Variations in the chondroitin sulfate-protein linkage region of aggrecans from bovine nasal and human articular cartilages

Aggrecan-derived chondroitin sulfate (CS) chains, released by beta-elimination, were derivatized with p-aminobenzoic acid or p-aminophenol; radioiodinated; and subjected to graded or complete degradations by chondroitin ABC lyase to generate linkage region fragments of the basic structure DeltaGlyUA-GalNAc-GlcUA-Gal-Gal-Xyl-R (where DeltaGlyUA represents 4, 5-unsaturated glycuronic acid, and R is the adduct), by chondroitin AC lyase to generate the shorter fragment DeltaGlyUA-Gal-Gal-Xyl-R, or by chondroitin C lyase to generate the same fragment when it was linked to a 6-O-sulfated or unsulfated GalNAc at the nonreducing end. Fragments were separated by size using gel chromatography, by charge using ion-exchange chromatography, and by size/charge using electrophoresis and then characterized by stepwise degradations from the nonreducing end by using mercuric acetate to remove all terminal DeltaGlyUA, by bacterial glycuronidase to remove the same residue when linked to unsulfated or 6-O-sulfated GalNAc/Gal, by mammalian 4-sulfatase to remove sulfate from terminal GalNAc 4-O-sulfate, by chondro-4-sulfatase to remove 4-O-sulfate from other GalNAc/Gal residues, and by beta-galactosidase to remove terminal Gal. Results with CS from bovine nasal cartilage aggrecan show that, in nearly all chains, Xyl and probably also the first Gal are unsubstituted, whereas the second Gal is 4-O-sulfated in one CS chain out of five. The first disaccharide repeat is sulfated at C-4 of GalNAc in one chain out of three and unsulfated in the other two. A sulfated first disaccharide is always joined to an unsulfated GlcUA-Gal-Gal sequence. In contrast, CS from human articular cartilage usually has a sulfated first disaccharide repeat. In CS from young human cartilage, sulfate groups are mostly at C-4 of GalNAc in the major part of the chain, but at C-6 in the nonreducing distal portion. In CS from old cartilage, sulfation at C-6 of GalNAc is a major feature from the nonreducing end down to approximately positions 4 and 5 from the linkage region, where GalNAc 4-O-sulfate is common.     

Structure determination for octasaccharides derived from the carbohydrate-protein linkage region of chondroitin sulphate chains in the proteoglycan aggrecan from bovine articular cartilage

Five octasaccharides derived from the protein carbohydrate linkage region of chondroitin sulphate (CS) have been isolated from the large aggregating proteoglycan (aggrecan) extracted from the bovine articular cartilage of 6-year-old to 8-year-old animals. Following the purification of aggrecan the attached CS chains were digested with CS ABC endolyase and subsequently released from the protein core by beta-elimination. The individual oligosaccharides were purified by strong anion-exchange chromatography and their structures determined by very high-field one-dimensional and two-dimensional 1H-NMR spectroscopy. They were found to be octasaccharides, comprised of tetrasaccharide repeat-region extensions to the core tetrasaccharide linkage region structure. They have the following structures: deltaUA(beta1-3)GalNAc(beta1-4)GlcA(beta1-3)GalNAc(beta1-4)+ ++GlcA(beta1-3)Gal(beta1-3)Gal(beta1-4)Xyl-ol, deltaUA(beta1-3)GalNAc(beta1-4)GlcA(beta1-3)GalNAc6S(b eta1-4)GlcA(beta1-3)Gal(beta1-3)Gal(beta1-4)Xyl-ol, deltaUA(beta1-3)GalNAc6S(beta1-4)GlcA(beta1-3)GalNAc(b eta1-4)GlcA(beta1-3)Gal(beta1-3)Gal(beta1-4)Xyl-ol, deltaUA(beta1-3)GalNAc6S(beta1-4)GlcA(beta1-3)GalNA c6S(beta1-4)GlcA(beta1-3)Gal(beta1-3)Gal(beta1-4)Xyl-ol and deltaUA(beta1-3)GalNAc4S(beta1-4)GlcA(beta1-3)GalNA c6S(beta1-4)GlcA(beta1-3)Gal(beta1-3)Gal(beta1-4)Xyl-ol. They differ only in the nature of the sulphation of the GalNAc residues of the tetrasaccharide-repeat-region extension, which forms the first two disaccharides of the repeat region. No sulphation of any of the uronic acid residues has been identified and in one oligosaccharide neither of the GalNAc residues were sulphated. The majority of the linkage regions contained GalNAc residues which were fully 6-sulphated. However, in a significant amount, only one of the residues was 6-sulphated while the other was either unsulphated or 4-sulphated. There was no evidence either for sulphation of the linkage region galactose residues or for phosphorylation of the xylose residue, through which the chain is attached to the core protein.     

Characterization of the specificities of human blood group H gene-specified alpha 1,2-L-fucosyltransferase toward sulfated/sialylated/fucosylated acceptors: evidence for an inverse relationship between alpha 1,2-L-fucosylation of Gal and alpha 1,6-L-fucosylation of asparagine-linked GlcNAc

The assembly of complex structures bearing the H determinant was examined by characterizing the specificities of a cloned blood group H gene-specified alpha 1,2-L-fucosyltransferase (FT) toward a variety of sulfated, sialylated, or fucosylated Gal beta 1,3/4GlcNAc beta- or Gal beta 1,3GalNAc alpha-based acceptor structures. (a) As compared to the basic type 2, Gal beta 1,4GlcNAc beta-(K(m) = 1.67 mM), the basic type 1 was 137% active (K(m) = 0.83 mM). (b) On C-6 sulfation of Gal, type 1 became 142.1% active and type 2 became 223.0% active (K(m) = 0.45 mM). (c) On C-6 sulfation of GlcNAc, type 2 showed 33.7% activity. (d) On C-3 or C-4 fucosylation of GlcNAc, both types 1 and 2 lost activity. (e) Type 1 showed 70.8% and 5.8% activity, respectively, on C-6 and C-4 O-methylation of GlcNAc. (f) Type 1 retained 18.8% activity on alpha 2,6-sialylation of GlcNAc. (g) Terminal type 1 or 2 of extended chain had lower activity. (h) With Gal in place of GlcNAc in type 1, the activity became 43.2%. (i) Compounds with terminal alpha 1,3-linked Gal were inactive. (j) Gal beta 1,3GalNAc alpha- (the T-hapten) was approximately 0.4-fold as active as Gal beta 1,4GlcNAc beta-. (k) C-6 sulfation of Gal on the T-hapten did not affect the acceptor activity. (l) C-6 sulfation of GalNAc decreased the activity to 70%, whereas on C-6 sulfation of both Gal and GalNAc the T-hapten lost the acceptor ability. (m) C-6 sialylation of GalNAc also led to inactivity. (n) beta 1,6 branching from GalNAc of the T-hapten by a GlcNAc residue or by units such as Gal beta 1, 4GlcNAc-, Gal beta 1,4(Fuc alpha 1,3)GlcNAc-, or 3-sulfoGal beta 1,4GlcNAc- resulted in 111.9%, 282.8%, 48.3%, and 75.3% activities, respectively. (o) The enhancement of enzyme affinity by a sulfo group on C-6 of Gal was demonstrated by an increase (approximately 5-fold) in the K(m) for Gal beta 1,4GlcNAc beta 1,6(Gal beta 1,3)GalNAc alpha-O-Bn in presence of 6-sulfoGal beta 1,- 4GlcNAc beta-O-Me (3.0 mM). (p) Among the two sites in Gal beta 1, 4GlcNAc beta 1,6(Gal beta 1,3) GalNAc alpha-O-Bn, the enzyme had a higher affinity ( > 3-fold) for the Gal linked to GlcNAc. (q) With respect to Gal beta 1,- 3GlcNAc beta-O-Bn (3.0 mM), fetuin triantennary asialo glycopeptide (2.4 mM), bovine IgG diantennary glycopeptide (2.8 mM), asialo Cowper's gland mucin (0.06 mM), and the acrylamide copolymers (0.125 mM each) containing Gal beta 1,3GlcNAc beta-, Gal beta 1,3(6-sulfo)GlcNAc beta-, Gal beta 1,3GalNAc alpha-, Gal beta 1,3Gal beta-, or Gal alpha 1,3Gal beta- units were 153.6%, 43.0%, 6.2%, 52.5%, 94.9%, 14.7%, 23.6%, and 15.6% active, respectively. (r) Fucosylation by alpha 1,2-L-FT of the galactosyl residue which occurs on the antennary structure of the bovine IgG glycopeptide was adversely affected by the presence of an alpha 1,6-L-fucosyl residue located on the distant glucosaminyl residue that is directly attached to the asparagine of the protein backbone. This became evident from the 4-fold activity of alpha 1,2-L-FT toward bovine IgG glycopeptide after approximately 5% removal of alpha 1,6-linked Fuo.     

Synthesis of oligosaccharides related to HNK-1 antigen. 2. Synthesis of 3'-O-(3-O-sulfo-beta-D-glucuronopyranosyl)-lacto-N-neotetrao se beta-propylglycoside

A derivative of allyl 3"-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-beta-lactoside with a free OH group at C-4GlcNAc was glycosylated with trichloroacetimidate of selectively protected GlcA(beta 1-->3)Gal alpha disaccharide in dichloromethane in the presence of trimethylsilyl triflate resulting in a pentasaccharide product with an 82% yield. This product was converted to monohydroxy derivative with a free OH group at C-3GlcA via the formation and the subsequent opening of the 6,3-lactone ring in the glucuronic acid residue. The 3"'-O-sulfation of the monohydroxy derivative, the removal of the protective groups, and the reduction of the allyl aglycon yielded the pentasaccharide propyl glycoside NaSO3-3GlcA(beta 1-->3)Gal(beta 1-->4)GlcNAc(beta 1-->3)Gal(beta 1-->4)Glc beta-Opr comprising the oligosaccharide chain of the SGGL-1 glycolipid, which is recognized by HNK-1 antibodies. NaSO3-3GlcA(beta 1--> 3)Gal beta OAll, GlcA(beta 1-->3)Gal(beta 1-->4)GlcNAc(beta 1-->3)Gal(beta 1-->4)Glc beta-OPr and GlcA(beta 1-->3)Gal beta OAll were synthesized in a similar way.     

Assessment of glycosaminoglycan-protein linkage tetrasaccharides as acceptors for GalNAc- and GlcNAc-transferases from mouse mastocytoma

Two glycosaminoglycan-protein linkage tetrasaccharide-serine compounds, GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser and GlcAbeta1-3Gal(4-O-sulfate)beta1-3Galbeta1-4Xylbeta1-O -Ser, were tested as hexosamine acceptors, using UDP-[3H]GlcNAc and UDP-[3H]GalNAc as sugar donors, and solubilized mouse mastocytoma microsomes as enzyme source. The nonsulfated Ser-tetrasaccharide was found to function as an acceptor for a GalNAc residue, whereas the Ser-tetrasaccharide containing a sulfated galactose unit was inactive. Characterization of the radio-labelled product by digestion with alpha-N-acetylgalactosaminidase and beta-N-acetylhexosaminidase revealed that the [3H]GalNAc unit was alpha-linked, as in the product previously synthesized using serum enzymes, and not beta-linked as found in the chondroitin sulfate polymer. Heparan sulfate/heparin biosynthesis could not be primed by either of the two linkage Ser-tetrasaccharides, since no transfer of [3H]GlcNAc from UDP-[3H]GlcNAc could be detected. By contrast, transfer of a [3H]GlcNAc unit to a [GlcAbeta1-4GlcNAcalpha1-4]2-GlcAbeta1-4-aMan hexasaccharide acceptor used to assay the GlcNAc transferase involved in chain elongation, was readily detected. These results are in agreement with the recent proposal that two different N-acetylglucosaminyl transferases catalyse the biosynthesis of heparan sulfate. Although the mastocytoma system contains both the heparan sulfate/heparin and chondroitin sulfate biosynthetic enzymes the Ser-tetrasaccharides do not seem to fulfil the requirements to serve as acceptors for the first HexNAc transfer reactions involved in the formation of these polysaccharides.     

Nonreducing end structures of chondroitin sulfate chains on aggrecan isolated from Swarm rat chondrosarcoma cultures

Chondrocyte cultures derived from the Swarm rat chondrosarcoma were metabolically labeled with [35S]sulfate or [6-3H]GlcN. Radiolabeled aggrecan was purified from the cell layer and exhaustively digested with chondroitin ABC lyase. Digestion products were resolved into disaccharide and monosaccharide residues using Toyopearl HW40S chromatography. The separated saccharide pools were reduced with NaBH4 and applied onto a CarboPac PA1 column to resolve all of the internal disaccharide alditols (unsaturated) from the nonreducing end disaccharide (saturated) and monosaccharide alditols. Mercuric acetate treatment was used prior to carbohydrate analysis to identify unambiguously the saturated from the unsaturated disaccharides. The chondroitin sulfate (CS) chains from these aggrecan preparations contained: (a) an internal disaccharide composition of unsulfated (3-4 per chain), 4-sulfated (approximately 32 per chain), 6-sulfated (approximately 1 per 14 chains), and 4,6-sulfated disaccharides (approximately 1 per 6 chains) and (b) a nonreducing terminal composition of 4-sulfated GalNAc (approximately 4 out of every 7 chains), 4,6-disulfated GalNAc (approximately 2 out of every 7 chains), and GlcUA adjacent to a 4-sulfated GalNAc residue (approximately 1 out of every 7 chains). Thus, the vast majority of these CS chains terminated with a sulfated GalNAc residue. The presence of 4,6-disulfated GalNAc at nonreducing termini is 60-fold more abundant than 4,6-disulfated GalNAc in interior disaccharides. This observation is consistent with the suggestion that disulfation of terminal GalNAc residues is involved in chain termination.     

Novel sulfated oligosaccharides containing 3-O-sulfated glucuronic acid from king crab cartilage chondroitin sulfate K. Unexpected degradation by chondroitinase ABC

We prepared a series of oligosaccharides from king crab cartilage chondroitin sulfate K after exhaustive digestion with testicular hyaluronidase, and determined the structures of four tetrasaccharides and a pentasaccharide by fast atom bombardment mass spectrometry, high performance liquid chromatography analysis of chondroitinase AC-II digests, and 500-MHz 1H NMR spectroscopy. The tetrasaccharides shared the common core structure GlcAbeta1-3GalNAcbeta1-4GlcAbeta1-3GalNAc with various sulfation profiles. One structure was GlcAbeta1-3GalNAc(4S)beta1-4GlcAbeta1-3GalNAc(4S), whereas three of them have the following hitherto unreported structures including a novel glucuronate 3-O-sulfate: GlcA(3S)beta1-3GalNAc(4S)beta1-4GlcAbeta1-3GalNAc(4S), GlcAbeta1-3GalNAc(4S)beta1-4GlcA(3S)beta1-3GalNAc(4S), and GlcA(3S)beta1-3GalNAc(4S)beta1-4GlcA(3S)beta1-3GalNAc(4S), where 3S or 4S represents 3-O- or 4-O-sulfate, respectively. The structure of the pentasaccharide was determined as GlcA(3S)beta1-3GalNAc(4S)beta1-4GlcA(3S)beta1- 3GalNAc(4S)beta1-4GlcA. Chondroitinase ABC digestion of the tetrasaccharides with GlcA(3S) at the internal position destroyed the disaccharide unit containing GlcA(3S) derived from the reducing side and resulted in only the disaccharide unit from the non-reducing side. In contrast, these tetrasaccharides remained totally resistant to chondroitinase AC-II. The results indicated that it is necessary to reevaluate the disaccharide composition of chondroitin sulfate poly- or oligosaccharides purified from various biological sources, since they were usually determined after chondroitinase ABC digestion. It is probable that the structures containing GlcA(3S) would not have been detected.     

Enzymatic transfer of a beta1,6-linked N-acetylglucosamine to the alpha-galactose of globo-N-tetraose: in vitro synthesis of a novel hybrid pentasaccharide of lacto-globo type

A novel saccharide was synthesized by incubating globo-N-tetraose, GalNAc beta1-3Gal alpha1-4Gal beta1-4Glc, and UDP[3H]GlcNAc with hog gastric mucosal microsomes, known to contain beta1,6-N-acetylglucosaminyltransferase activity of a broad acceptor specificity. Chromatography and MALDI-TOF mass spectrometry of the product, as well as the amount of incorporated radioactivity indicated that one [3H]GlcNAc residue was transferred to the acceptor saccharide. One- and two-dimensional 1H NMR-spectroscopic analysis of the product and ESI-CID mass spectrometry of the pentasaccharide in permethylated form established its structure as GalNAc beta1-3([3H]GlcNAc beta1-6)Gal alpha1-4Gal beta1-4Glc. The new enzyme activity possesses substrate specificity features common to a purified beta1,6-GlcNAc-transferase from bovine tracheal epithelium, which forms branches at the subterminal beta1,3-substituted galactose and accepts both GlcNAc- and Gal-configuration at the terminal residue of the acceptor (Ropp et al. (1991) J. Biol. Chem., 266, 23863-23871). The new beta1,6-GlcNAc-branch was readily galactosylated by bovine milk beta1,4-galactosyltransferase, revealing a pathway to novel hybrid type glycans with N-acetyllactosamine chains on globotype saccharides. This pathway may lead to the rare IP blood-group antigen and to globoside-like molecules mediating cell adhesion.     

The enzymatic sulfation of glycoprotein carbohydrate units: blood group T-hapten specific and two other distinct Gal:3-O-sulfotransferases as evident from specificities and kinetics and the influence of sulfate and fucose residues occurring in the carbohydrate chain on C-3 sulfation of terminal Gal

Enzymatic 3-O-sulfation of terminal beta-Gal residues was investigated by screening sulfotransferase activity present in 37 human tissue specimens toward the following synthesized acceptor moieties: Galbeta1,3GalNAc alpha-O-Al, Galbeta1,4GlcNAcbeta-O-Al, Galbeta1,3GlcNAcbeta-O-Al, and mucin-type Galbeta1,4GlcNAcbeta1,6(Galbeta1,3)GalNAc alpha-O-Bn structures containing a C-3 methyl substituent on either Gal. Two distinct types of Gal: 3-O-sulfotransferases were revealed. One (Group A) was specific for the Galbeta1, 3GalNAc alpha- linkage and the other (Group B) was directed toward the Galbeta1,4GlcNAc branch beta1,6 linked to the blood group T hapten. Enzyme activities found in breast tissues were unique in showing a strict specificity for the T-hapten. Galbeta-O-allyl or benzyl did not serve as acceptors for Group A but were very active with Group B. An examination of activity present in six human sera revealed a specificity of the serum enzyme toward beta1,3 linked Gal, particularly, the T-hapten without beta1,6 branching. Group A was highly active toward T-hapten/acrylamide copolymer, anti-freeze glycoprotein, and fetuin O-glycosidic asialo glycopeptide; less active toward fetuin triantennary asialo glycopeptide; and least active toward bovine IgG diantennary glycopeptide. Group B was moderately and highly active, respectively, with the latter two glycopeptides noted and least active with the first two. Competition experiments performed with Galbeta1,3GalNAc alpha-O-Al and Galbeta1,4GlcNAcbeta1,6(Galbeta1,3)GalNAc alpha-O-Bn having a C-3 substituent (methyl or sulfate) on either Gal reinforced earlier findings on the specificity characteristics of Group A and Group B. Group A displayed a wider range of optimal activity (pH 6.0-7.4), whereas Group B possessed a peak of activity at pH 7.2. Mg2+ stimulated Group A 55% and Group B 150%, whereas Mn+2 stimulated Group B 130% but inhibited Group A 75%. Ca2+ stimulated Group B 100% but inhibited Group A 35%. Group A and Group B enzymes appeared to be of the same molecular size (<100,000 Da) as observed by Sephacryl S-100 HR column chromatography. The following effects upon Gal: 3-O-sulfotransferase activities by fucose, sulfate, and other substituents on the carbohydrate chains were noted. (1) A methyl or GlcNAc substituent on C-6 of GalNAc diminished the ability of Galbeta1,3GalNAc alpha-O-Al to act as an acceptor for Group A. (2) An alpha1,3-fucosyl residue on the beta1,6 branch in the mucin core structure did not affect the activity of Group A toward Gal linked beta1,3 to GalNAc alpha-. (3) Lewis x and Lewis a terminals did not serve as acceptors for either Group A or B enzymes. (4) Elimination of Group B activity on Gal in the beta1,6 branch owing to the presence of a 3-fucosyl or 6-sulfo group on GlcNAc did not hinder any action toward Gal linked beta1,3 to GalNAc alpha. (5) Group A activity on Gal linked beta1,3 to GalNAc remained unaffected by 3'-sulfation of the beta1,6 branch. The reverse was true for Group B. (6) The acceptor activity of the T-hapten was increased somewhat upon C-6 sulfation of GalNAc, whereas, C-6 sialylation resulted in an 85% loss of activity. (7) A novel finding was that Galbeta1,4GlcNAcbeta-O-Al and Galbeta1,3GlcNAcbeta-O-Al, upon C-6 sulfation of the GlcNAc moiety, became 100% inactive and 5- to 7-fold active, respectively, in their ability to serve as acceptors for Group B.     

Cardiovascular effects of microinjections of opioid agonists into the ''Depressor Region'' of the ventrolateral periaqueductal gray region

Differentiating the binding properties of applied lectins should facilitate the selection of lectins for characterization of glycoreceptors on the cell surface. Based on the binding specificities studied by inhibition assays of lectin-glycan interactions, over twenty Gal and/or GalNAc specific lectins have been divided into eight groups according to their specificity for structural units (lectin determinants), which are the disaccharide as all or part of the determinants and of GalNAc alpha 1-->Ser (Thr) of the peptide chain. A scheme of codes for lectin determinants is illustrated as follows: (1) F (GalNAc alpha 1-->3GalNAc), Forssman specific disaccharide--Dolichos biflorus (DBL), Helix pomatia (HPL) and Wistaria floribunda (WFL) lectins. (2) A (GalNAc alpha 1-->3 Gal), blood group A specific disaccharide--Codium fragile subspecies tomentosoides (CFT), Soy bean (SBL), Vicia villosa-A4 (VVL-A4), and Wistaria floribunda (WFL) lectins. (3) Tn (GalNAc alpha 1-->Ser (Thr) of the protein core)--Vicia villosa B4 (VVL-B4), Salvia sclarea (SSL), Maclura pomifera (MPL), Bauhinia purpurea alba (BPL) and Artocarpus integrifolia (Jacalin, AIL). (4) T (Gal beta 1-->3GalNAc), the mucin type sugar sequences on the human erythrocyte membrane(T alpha), T antigen or the disaccharides at the terminal nonreducing end of gangliosides (T beta)--Peanut (PNA), Bauhinia purpurea alba (BPL), Maclura pomifera (MPL), Sophora japonica (SJL), Artocarpus lakoocha (Artocarpin) lectins and Abrus precatorius agglutinin (APA).(5) I and II (Gal beta 1-->3(4)GlcNAc)--the disaccharide residue at the nonreducing end of the carbohydrate chains derived from either N- or O-glycosidic linkage--Ricinus communis agglutinin (RCA1), Datura stramonium (TAL, Thorn apple), Erythrina cristagalli (ECL, Coral tree), and Geodia cydonium (GCL). (6) B (Gal alpha 1-->3Gal), human blood group B specific disaccharide--Griffonia(Banderiaea) simplicifolia B4 (GSI-B4). (7) E (Gal alpha 1-->4Gal), receptors for pathogenic E. coli agglutinin, Shiga toxin and Mistletoe toxic lectin-I (ML-I) and abrin-a.     

Study of the amidase signature group

A novel mono-sulfated glycosphingolipid based on the gangliotriaose core structure was isolated from rat kidney. The isolation procedure involved extraction of lipids with chloroform/methanol, mild alkaline methanolysis, column chromatographies with anion exchangers and silica beads. The structure was characterized by compositional analysis, FTIR spectroscopy, methylation analysis, 1H-NMR spectroscopy and negative-ion liquid secondary ion mass spectrometry (LSMIS) using the intact glycolipid and its desulfation product. The two dimensional chemical shift correlated spectroscopy provided information on the sugar sequence as well as anomeric configurations, and indicated the presence of a 3-O-sulfated N-acetylgalactosamine within the molecule. Negative-ion LSIMS with high- and low-energy collision-induced dissociation defined the sugar sequence and ceramide composition, confirming the presence of a sulfated N-acetylgalactosamine at the non-reducing terminus. From these results, the complete structure was proposed to be HSO3-3GalNAc beta 1-4Gal beta 1-4Glc beta 1-1Cer (Gg3Cer III3-sulfate, SM2b).     

Sulfated di-, tri- and tetraantennary N-glycans in human Tamm-Horsfall glycoprotein

The primary structures of 32 sulfated di-, tri- and tetraantennary N-glycans of human Tamm-Horsfall glycoprotein (THP) have been determined. THP was isolated from the urine of one healthy male donor. The intact carbohydrate chains were released by PNGase-F and fractionated via FPLC on Resource Q, HPLC on LiChrosorb-NH2, and high-pH anion-exchange chromatography on CarboPac PA-1. Characterizations were performed using 500-MHz and 600-MHz 1H-NMR spectroscopy, in combination with sialidase treatments. The type of characterized N-glycans ranged from monosulfated to trisulfated N-glycans, whereby the sulfate groups were present as 3-O-sulfated Gal (Gal3S) and 4-O-sulfated GalNAc (GalNAc4S). A compilation of the established structures is shown below. [structure in text]     

A novel method to co-localize glycosaminoglycan-core oligosaccharide glycosyltransferases in rat liver Golgi. Co-localization of galactosyltransferase I with a sialyltransferase

4-Methylumbelliferyl-beta-xyloside (Xyl beta MU) primes glycosaminoglycan synthesis by first serving as an acceptor for the addition of 2 galactoses and 1 glucuronic acid residue to make the typical core structure, GlcUA beta 1, 3Gal beta 1,3Gal beta 1,4Xyl beta MU. To investigate the relative localization of these biosynthetic enzymes, intact and properly oriented rat liver Golgi preparations were incubated with Xyl beta MU and 1 microM UDP-[3H]Gal and then chased with 5 microM of unlabeled UDP-Gal, UDP-GlcUA, UDP-GlcNAc, UDP-GalNAc, and CMP-Neu5Ac. Under these conditions, no intervesicular transport occurs and acceptor labeling depends entirely upon transporter-mediated delivery of the labeled sugar nucleotides into the lumen of a vesicle and co-localization of the appropriate glycosyltransferases. The labeled products were isolated from the incubation medium and from within the Golgi and their structures analyzed by C18, anion-exchange, and amine adsorption high performance liquid chromatography in combination with glycosidase digestions. Surprisingly, the major products within the Golgi were two sialylated xylosides (Sia alpha 2,3Gal beta 1,4Xyl-beta MU and Sia alpha 2,8Sia alpha 2,3Gal beta 1,4Xyl beta MU) rather than the expected group of partially completed GAG core structures. Less than 10% of the products within the Golgi are the expected core structures containing a second Gal residue or, in addition, GlcUA. The amount of the sialylated products is only partially decreased if the chase is omitted or if the chase is done in the absence of added CMP-Sia, suggesting a pool of previously transported CMP-Sia drives synthesis of the major products. Conversely, when detergent permeabilized vesicles are provided with high concentration of the same sugar nucleotides, the ratio of sialylated products is reduced and replaced by an increase in GAG-like products. These results argue that GAG core-specific Ga1 transferase I and II are not extensively co-localized within the same Golgi compartment. By contrast, glycosaminoglycan core Gal transferase I is substantially co-localized with an alpha-2,3-sialyltransferase and an alpha-2,8-sialyltransferase. Incubating intact Golgi vesicles with exogenous diffusible acceptors offers a novel method to assess the functional co-localization of glycosyltransferases of multiple pathways within the Golgi compartments.     

Enzymatic synthesis of N-linked oligosaccharides terminating in multiple sialyl-Lewis(x) and GalNAc-Lewis(x) determinants: clustered glycosides for studying selectin interactions

Galactosyltransferase, sialyltransferase, and fucosyltransferase were used to create a panel of complex oligosaccharides that possess multiple terminal sialyl-Le(x) (NeuAc alpha 2-3Gal[Fuc alpha 1-3] beta 1-4GlcNAc) and GalNAc-Le(x) (GalNAc[Fuc alpha 1-3]beta 1-4GlcNAc). The enzymatic synthesis of tyrosinamide biantennary, triantennary, and tetraantennary N-linked oligosaccharides bearing multiple terminal sialyl-Le(x) was accomplished on the 0.5 mumol scale and the purified products were characterized by electrospray MS and 1H NMR. Likewise, biantennary and triantennary tyrosinamide oligosaccharides bearing multiple terminal GalNAc-Le(x) determinants were synthesized and similarly characterized. The transfer kinetics of human milk alpha 3/4-fucosyltransferase were compared for biantennary oligosaccharide acceptor substrates possessing Gal beta 1-4GlcNAc, GalNAc beta 1-4GlcNAc, and NeuAc alpha 2-3Gal beta 1-4GlcNAc which established NeuAc alpha 2-3Gal beta 1-4GlcNAc as the most efficient acceptor substrate. The resulting complex oligosaccharides were chemically tethered through the tyrosinamide aglycone to the surface of liposomes containing phosphatidylthioethanol, resulting in the generation of glycoliposomes probe which will be useful to study relationships between binding affinity and the micro- and macro-clustering of selectin ligand.     

Molecular species analysis of glycosphingolipids from small intestine of Japanese quail, Coturnix coturnix Japonica by HPLC/FAB/MS

Neutral glycosphingolipids were isolated from quail small intestine and their structures were analysed. They contained: Gal beta 1-4GlcCer(LacCer), Gal alpha 1-4GalCer(Ga2Cer), Gal alpha 1-4Gal beta 1-4GlcCer(Gb3Cer), GlcNAc beta 1-3Gal beta 1-4GlcCer(Lc3Cer), GalNAc beta 1-4Gal beta 1-4GlcCer(Gg3Cer), GalNAc beta 1-4[GalNAc beta 1-3] Gal beta 1-4GlcCer(LcGg4Cer), and GalNAc alpha 1-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4GlcCer (Forssman glycolipid) as well as glucosylceramide, galactosylceramide (Nishimura K et al. 1984) Biochim Biophys Acta 796:269-76) and the LeX glycolipid, III3 Fuc alpha-nLc4Cer (Nishimura K et al. (1989) J. Biochem (Tokyo) 101:1315-18). The molecular species compositions of these glycosphingolipids were examined using fast atom bombardment-mass spectrometry linked with reversed-phase high-performance liquid chromatography. By such analysis, we could classify the quail glycosphingolipids into at least three classes: glycolipids rich in species having four hydroxyl groups in the ceramides (GalCer, Gg3Cer, LcGg4Cer and LeX), those rich in the ceramides of N-acyl trihydroxysphinganine with normal fatty acids (Lc3Cer), and glycolipids rich in the ceramides of N-acyl sphingenine with normal fatty acids (LacCer, Gb3Cer and Forssman glycolipid). Immunohistochemical observation implies that the differences in the hydrophobic moieties specified the localization of glycosphingolipids in the tissue.     

Enzymatic synthesis of oligosaccharides containing Gal beta-->4Gal disaccharide at the non-reducing end using beta-galactanase from Penicillium citrinum

The transglycosylation reaction was done with a beta-galactanase from Penicillium citrinum. The regioselectivity in the transglycosylation reaction was studied using soy bean arabinogalactan as a donor and mono- or disaccharide derivatives containing beta-galactosyl residue as acceptors. We also synthesized oligosaccharides containing Gal beta 1-->4Gal sequence such as Gal beta 1-->4Gal beta1-->4Glc, Gal beta 1-->4Gal beta 1-->3GlcNac, Gal beta 1-->4Gal beta 1-->4GlcNAc, Gal beta 1-->4Gal beta 1-->6GlcNAc, and Gal beta 1-->4Gal beta 1-->3GalNAc for use in the total synthesis of complex sugar chains.     

Characterization of an N-acetylglucosamine-6-O-sulfotransferase from human respiratory mucosa active on mucin carbohydrate chains

A microsomal GlcNAc-6-O-sulfotransferase activity from human bronchial mucosa, able to transfer a sulfate group from adenosine 3'-phosphate 5'-phosphosulfate onto methyl-N-acetylglucosaminides or terminal N-acetylglucosamine residues of carbohydrate chains from human respiratory mucins, has been characterized. The reaction products containing a terminal HO3S-6GlcNAc were identified by high performance anion-exchange chromatography. Using methyl-beta-N-acetylglucosaminide as a substrate, the optimal activity was obtained with 0.1% Triton X-100, 30 mM NaF, 20 mM Mn2+, 5 mM AMP in a 30 mM MOPS (3-(N-morpholino) propanesulfonic acid) buffer at pH 6.7. The apparent Km values for adenosine 3'-phosphate 5'-phosphosulfate and methyl-beta-N-acetylglucosaminide were observed at 9.1 x 10(-6) M and 0.54 x 10(-3) M, respectively. The enzyme had more affinity for carbohydrate chains with a terminal GlcNAc residue than for methyl-beta-N-acetylglucosaminide; it was unable to catalyze the transfer of sulfate to position 6 of the GlcNAc residue contained in a terminal Galbeta1-4GlcNAc sequence. However, oligosaccharides with a nonreducing terminal HO3S-6GlcNAc were substrates for a beta1-4 galactosyltransferase from human bronchial mucosa. These data point out that GlcNAc-6-O-sulfotransferase must act before beta1-4 galactosylation in mucin-type oligosaccharide biosynthesis.     

Purification and properties of human N-acetylgalactosamine-6-sulfate sulfatase

1. Human N-acetylgalactosamine-6-sulfate sulfatase (EC 3.1.6.-) from human placenta has been purified more than 3000-fold by gel filtration, ion-exchange and substrate affinity chromatography. The enzyme has a molecular weight of 90 000 by gel filtration chromatography and 85 000 by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Enzyme purified from cultured human skin fibroblasts has similar properties. 2. The tritium-labeled chrondroitin 6-sulfate trisaccharide N-acetylgalactosamine 6-sulfate-(beta, 1-4)-glucuronic acid-(beta, 1-3(-N-acetyl[1-3H]galactosaminitol 6-sulfate as substrate demonstrated a Km of 0.12 mM at pH 4.5. Sulfate was hydrolyzed only from the non-reducing terminal of this disulfated trisaccharide. Hyaluronic acid, dermatan sulfate, chondroitin 4-sulfate, heparin and chondroitin 6-sulfate tetrasaccharide were slightly inhibitory, whereas 6-sulfated pentasaccharides and heptasaccharides were strongly inhibitory. The enzyme dose not hydrolyze sulfate from N-acetylglucosamine 6-sulfate.     

Intestinal immune system modulating polysaccharides from rhizomes of Atractylodes lancea

Hot water extract (ALR-0) of rhizomes of Atractylodes lanceo DC. was fractionated into MeOH-soluble fraction (ALR-1), supernatant fraction of EtOH precipitation (ALR-3 + 4), and crude polysaccharide fraction (ALR-5). Among these fractions, only ALR-5 showed potent stimulating activity for proliferation of bone marrow cells mediated by Peyer's patch cells. ALR-5 gave three potently active carbohydrate-rich fractions (ALR-5IIa, 5IIb, and 5IIc) by anion-exchange chromatography on DEAE-Sepharose CL-6B, and three active polysaccharides (ALR-5IIa-1-1, ALR-5IIb-2-2, and ALR-5IIc-3-1) were further purified from the respective fractions. The order of activity was revealed to be ALR-5IIb-2-2 > or = ALR-5IIa-1-1 > ALR-5IIc-3-1, ALR-5IIa-1-1, 5IIb-2-2, and 5IIc-3-1 each was eluted as a single peak on HPLC and their molecular weights were estimated to be 74,000, 3,100, 16,000, respectively. ALR-5IIa-1-1 consisted mainly of Ara and Gal (molar ratio; 0.6: 1.0) in addition to a trace amount of uronic acid whereas ALR-5IIb-2-2 and ALR-5IIc-3-1 mainly comprised Ara, Gal, GlcA, and GalA (molar ratio; 0.2: 1.0: 0.2: 0.8, and 0.5: 1.0: 0.7: 1.5, respectively). Methylation analysis indicated that ALR-5IIa-1-1 consisted mainly of terminal Araf, 4- or 5-linked Ara, 3.4- or 3.5-branched Ara, and 3-linked, 4-linked, and 3,6-branched Gal. ALR-5IIb-2-2 and ALR-5IIc-3-1 were composed mainly of terminal Araf, 4- or 5-linked Ara, 4-linked Gal, 4-linked GalA, and terminal GlcA. In addition, ALR-5IIb-2-2 mainly comprised 4-linked Xyl whereas ALR-5IIc-3-1 consisted mainly of 2,4-branched Rha. Single radial gel diffusion indicated that ALR-5IIa-1-1 showed a strong reactivity with beta-glucosyl-Yariv antigen, whereas ALR-5IIb-2-2 and ALR-5IIc-3-1 did not show the reactivity with the antigen. Treatments of ALR-5IIa with NalO4, NaClO2 and pronase did not reduce the stimulating activity for Peyer's patch cells, however combination of exo-alpha-L-arabinofuranosidase and exo-beta-D-(1-->3)-galactanase digestions of ALR-5IIa-1-1 significantly decreased its activity.