revealed the presence of both high mannose and complex-type N-glycans in murine zona pellucida. The predominant high mannose-type glycan had the composition Man(5)GlcNAc(2), but larger oligosaccharides of this type were also detected. Biantennary, triantennary, and tetraantennary complex-type N-glycans were found to be terminated with the following antennae: Galbeta1-4GlcNAc, NeuAcalpha2-3Galbeta1-4GlcNAc, NeuGcalpha2-3Galbeta1-4GlcNAc, the Sd(a) antigen (NeuAcalpha2-3[GalNAcbeta1-4]Galbeta1-4GlcNAc, NeuGcalpha2-3[GalNAcbeta1-4]Galbeta1-4GlcNAc), and terminal GlcNAc. Polylactosamine-type sequence was also detected on a subset of the antennae. Analysis of the O-glycans indicated that the majority were core 2-type (Galbeta1-4GlcNAcbeta1-6[Galbeta1-3]GalNAc). The beta1-6-linked branches attached to these O-glycans were terminated with the same sequences as the N-glycans, except for terminal GlcNAc. Glycans bearing Galbeta1-4GlcNAcbeta1-6 branches have previously been suggested to mediate initial murine gamete binding. Oligosaccharides terminated with GalNAcbeta1-4Gal have been implicated in the secondary binding interaction that occurs following the acrosome reaction. The significant implications of these observations are discussed.     

Galactosylation of N-linked oligosaccharides by human beta-1,4-galactosyltransferases I, II, III, IV, V, and VI expressed in Sf-9 cells

Several studies showed that Sf-9 cells can synthesize the galactosylated N-linked oligosaccharides if beta-1,4-galactosyltransferase (beta-1,4-GalT) is supplied. The full-length human beta-1,4-GalT I, II, III, IV, V, and VI cDNAs were independently transfected into Sf-9 cells, and the galactosylation of endogenous membrane glycoproteins was examined by lectin blot analysis using Ricinus communis agglutinin-I (RCA-I), which preferentially interacts with oligosaccharides terminated with Galbeta1-->4GlcNAc group. Several RCA-I-reactive bands appeared in all of the gene-transfected cells, and disappeared on treatment of blots with beta-1,4-galactosidase or N-glycanase prior to incubation with lectin. Introduction of the antisense beta-1,4-GalT II and V cDNAs separately into human colorectal adenocarcinoma SW480 cells, in which beta-1,4-GalT I, II, and V genes were expressed, resulted in the reduction of RCA-I binding toward N-linked oligosaccharides of the membrane glycoproteins. Differences were found in their K(m) values toward UDP-Gal and GlcNAcbeta-S-pNP and in their acceptor specificities toward oligosaccharides with the GlcNAcbeta1-->4(GlcNAcbeta1-->2)Man branch and with the GlcNAcbeta1-->6(GlcNAcbeta1-->2)Man branch. These results indicate that beta-1,4-GalTs II, III, IV, V, and VI are involved in the N-linked oligosaccharide biosynthesis cooperatively but not in a redundant manner with beta-1,4-GalT I within cells.     

The sialylated oligosaccharides of recombinant bovine lutropin modulate hormone bioactivity

The Asn-linked oligosaccharides from bovine lutropin (bLH(Pit] are predominantly dibranched complex-type structures with the terminal sequence SO4-4GalNAc beta 1,4GlcNAc beta 1,2Man alpha. Recombinant bLH expressed in Chinese hamster ovary cells (bLH(CHO] bears di- (60%) and tribranched (30%) complex-type oligosaccharides; however, these terminate in the sequence Sia alpha 2,3Gal beta 1,4GlcNAc beta 1,2Man alpha. In contrast to the limited spectrum of oligosaccharide structures present on recombinant bLH(CHO), the endogenous glycoproteins synthesized by CHO cells bear a heterogeneous array of Asn-linked oligosaccharides with 0, 1, 2, 3, or 4 sialic acid moieties. The sialic acid moieties on the Asn-linked oligosaccharides of both endogenous glycoproteins and recombinant bLH(CHO) are exclusively alpha 2,3-linked, suggesting that the alpha 2,6-sialyl-transferase is not active in CHO cells. The bioactivities of bLH(Pit) and bLH(CHO) were compared using MA-10 cells following sequential digestion with neuraminidase and beta-galactosidase. Neither the ED50 (dose producing 50% of the maximum response) for progesterone production (7.2 ng/ml) nor the Pmax (maximum level of progesterone produced) (470 ng/ml) was altered for bLH(Pit) by these treatments, consistent with the absence of either sialic acid or Gal on bLH(Pit). The ED50 for progesterone production by recombinant bLH(CHO) (16.4 ng/ml) was significantly greater than for bLH(Pit) but was reduced to 5.3 ng/ml following removal of terminal sialic acid. Removal of the subterminal Gal was without further effect. The Pmax for bLH(CHO) (180 ng/ml) was not altered by these treatments. The reduction in bLH(CHO) bioactivity caused by the presence of terminal sialic acid suggests that the presence of terminal sulfate on bLH(Pit) oligosaccharides may also reduce its bioactivity and may play a modulatory role in regulating hormone bioactivity.     

Novel poly-GalNAcbeta1-4GlcNAc (LacdiNAc) and fucosylated poly-LacdiNAc N-glycans from mammalian cells expressing beta1,4-N-acetylgalactosaminyltransferase and alpha1,3-fucosyltransferase

Glycans containing the GalNAcbeta1-4GlcNAc (LacdiNAc or LDN) motif are expressed by many invertebrates, but this motif also occurs in vertebrates and is found on several mammalian glycoprotein hormones. This motif contrasts with the more commonly occurring Galbeta1-4GlcNAc (LacNAc or LN) motif. To better understand LDN biosynthesis and regulation, we stably expressed the cDNA encoding the Caenorhabditis elegans beta1,4-N-acetylgalactosaminyltransferase (GalNAcT), which generates LDN in vitro, in Chinese hamster ovary (CHO) Lec8 cells, to establish L8-GalNAcT CHO cells. The glycan structures from these cells were determined by mass spectrometry and linkage analysis. The L8-GalNAcT cell line produces complex-type N-glycans quantitatively bearing LDN structures on their antennae. Unexpectedly, most of these complex-type N-glycans contain novel "poly-LDN" structures consisting of repeating LDN motifs (-3GalNAcbeta1-4GlcNAcbeta1-)n. These novel structures are in contrast to the well known poly-LN structures consisting of repeating LN motifs (-3Galbeta1-4GlcNAcbeta1-)n. We also stably expressed human alpha1,3-fucosyltransferase IX in the L8-GalNAcT cells to establish a new cell line, L8-GalNAcT-FucT. These cells produce complex-type N-glycans with alpha1,3-fucosylated LDN (LDNF) GalNAcbeta1-4(Fucalpha1-3)GlcNAcbeta1-R as well as novel "poly-LDNF" structures (-3GalNAcbeta1-4(Fucalpha 1-3)GlcNAcbeta1-)n. The ability of these cell lines to generate glycoprotein hormones with LDN-containing N-glycans was studied by expressing a recombinant form of the common alpha-subunit in L8-GalNAcT cells. The alpha-subunit N-glycans carried LDN structures, which were further modified by co-expression of the human GalNAc 4-sulfotransferase I, which generates SO4-4GalNAcbeta1-4GlcNAc-R. Thus, the generation of these stable mammalian cells will facilitate future studies on the biological activities and properties of LDN-related structures in glycoproteins.     

Oligosaccharide preferences of beta1,4-galactosyltransferase-I: crystal structures of Met340His mutant of human beta1,4-galactosyltransferase-I with a pentasaccharide and trisaccharides of the N-glycan moiety

beta-1,4-Galactosyltransferase-I (beta4Gal-T1) transfers galactose from UDP-galactose to N-acetylglucosamine (GlcNAc) residues of the branched N-linked oligosaccharide chains of glycoproteins. In an N-linked biantennary oligosaccharide chain, one antenna is attached to the 3-hydroxyl-(1,3-arm), and the other to the 6-hydroxyl-(1,6-arm) group of mannose, which is beta-1,4-linked to an N-linked chitobiose, attached to the aspargine residue of a protein. For a better understanding of the branch specificity of beta4Gal-T1 towards the GlcNAc residues of N-glycans, we have carried out kinetic and crystallographic studies with the wild-type human beta4Gal-T1 (h-beta4Gal-T1) and the mutant Met340His-beta4Gal-T1 (h-M340H-beta4Gal-T1) in complex with a GlcNAc-containing pentasaccharide and several GlcNAc-containing trisaccharides present in N-glycans. The oligosaccharides used were: pentasaccharide GlcNAcbeta1,2-Manalpha1,6 (GlcNAcbeta1,2-Manalpha1,3)Man; the 1,6-arm trisaccharide, GlcNAcbeta1,2-Manalpha1,6-Manbeta-OR (1,2-1,6-arm); the 1,3-arm trisaccharides, GlcNAcbeta1,2-Manalpha1,3-Manbeta-OR (1,2-1,3-arm) and GlcNAcbeta1,4-Manalpha1,3-Manbeta-OR (1,4-1,3-arm); and the trisaccharide GlcNAcbeta1,4-GlcNAcbeta1,4-GlcNAc (chitotriose). With the wild-type h-beta4Gal-T1, the K(m) of 1,2-1,6-arm is approximately tenfold lower than for 1,2-1,3-arm and 1,4-1,3-arm, and 22-fold lower than for chitotriose. Crystal structures of h-M340H-beta4Gal-T1 in complex with the pentasaccharide and various trisaccharides at 1.9-2.0A resolution showed that beta4Gal-T1 is in a closed conformation with the oligosaccharide bound to the enzyme, and the 1,2-1,6-arm trisaccharide makes the maximum number of interactions with the enzyme, which is in concurrence with the lowest K(m) for the trisaccharide. Present studies suggest that beta4Gal-T1 interacts preferentially with the 1,2-1,6-arm trisaccharide rather than with the 1,2-1,3-arm or 1,4-1,3-arm of a bi- or tri-antennary oligosaccharide chain of N-glycan.     

Tumor antigen occurs in N-glycan of royal jelly glycoproteins: honeybee cells synthesize T-antigen unit in N-glycan moiety

In our previous paper (Kimura, Y., et al., Biosci. Biotechnol. Biochem., 67, 1852-1856, 2003), we found that a complex type N-glycans containing beta1-3 galactose residue occurs on royal jelly glycoproteins. During structural analysis of minor components of royal jelly N-glycans, we found complex type N-glycans bearing both galactose and N-acetylgalactosamine residues. Detailed structural analysis of pyridylaminated oligosaccharide revealed that the newly found N-glycan had a complex type structure harboring a tumor marker (T-antigen) unit: Galbeta1-3GalNAcbeta1-4GlcNAcbeta1-2Manalpha1-6 (Galbeta1-3GalNAcbeta1-4GlcNAcbeta1-2Manalpha1-3) Manbeta1-4GlcNAcbeta1-4GlcNAc. To our knowledge, this may be the first report of the presence of the T-antigen unit in the N-glycan moiety of eucaryotic glycoproteins.     

Isolation and characterization of endogenous ligands for liver mannan-binding protein

Endogenous ligands for the hepatic lectin which is specific for mannose and N-acetylglucosamine (mannan-binding protein, MBP) were isolated from rat liver rough microsomes and primary cultured hepatocytes by affinity chromatography on an immobilized MBP column. Western blotting using specific antisera revealed that serum glycoproteins, alpha 1-macroglobulin, alpha 1-antitrypsin, and alpha 1-acid glycoprotein, and a lysosomal enzyme, beta-glucuronidase were the major constituents of the endogenous ligands. These endogenous ligands consisted of high mannose-type oligosaccharides of Man9GlcNAc2 and Man8GlcNAc2, and had rapid turnover rates with an average half-life of 45 min, indicating that they were mainly composed of biosynthetic intermediates of glycoproteins. In view of the identification of the endogenous ligands as the biosynthetic intermediates of glycoproteins, the possible functions of the intracellular lectin are discussed in relation to the intracellular transport of glycoproteins.     

Molecular cloning and characterization of human GnT-IX,a novel beta1,6-N-acetylglucosaminyltransferase that is specifically expressed in the brain

A novel beta1,6-N-acetylglucosaminyltransferase (beta1, 6GnT) cDNA was identified by a BLAST search using the amino acid sequence of human GnT-V as a query. The full-length sequence was determined by a combination of 5'-rapid amplification of cDNA end analysis and a further data base search. The open reading frame encodes a 792 amino acid protein with a type II membrane protein structure typical of glycosyltransferases. The entire sequence identity to human GnT-V is 42%. When pyridylaminated (PA) agalacto biantennary N-linked oligosaccharide was used as an acceptor substrate, the recombinant enzyme generated a novel product other than the expected GnT-V product, (GlcNAcbeta1,2-Manalpha1,3-)[GlcNAcbeta1,2-(GlcNAcbeta1,6-)Manalpha1,6-]Manbeta1,4-GlcNAcbeta1,4-GlcNAc-PA. This new product was identified as [GlcNAcbeta1,2-(GlcNAcbeta1,6-)Manalpha1,3-][Glc-NAcbeta1,2-(GlcNAcbeta1,6-)Manalpha1,6-]Manbeta1,4-GlcNAcbeta1,4-GlcNAc-PA by mass spectrometry and 1H NMR. Namely, the new GnT (designated as GnT-IX) has beta1,6GnT activity not only to the alpha1,6-linked mannose arm but also to the alpha1,3-linked mannose arm of N-glycan, forming a unique structure that has not been reported to date. Northern blot analysis showed that the GnT-IX gene is exclusively expressed in the brain, whereas the GnT-V gene is expressed ubiquitously. These results suggest that GnT-IX is responsible for the synthesis of a unique oligosaccharide structure in the brain.     

The hepatic asialoglycoprotein receptor selectively binds to some endogenous tissues

The hepatic asialoglycoprotein receptor (ASGP-R) was isolated from various rat tissues or freshly prepared single cell suspensions and tested for the binding to endogenous tissues or specific cell types by indirect immunofluorescence. Inhibition with N-acetyl-D-galactosamine demonstrated specificity of binding. ASGP-R binds to mesodermal tissues and to selected cells of the majority of glandular tissues but not to lining epithelia. ASGP-R stains heart muscle but not skeletal muscle. In addition, ASGP-R stains spleen cells (52%), bone marrow cells (55%), thymocytes (62%), and a fraction of peripheral blood lymphocytes (29%), which was identified as B-lymphocytes. Five different rat tumors also showed binding of ASGP-R. The binding pattern and staining intensity of peanut agglutinin and soybean agglutinin were strikingly different although the binding specificity of these lectins is related to the ASPG-R. It is concluded that considerable numbers of endogenous binding sites for the hepatic ASGP-R exist in normal tissue, even on cells which pass the liver on circulation.     

Tumor-related expression of alpha1,2fucosylated antigens on colorectal carcinoma cells and its suppression by cell-mediated priming using sugar acceptors for alpha1,2fucosyltransferase

The accumulation of alpha1,2fucosylated antigens, such as Y (Fucalpha1,2Galbeta1,4 [Fucalpha1,3]GlcNAcbeta), Le(b) (Fucalpha1,2Galbeta1,3-[Fucalpha1,4]GlcNAcbeta), and H type 2 (Fucalpha1,2 Galbeta1,4GlcNAcbeta) occurs specifically within human colorectal tumor tissues and can be detected by an antifucosylated antigen antibody, such as the YB-2 antibody. In the present investigation, we found that the expression of these antigens bearing an alpha1,2-linked fucose correlated with the resistance of the tumor cells to anticancer treatments. Addition of an exogenous sugar acceptor for alpha1,2fucosyltransferase to the cell medium resulted in suppression of alpha1,2fucosylated antigen expression on the tumor cells and increased susceptibility to anticancer treatment. The increased susceptibility may be attributed to cancer cell-mediated priming by sugar acceptors for alpha1,2fucosyltransferase added to the medium.     

Lectin affinity high-performance liquid chromatography. Interactions of N-glycanase-released oligosaccharides with Ricinus communis agglutinin I and Ricinus communis agglutinin II

The structural determinants required for interaction of oligosaccharides with Ricinus communis agglutinin I (RCAI) and Ricinus communis agglutinin II (RCAII) have been studied by lectin affinity high-performance liquid chromatography (HPLC). Homogeneous oligosaccharides of known structure, purified following release from Asn with N-glycanase and reduction with NaBH4, were tested for their ability to interact with columns of silica-bound RCAI and RCAII. The characteristic elution position obtained for each oligosaccharide was reproducible and correlated with specific structural features. RCAI binds oligosaccharides bearing terminal beta 1,4-linked Gal but not those containing terminal beta 1,4-linked GalNAc. In contrast, RCAII binds structures with either terminal beta 1,4-linked Gal or beta 1,4-linked GalNAc. Both lectins display a greater affinity for structures with terminal beta 1,4-rather than beta 1,3-linked Gal, although RCAII interacts more strongly than RCAI with oligosaccharides containing terminal beta 1,3-linked Gal. Whereas terminal alpha 2,6-linked sialic acid partially inhibits oligosaccharide-RCAI interaction, terminal alpha 2,3-linked sialic acid abolishes interaction with the lectin. In contrast, alpha 2,3- and alpha 2,6-linked sialic acid equally inhibit but do not abolish oligosaccharide interaction with RCAII. RCAI and RCAII discriminate between N-acetyllactosamine-type branches arising from different core Man residues of dibranched complex-type oligosaccharides; RCAI has a preference for the branch attached to the alpha 1,3-linked core Man and RCAII has a preference for the branch attached to the alpha 1,6-linked core Man. RCAII but not RCAI interacts with certain di- and tribranched oligosaccharides devoid of either Gal or GalNAc but bearing terminal GlcNAc, indicating an important role for GlcNAc in RCAII interaction. These findings suggest that N-acetyllactosamine is the primary feature required for oligosaccharide recognition by both RCAI and RCAII but that lectin interaction is strongly modulated by other structural features. Thus, the oligosaccharide specificities of RCAI and RCAII are distinct, depending on many different structural features including terminal sugar moieties, peripheral branching pattern, and sugar linkages.     

Structure elucidation of NeuAc, NeuGc and Kdn-containing O-glycans released from Triturus alpestris oviductal mucins

Eggs from Amphibia are surrounded by several layers of jelly that are needed for proper fertilization. Jelly coat is composed of highly glycosylated mucin-type glycoproteins containing up to 60% of carbohydrates, which display a remarkable species-specificity. This material obtained from Triturus alpestris was submitted to reductive beta-elimination and the released oligosaccharide-alditols were further fractionated by HPLC. Structural characterization was performed through a combination of two dimensional (1)H-(1)H and (1)H-(13)C NMR and ESI-MS/MS analysis. Numerous carbohydrate chains are characterized by the presence of the Cad (Sd(a)) determinant, including respectively NeuAc, NeuGc or Kdn as a sialic acid. But the most significant O-glycan sequences which mark the difference between the jelly of T. alpestris and other studies amphibian jellies are polymers of GalNAc(beta 1-4)GlcNAc (LacdiNAc) which form part of the following sequence: HSO(3)(4)(GalNAcbeta 1-4GlcNAcbeta 1-3)(1-3)GalNAcbeta 1-4(GlcNAcbeta 1-3)(0-1)GlcNAcbeta 1-6GalNAc-ol.     

Functional expression and genomic structure of human N-acetylglucosamine-6-O-sulfotransferase that transfers sulfate to beta-N-acetylglucosamine at the nonreducing end of an N-acetyllactosamine sequence

The cDNA and gene encoding human N-acetylglucosamine-6-O-sulfotransferase (Gn6ST) have been cloned. Comparative analysis of this cDNA with the mouse Gn6ST sequence indicates 96% amino acid identity between the two sequences. The expression of a soluble recombinant form of the protein in COS-1 cells produced an active sulfotransferase, which transferred sulfate to the terminal GlcNAc in GlcNAcbeta1-O-CH(3), GlcNAcbeta1-3Galbeta1-O-CH(3) and GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4Gl cNAc but not in GlcNAcalpha1-4GlcAbeta1-3Galbeta1-3Galbeta1-4 Xylbeta1-O-Ser. In addition, neither Galbeta1-4GlcNAcbeta1-O-naphthalenemethanol nor GalNAcbeta1-4GlcAbeta1-3Galbeta1-3Galbeta1-4X ylbeta1-O-Ser were utilized as acceptors. These findings indicate that a terminal beta-linked GlcNAc residue is necessary for acceptor substrates of Gn6ST. The human Gn6ST gene spans about 7 kb, consists of two exons and exhibits an intron-less coding region.     

A 3D model for the human hepatic asialoglycoprotein receptor (ASGP-R)

The human hepatic Asialoglycoprotein Receptor (ASGP-R) consists of two different types of liver specific membrane glycoproteins that bind to terminal galactose and N-acetylgalactosamine residues of serum glycoproteins. The two different polypeptide chains are referred to as two receptor subunits, HH1 and HH2, which are both involved in the activity of the functional receptor. This receptor has served as a model for understanding receptor-mediated endocytosis and carbohydrate mediated recognition phenomena. Here models for the C-terminal extracellular region of both HH1 and HH2 subunit are presented. The standard homology building procedure was modified in order to make it suitable for the modeling problem at hand. The models for the extracellular regions of HH1 and HH2 were initially constructed by exploiting several fragments, belonging to proteins of known 3D structure, and showing high local sequence similarity with respect to the glycoproteins of interest. Putative binding sites were first hypothesized on the basis of the comparison with other complexes of lectins, the crystal structure of which was available in the Protein Data Bank. A model for the complex involving the HH2 subunit and the typical high affinity ligand N-acetylgalactosamine (NacGal) was refined as the first by a suitable combination of MD simulations and Energy Minimization calculations, since it seemed to quickly converge to a plausible structure. An intermediate model for HH1 was then rebuilt on the basis of the refined model for HH2. It was then submitted to a sequence of molecular dynamics simulations with templates which took into account the secondary structure prediction for a final refinement. The structures of small regions of the models, located around the binding sites, were compared with more recent crystallographic data regarding a complex involving the mutant of Mannose Binding Protein QPDWGH (1BCH entry in the Protein Data Bank) and NacGal. This mutant shows high local sequence similarity with HH1 and HH2 at the binding sites. On the basis of the above comparison, different locations of the binding sites were also considered. In addition to other expected interactions, two hydrophobic interactions were observed in the models with Trp residues (positions 243 in HH1 and 181 or 267 in HH2 respectively) and His residues (positions 256 in HHI and 184 in HH2.respectively). The quality of the models was evaluated by the Procheck program and they seemed plausible. This observation together with analogies found between binding sites of the models and IBCH supported the validity of the models. A further validation element arose by comparison between experimental binding data available in the literature about the homologous rat receptor subunits and theoretical interaction energies evaluated, by means of the DOCK 3.5 program, in models for the rat subunits obtained from the corresponding human ones. The new modeling procedure used here appears to be a well-suited method for structural analysis of small regions, located around the ligands, in proteins of unknown 3D structure.     

Glycoprotein hormone GalNAc-4-sulphotransferase

The glycoprotein hormones lutropin (LH) and thyrotropin and a limited number of additional glycoproteins bear carbohydrate structures terminating with the unique sequence SO(4)-4-GalNAcbeta1,4GlcNAcbeta that has been conserved in the glycoprotein hormones of all vertebrate species. Synthesis of these structures is mediated by a protein-specific beta1,4GalNAc-transferase and a GalNAc-4-sulphotransferase (GalNAc-4-ST1). GalNAc-4-ST1 is a member of a family of sulphotransferases that includes HNK-1 sulphotransferase, chondroitin-4-sulphotransferases-1-3 and dermatan-4-sulphotransferase-1. With the exception of HNK-1-ST, these sulphotransferases add sulphate to the C-4 hydroxy group of either terminal or non-terminal beta1,4-linked GalNAc. GalNAc-4-ST1 is most highly expressed in pituitary, cerebellum and other regions of the brain. The terminal GalNAcSO(4) on LH is recognized by the cysteine-rich domain of the mannose/GalNAc-4-SO(4) receptor located in hepatic endothelial cells. Each cysteine-rich domain binds a single terminal GalNAc-4-SO(4), and the receptor must form non-covalently associated homodimers in order to simultaneously engage two GalNAc-4-SO(4) moieties on separate oligosaccharides with sufficient affinity to form stable complexes. The receptor mediates the clearance of LH from the blood. This clearance, in conjunction with the stimulated release of hormone from dense core granules in pituitary gonadotroph cells, is required to produce the episodic rise and fall in LH levels needed for optimal oestrogen production during the implantation of embryos in the uterus.