The unusual tetrasaccharide sequence GlcA{beta}-3GalNAc(4-sulfate)({beta}1-4GlcA(2-sulfate){beta}1-3GalNAc(6-sulfate) found in the hexasaccharides prepared by testicular hyaluronidase digestion of shark cartilage chondroitin sulfate D

Eight hexasaccharide fractions were isolated from commercialshark cartilage chondroitin sulfate D by means of gel nitrationchromatography and HPLC on an amine-bound silica column afterexhaustive digestion with sheep testicular hyaluronidase. Capillaryelectrophoresis of the enzymatic digests as well as one- andtwo-dimensional 500 MHz ¹H-NMR spectroscopy demonstrated thatthese hexasaccharides share the common core saccharide structureGlcAß1-3GalNAcß1-4GlcAß1-3GalNAcß1-4GlcAß1-3GalNAcwith three, four, or five sulfate groups in different combinations.Six structures had the same sulfation profiles as those of theunsaturated hexasaccharides isolated from the same source afterdigestion with chondroitinase ABC (Sugahara et al., Eur. J.Biochem., 293, 871–880, 1996) and the other two have notbeen reported so far. In the new components, a D disaccharideunit, GlcA(2-sulfate)ß1-3GalNAc(6-sulfate), characteristicof chondroitin sulfate D was arranged on the reducing side ofan A disaccharide unit, GlcAß1-3GalNAc(4-sulfate),forming an unusual A-D tetrasaccharide sequence, GlcAß1-3GalNAc(4-sulfate)-4GlcA(2-sulfate)ß1-3GaINAc(6-sulfate)which is known to be recognized by the monoclonal antibody MO225.These findings support the notion that the tetrasaccharide sequence,GlcAß1-3GalNAc(4-sulfate)ß1-4GlcAß1-3GalNAc(6-sulfate)is included in the acceptor site of a hitherto unreported 2-O-sulfotransferaseresponsible for its synthesis. The sulfated hexasaccharidesisolated in this study will be useful as authentic oligosaccharideprobes and enzyme substrates in studies of sulfated glycosaminogly-cans. sulfated hexasaccharides chondroitin sulfate D hyaluronidase ¹ H-NMR     

Chondroitinase-mediated degradation of rare 3-O-sulfated glucuronic acid in functional oversulfated chondroitin sulfate K and E

Chondroitin sulfate K (CS-K) from king crab cartilage rich in rare 3-O-sulfated glucuronic acid (GlcUA(3S)) displayed neuritogenic activity and affinity toward various growth factors like CS-E from squid cartilage. CS-K-mediated neuritogenesis of mouse hippocampal neurons in culture was abolished by digestion with chondroitinase (CSase) ABC, indicating the possible involvement of GlcUA(3S). However, identification of GlcUA(3S) in CS chains by conventional high performance liquid chromatography has been hampered by its CSase ABC-mediated degradation. To investigate the degradation process, an authentic CS-E tetrasaccharide, Delta4,5HexUA-GalNAc(4S)-GlcUA(3S)-GalNAc(4S), was digested with CSase ABC, and the end product was identified as GalNAc(4S) by electrospray ionization mass spectrometry (ESI-MS). Putative GalNAc(6S) and GalNAc(4S,6S), derived presumably from GlcUA(3S)-GalNAc(6S) and GlcUA(3S)-GalNAc(4S,6S), respectively, were also detected by ESI-MS in the CSase ABC digest of a CS-E oligosaccharide fraction resistant to CSases AC-I and AC-II. Intermediates during the CSase ABC-mediated degradation of Delta4,5HexUA(3S)-GalNAc(4S) to GalNAc(4S) were identified through ESI-MS of a partial CSase ABC digest of a CS-K tetrasaccharide, GlcUA(3S)-GalNAc(4S)-GlcUA(3S)-GalNAc(4S), and the conceivable mechanism behind the degradation of the GlcUA(3S) moiety was elucidated. Although a fucose branch was also identified in CS-K, defucosylated CS-K exhibited greater neuritogenic activity than the native CS-K, excluding the possibility of the involvement of fucose in the activity. Rather, (3S)-containing disaccharides are likely involved. These findings will enable us to detect GlcUA(3S)-containing disaccharides in CS chains to better understand CS-mediated biological processes.     

Neuritogenic activity of chondroitin/dermatan sulfate hybrid chains of embryonic pig brain and their mimicry from shark liver. Involvement of the pleiotrophin and hepatocyte growth factor signaling pathways

Accumulating evidence suggests the involvement of chondroitin sulfate (CS) and dermatan sulfate (DS) hybrid chains in the brain's development and critical roles for oversulfated disaccharides and IdoUA residues in the growth factor-binding and neuritogenic activities of these chains. In the pursuit of sources of CS/DS with unique structures, neuritogenic activity, and therapeutic potential, two novel CS/DS preparations were isolated from shark liver by anion exchange chromatography. The major (80%) low sulfated and minor (20%) highly sulfated fractions had an average molecular mass of 3.8-38.9 and 75.7 kDa, respectively. Digestion with various chondroitinases (CSases) revealed a large panel of disaccharides with either GlcUA or IdoUA scattered along the polysaccharide chains in both of the fractions. The higher M(r) fraction, richer in IdoUA(2-O-sulfate)alpha1-3GalNAc(4-O-sulfate) and GlcUAbeta/IdoUAalpha1-3GalNAc(4,6-O-disulfate) units, exerted greater neurite outgrowth-promoting (NOP) activity and better promoted the binding of various heparin-binding growth factors, including pleiotrophin (PTN), midkine, recombinant human heparin-binding epidermal growth factor-like growth factor, VEGF(165), fibroblast growth factor-2, fibroblast growth factor-7, and hepatocyte growth factor (HGF). These activities were largely abolished by digestion with CSase ABC or B but only moderately affected by a mixture of CSases AC-I and AC-II. In addition, the NOP activity of the larger fraction was markedly reduced by desulfation with alkali, suggesting a role for the 2-O-sulfate of IdoUA(2-O-sulfate)alpha1-3GalNAc(4-O-sulfate). The NOP activity of the higher molecular weight fraction and that of the embryonic pig brain-derived CS/DS fraction were also sup pressed to a large extent by antibodies against HGF, PTN, and their individual receptors cMet and anaplastic lymphoma kinase, revealing the involvement of the HGF and PTN signaling pathways in the activity.     

A Novel Eliminase from a Marine Bacterium That Degrades Hyaluronan and Chondroitin Sulfate

Lyases cleave glycosaminoglycans (GAGs) in an eliminative mechanism and are important tools for the structural analysis and oligosaccharide preparation of GAGs. Various GAG lyases have been identified from terrestrial but not marine organisms even though marine animals are rich in GAGs with unique structures and functions. Herein we isolated a novel GAG lyase for the first time from the marine bacterium Vibrio sp. FC509 and then recombinantly expressed and characterized it. It showed strong lyase activity toward hyaluronan (HA) and chondroitin sulfate (CS) and was designated as HA and CS lyase (HCLase). It exhibited the highest activities to both substrates at pH 8.0 and 0.5 m NaCl at 30 °C. Its activity toward HA was less sensitive to pH than its CS lyase activity. As with most other marine enzymes, HCLase is a halophilic enzyme and very stable at temperatures from 0 to 40 °C for up to 24 h, but its activity is independent of divalent metal ions. The specific activity of HCLase against HA and CS reached a markedly high level of hundreds of thousands units/mg of protein under optimum conditions. The HCLase-resistant tetrasaccharide Δ^4,5HexUAα1-3GalNAc(6-O-sulfate)β1-4GlcUA(2-O-sulfate)β1-3GalNAc(6-O-sulfate) was isolated from CS-D, the structure of which indicated that HCLase could not cleave the galactosaminidic linkage bound to 2-O-sulfated d-glucuronic acid (GlcUA) in CS chains. Site-directed mutagenesis indicated that HCLase may work via a catalytic mechanism in which Tyr-His acts as the Brønsted base and acid. Thus, the identification of HCLase provides a useful tool for HA- and CS-related research and applications.     

Studies on the binding of wheat germ agglutinin (Triticum vulgaris) to O-glycans

The binding profile of Triticum vulgaris (WGA, wheat germ) agglutinin to 23 O-glycans (GalNAcα1→Ser/Thr containing glycoproteins, GPs) was quantitated by the precipitin assay and its specific interactions with O-glycans were confirmed by the precipitin inhibition assay. Of the 28 glycoforms tested, six complex O-glycans (hog gastric mucins, one human blood group A active and two precursor cyst GPs) reacted strongly with WGA and completely precipitated the lectin added. All of the other human blood group A active O-glycans and human blood group precursor GPs also reacted well with the lectin and precipitated over two-thirds of the agglutinin used. They reacted 4–50 times stronger than N-glycans (asialo-fetuin and asialo-human α₁ acid GP). The binding of WGA to O-glycans was inhibited by either p-NO₂-phenyl α,βGlcNAc or GalNAc. From these results, it is highly possible that cluster (multivalent) effects through the high density of weak inhibitory determinants on glycans, such as GalNAcα1→Ser/Thr (Tn), GalNAc at the non-reducing terminal, GlcNAcβ1→ at the non-reducing end and/or as an internal residue, play important roles in precipitation, while the GlcNAcβ1→4GlcNAc disaccharide may play a minor role in the precipitation of mammalian glycan-WGA complexes.     

Structural and functional characterization of oversulfated chondroitin sulfate/dermatan sulfate hybrid chains from the notochord of hagfish. Neuritogenic and binding activities for growth factors and neurotrophic factors

Oversulfated chondroitin sulfate (CS)/dermatan sulfate (DS) hybrid chains were purified from the notochord of hagfish. The chains (previously named CS-H for hagfish) have an average molecular mass of 18 kDa. Composition analysis using various chondroitinases demonstrated a variety of D-glucuronic acid (GlcUA)- and L-iduronic acid (IdoUA)-containing disaccharides variably sulfated with a higher proportion of GlcUA/IdoUA-GalNAc 4,6-O-disulfate, revealing complex CS/DS hybrid features. The hybrid chains showed neurite outgrowth-promoting activity of an axonic nature, which resembled the activity of squid cartilage CS-E and which was abolished fully by chondroitinase ABC digestion and partially by chondroitinase AC-I or B digestion, suggesting the involvement of both GlcUA and IdoUA in neuritogenic activity. Purified CS-H exhibited interactions in a BIAcore system with various heparin-binding proteins and neurotrophic factors (viz. fibroblast growth factor-2, -10, -16, and -18; midkine; pleiotrophin; heparin-binding epidermal growth factor-like growth factor; vascular endothelial growth factor; brain-derived neurotrophic factor; and glial cell line-derived neurotrophic factor), most of which are expressed in the brain, although fibroblast growth factor-1 and ciliary neurotrophic factor showed no binding. Kinetic analysis revealed high affinity binding of these growth factors and, for the first time, of the neurotrophic factors. Competitive inhibition revealed the involvement of both IdoUA and GlcUA in the binding of these growth factors, suggesting the importance of the hybrid nature of CS-H for the efficient binding of these growth factors. These findings, together with those from the recent analysis of brain CS/DS chains from neonatal mouse and embryonic pig (Bao, X., Nishimura, S., Mikami, T., Yamada, S., Itoh, N., and Sugahara, K. (2004) J. Biol. Chem. 279, 9765-9776), suggest physiological roles of the hybrid chains in the development of the brain.     

Peanut (Arachis hypogaea) lectin recognizes α-linked galactose, but not N-acetyl lactosamine in N-linked oligosaccharide terminals

Peanut (Arachis hypogaea) agglutinin (PNA) is extensively used as tumour marker as it strongly recognises the cancer specific T antigen (Galβ1→3GalNAc-), but not its sialylated version. However, an additional specificity towards Galβ1→4GlcNAc (LacNAc), which is not tumour specific, had been attributed to PNA. For correct interpretation of lectin histochemical results we examined PNA sugar specificity using naturally occurring or semi-synthetic glycoproteins, matrix-immobilised galactosides and lectin-binding tissue glycoproteins, rather than mono- or disaccharides as ligands. Dot-blots, transfer blots or polystyrene plate coatings of the soluble glycoconjugates were probed with horse-radish peroxidase (HRP) conjugates of PNA and other lectins of known specificity. Modifications of PNA-binding glycoproteins, including selective removal of O-linked oligosaccharides and treatment with glycosidases revealed that Galβ1→4GlcNAc (LacNAc) was ineffective while terminal α-linked galactose (TAG) as well as exposed T antigen (Galβ1→3 GalNAc-) was excellent as sugar moiety in glycoproteins for their recognition by PNA. When immobilised, melibiose was superior to lactose in PNA binding. Results were confirmed using TAG-specific human serum anti-α-galactoside antibody.     

Characterization of serum {beta}-glucuronyltransferase involved in chondroitin sulfate biosynthesis

We studied a glucuronyltransferase involved in chondroitin sulfate(CS) biosynthesis in a preparation obtained from fetal bovineserum by heparin-Sepharose affinity chromatography. This enzymetransferred GlcA from UDP-GlcA to the nonreducing GalNAc residuesof polymeric chondroitin. It required Mn²⁺ for maximal activityand showed a sharp pH optimum between pH 5.5 and 6.0. The apparentKm value of the glucuronyltransferase for UDP-GlcA was 51 µM.The specificity was investigated using structurally definedacceptor substrates, which consisted of chemically synthesizedtri-, penta-, and heptasaccharide-serines and various odd-numberedoligosaccharides with a GalNAc residue at the nonreducing terminus,prepared from chondroitin and CS by chondroitinase ABC digestionfollowed by mercuric acetate treatment. The enzyme utilizeda heptasaccharide-serine GalNAcß1-4GlcAß1-3GalNAcß1-4GlcAß1-3Galß1-3Galß1-4Xylß1-O-Serand a pentasaccharide-serine GalNAcß 4GlcAß1-3Galß1-3Galß1-4Xylß1-O-Seras acceptors. In contrast, neither a trisaccharide-serine Galß1-3Galß1-4Xylß1-O-Sernor an     

A fingerprinting method for chondroitin/dermatan sulfate and hyaluronan oligosaccharides

A previously published method for the analysis of glycosaminoglycan disaccharides by high pH anion exchange chromatography (Midura,R.J., Salustri,A., Calabro,A., Yanagishita,M. and Hascall,V.C. (1994), Glycobiology,4, 333-342) has been modified and calibrated for chondroitin and dermatan sulfate oligosaccharides up to hexasaccharide in size and hyaluronan oligosaccharides up to hexadecasaccharide. For hyaluronan oligosaccharides chain length controls elution position; however, for chondroitin and dermatan sulfate oligosaccharides elution times primarily depend upon the level of sulfation, although chain length and hence charge density plays a role. The sulfation position of GalNAc residues within an oligosaccharide is also important in determining its elution position. Compared to 4-sulfation a reducing terminal 6-sulfate retards elution; however, when present on an internal GalNAc residue it is the 4-sulfate containing oligosaccharide which elutes later. These effects allow discrimination between oligosaccharides differing only in the position of GalNAc sulfation. Using this simple methodology, a Dionex CarboPac PA-1 column with NaOH/NaCl eluents and detection by absorbance at 232 nm, a quantitative analytical fingerprint of a chondroitin/dermatan sulfate chain may be obtained, allowing a determination of the abundance of chondroitin sulfate, dermatan sulfate, and hyaluronan along with an analysis of structural features with a linear response to approximately 0.1 nmol. The method may readily be calibrated using either commercial disaccharides or the di- and tetrasaccharide products of a limit digest of commercial chondroitin sulfate by chondroitin ABC endolyase. Commercially available and freshly prepared shark, whale, bovine, and human cartilage chondroitin sulfates have been examined by this methodology and we have confirmed that freshly isolated shark cartilage CS contains significant amounts of the biologically important GlcA2Sbeta(1-3)GalNAc6S structure.     

Heparan Sulfate Containing Unsubstituted Glucosamine Residues: BIOSYNTHESIS AND HEPARANASE-INHIBITORY ACTIVITY*

Degradation of heparan sulfate (HS) in the extracellular matrix by heparanase is linked to the processes of tumor invasion and metastasis. Thus, a heparanase inhibitor can be a potential anticancer drug. Because HS with unsubstituted glucosamine residues accumulates in heparanase-expressing breast cancer cells, we assumed that these HS structures are resistant to heparanase and can therefore be utilized as a heparanase inhibitor. As expected, chemically synthetic HS-tetrasaccharides containing unsubstituted glucosamine residues, GlcAβ1–4GlcNH₃⁺(6-O-sulfate)α1–4GlcAβ1–4GlcNH₃⁺(6-O-sulfate), inhibited heparanase activity and suppressed invasion of breast cancer cells in vitro. Bifunctional NDST-1 (N-deacetylase/N-sulfotransferase-1) catalyzes the modification of N-acetylglucosamine residues within HS chains, and the balance of N-deacetylase and N-sulfotransferase activities of NDST-1 is thought to be a determinant of the generation of unsubstituted glucosamine. We also report here that EXTL3 (exostosin-like 3) controls N-sulfotransferase activity of NDST-1 by forming a complex with NDST-1 and contributes to generation of unsubstituted glucosamine residues.     

Mice Deficient in N-Acetylgalactosamine 4-Sulfate 6-O-Sulfotransferase Are Unable to Synthesize Chondroitin/Dermatan Sulfate containing N-Acetylgalactosamine 4,6-Bissulfate Residues and Exhibit Decreased Protease Activity in Bone Marrow-derived Mast Cells

Chondroitin sulfate (CS) and dermatan sulfate (DS) containing N-acetylgalactosamine 4,6-bissulfate (GalNAc(4,6-SO₄)) show various physiological activities through interacting with numerous functional proteins. N-Acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) transfers sulfate from 3′-phosphoadenosine 5′-phosphosulfate to position 6 of N-acetylgalactosamine 4-sulfate in CS or DS to yield GalNAc(4,6-SO₄) residues. We here report generation of transgenic mice that lack GalNAc4S-6ST. GalNAc4S-6ST-null mice were born normally and fertile. In GalNAc4S-6ST-null mice, GalNAc(4,6-SO₄) residues in CS and DS disappeared completely, indicating that GalNAc4S-6ST should be a sole enzyme responsible for the synthesis of GalNAc(4,6-SO₄) residues in both CS and DS. IdoA-GalNAc(4,6-SO₄) units that account for ∼40% of total disaccharide units of DS in the liver of the wild-type mice disappeared in the liver DS of GalNAc4S-6ST-null mice without reduction of IdoA content. Bone marrow-derived mast cells (BMMCs) derived from GalNAc4S-6ST-null mice contained CS without GlcA-GalNAc(4,6-SO₄) units. Tryptase and carboxypeptidase A activities of BMMCs derived from GalNAc4S-6ST-null mice were lower than those activities of BMMCs derived from wild-type mice, although mRNA expression of these mast cell proteases was not altered. Disaccharide compositions of heparan sulfate/heparin contained in the mast cells derived from BMMCs in the presence of stem cell factor were much different from those of heparan sulfate/heparin in BMMCs but did not differ significantly between wild-type mice and GalNAc4S-6ST-null mice. These observations suggest that CS containing GalNAc(4,6-SO₄) residues in BMMCs may contribute to retain the active proteases in the granules of BMMCs but not for the maturation of BMMCs into connective tissue-type mast cells.     

Oversulfated chondroitin/dermatan sulfates containing GlcAbeta1/IdoAalpha1-3GalNAc(4,6-O-disulfate) interact with L- and P-selectin and chemokines

We previously reported that versican, a large chondroitin/dermatan sulfate (CS/DS) proteoglycan, interacts through its CS/DS chains with adhesion molecules L- and P-selectin and CD44, as well as chemokines. Here, we have characterized these interactions further. Using a metabolic inhibitor of sulfation, sodium chlorate, we show that the interactions of the CS/DS chains of versican with L- and P-selectin and chemokines are sulfation-dependent but the interaction with CD44 is sulfation-independent. Consistently, versican's binding to L- and P-selectin and chemokines is specifically inhibited by oversulfated CS/DS chains containing GlcAbeta1-3GalNAc(4,6-O-disulfate) or IdoAalpha1-3GalNAc(4,6-O-disulfate), but its binding to CD44 is inhibited by all the CS/DS chains, including low-sulfated and unsulfated ones. Affinity and kinetic analyses using surface plasmon resonance revealed that the oversulfated CS/DS chains containing GlcAbeta1/IdoAalpha1-3GalNAc(4,6-O-disulfate) bind directly to selectins and chemokines with high affinity (K(d) 21.1 to 293 nm). In addition, a tetrasaccharide fragment of repeating GlcAbeta1-3GalNAc(4,6-O-disulfate) units directly interacts with L- and P-selectin and chemokines and oversulfated CS/DS chains containing GlcAbeta1/IdoAalpha1-3GalNAc(4,6-O-disulfate) inhibit chemokine-induced Ca(2+) mobilization. Taken together, our results show that oversulfated CS/DS chains containing GlcAbeta1/IdoAalpha1-3GalNAc(4,6-O-disulfate) are recognized by L- and P-selectin and chemokines, and imply that these chains are important in selectin- and/or chemokine-mediated cellular responses.     

Novel 70-kDa chondroitin sulfate/dermatan sulfate hybrid chains with a unique heterogeneous sulfation pattern from shark skin, which exhibit neuritogenic activity and binding activities for growth factors and neurotrophic factors

Chondroitin sulfate (CS) and dermatan sulfate (DS) hybrid chains of proteoglycans are critical in growth factor binding, neuritogenesis, and brain development. Here we isolated CS/DS hybrid chains from shark skin aiming to develop therapeutic agents. Digestion with various chondroitinases showed that both GlcUA- and IdoUA-containing disaccharides are scattered along the polysaccharide chains with an unusually large average molecular mass of 70 kDa. The CS/DS chains were separated into major (80%) and minor (20%) fractions by anion-exchange chromatography. Both fractions had relatively low degrees of sulfation (sulfate/disaccharide molar ratio=1.17 versus 0.87), showing a unique feature compared with the marine CS and DS isolated to date, most of which are oversulfated. They were highly heterogeneous and characterized by multiple disaccharides including GlcUA-GalNAc, GlcUA-GalNAc(6S), GlcUA-GalNAc(4S), IdoUA-GalNAc(4S), GlcUA-GalNAc(4S,6S), IdoUA-GalNAc(4S,6S), GlcUA(2S)-GalNAc(6S), and/or IdoUA(2S)-GalNAc(6S), IdoUA(2S)-GalNAc(4S) and novel GlcUA(2S)-GalNAc(4S), where 2S, 4S, and 6S represent 2-O-, 4-O- and 6-O-sulfate, respectively. The CS/DS chains bound two neurotrophic factors and various growth factors expressed in the brain with high affinity as evaluated for the major fraction by kinetic analysis using a surface plasmon resonance detector, and also promoted the outgrowth of neurites of both an axonic and a dendritic nature. The neuritogenic activity was abolished completely by digestion with chondroitinase ABC, AC-I, or B, suggesting the importance of both GlcUA- and IdoUA-containing moieties. It also showed anti-heparin cofactor II activity comparable to that exhibited by DS from porcine skin. Thus, by virtue of its unique structure and biological activities, DS will find a potential use in therapeutics.     

Construction of a Chondroitin Sulfate Library with Defined Structures and Analysis of Molecular Interactions

Chondroitin sulfate (CS) is a linear acidic polysaccharide, composed of repeating disaccharide units of glucuronic acid and N-acetyl-d-galactosamine and modified with sulfate residues at different positions, which plays various roles in development and disease. Here, we chemo-enzymatically synthesized various CS species with defined lengths and defined sulfate compositions, from chondroitin hexasaccharide conjugated with hexamethylenediamine at the reducing ends, using bacterial chondroitin polymerase and recombinant CS sulfotransferases, including chondroitin-4-sulfotransferase 1 (C4ST-1), chondroitin-6-sulfotransferase 1 (C6ST-1), N-acetylgalactosamine 4-sulfate 6-sulfotransferase (GalNAc4S-6ST), and uronosyl 2-sulfotransferase (UA2ST). Sequential modifications of CS with a series of CS sulfotransferases revealed their distinct features, including their substrate specificities. Reactions with chondroitin polymerase generated non-sulfated chondroitin, and those with C4ST-1 and C6ST-1 generated uniformly sulfated CS containing >95% 4S and 6S units, respectively. GalNAc4S-6ST and UA2ST generated highly sulfated CS possessing ∼90% corresponding disulfated disaccharide units. Sequential reactions with UA2ST and GalNAc4S-6ST generated further highly sulfated CS containing a mixed structure of disulfated units. Surprisingly, sequential reactions with GalNAc4S-6ST and UA2ST generated a novel CS molecule containing ∼29% trisulfated disaccharide units. Enzyme-linked immunosorbent assay and surface plasmon resonance analysis using the CS library and natural CS products modified with biotin at the reducing ends, revealed details of the interactions of CS species with anti-CS antibodies, and with CS-binding molecules such as midkine and pleiotrophin. Chemo-enzymatic synthesis enables the generation of CS chains of the desired lengths, compositions, and distinct structures, and the resulting library will be a useful tool for studies of CS functions.     

Chondroitin sulfate and keratan sulfate are the major glycosaminoglycans present in the adult zebrafish Danio rerio (Chordata-Cyprinidae)

The zebrafish Danio rerio (Chordata-Cyprinidae) is a model organism frequently used to study the functions of proteoglycans and their glycosaminoglycan (GAG) chains. Although several studies clearly demonstrate the participation of these polymers in different biological and cellular events that take place during embryonic development, little is known about the GAGs in adult zebrafish. In the present study, the total GAGs were extracted from the whole fish by proteolytic digestion, purified by anion-exchange chromatography and characterized by electrophoresis after degradation with specific enzymes and/or by high-performance liquid chromatography (HPLC) analysis of the disaccharides. Two GAGs were identified: a low-molecular-weight chondroitin sulfate (CS) and keratan sulfate (KS), corresponding to ∼80% and 20% of the total GAGs, respectively. In the fish eye, KS represents ∼ 80% of total GAGs. Surprisingly, no heparinoid was detected, but may be present in the fish at concentrations lower than the limit of the method used. HPLC of the disaccharides formed after chondroitin AC or ABC lyase degradation revealed that the zebrafish CS is composed by ΔUA-1→3-GalNAc(4SO₄) (59.4%), ΔUA-1→3-GalNAc(6SO₄) (23.1%), and ΔUA-1→3-GalNAc (17.5%) disaccharide units. No disulfated disaccharides were detected. Immunolocalization on sections from zebrafish retina using monoclonal antibodies against CS4- or 6-sulfate showed that in the retina these GAGs are restricted to the outer and inner plexiform layers. This is the first report showing the presence of KS in zebrafish eye, and the structural characterization of CS and its localization in the zebrafish retina. Detailed information about the structure and tissue localization of GAGs is important to understand the functions of these polymers in this model organism. The contributions of Aline R.C. Souza and Eliene O. Kozlowski should be considered equal.     

Two novel oligosaccharides from Solanum nigrum

Phytochemical analysis of Solanum nigrum has resulted in the isolation of two novel disaccharides. Their structures were determined as ethyl β-d-thevetopyranosyl-(1→4)-β-d-oleandropyranoside (1) and ethyl β-d-thevetopyranosyl-(1→4)-α-d-oleandropyranoside (2), respectively, by chemical and spectroscopic methods.     

Analysis of the structure and neuritogenic activity of chondroitin sulfate/dermatan sulfate hybrid chains from porcine fetal membranes

The amniotic membrane (AM) is the innermost layer of fetal membranes and possesses various biological activities. Although the mechanism underlying these biological activities remains unclear, unique components seem to be involved. AM contains various extracellular matrix components such as type I collagen, laminin, fibronectin, hyaluronan, and proteoglycans bearing chondroitin sulfate/dermatan sulfate (CS/DS) glycosaminoglycan side chains. Since CS/DS have been implicated in various biological processes, we hypothesized that CS/DS in AM may play a major role in the biological activities of AM. Therefore, the structure and bioactivity of the CS/DS chains from porcine fetal membranes (FM-CS/DS) were investigated. A compositional analysis using various chondroitinases revealed that the characteristic DS domain comprised of iduronic acid-containing disaccharide units is embedded in FM-CS/DS, along with predominant disaccharide units, GlcA-GalNAc, GlcA-GalNAc(4-O-sulfate), and GlcA-GalNAc(6-O-sulfate), where GlcA and GalNAc represent D-glucuronic acid and N-acetyl-D-galactosamine, respectively. The average molecular mass of FM-CS/DS chains was unusually large and estimated to be 250 – 300 kDa. The FM-CS/DS chains showed neurite outgrowth-promoting activity, which was eliminated by digestion with chondroitinase ABC of the CS/DS chains. This activity was suppressed by antibodies against growth factors including pleiotrophin, midkine, and fibroblast growth factor-2, suggesting the involvement of these growth factors in the neurite outgrowth-promoting activity. The binding of these growth factors to FM-CS/DS was also demonstrated by surface plasmon resonance spectroscopy.     

Product-identification and substrate-specificity studies of the GDP-l-fucose: 2-acetamido-2-deoxy-β-d-glucoside (fuc→asn-linked GlcNAc) 6-α-l-fucosyltransferase in a golgi-rich fraction from porcine liver

Golgi-rich membranes from porcine liver have been shown to contain an enzyme that transfers l-fucose in α-(1→6) linkage from GDP-l-fucose to the asparagine-linked 2-acetamido-2-deoxy-d-glucose r residue of a glycopeptide derived from human α₁-acid glycoprotein. Product identification was performed by high-resolution, ¹H-n.m.r. spectroscopy at 360 MHz and by permethylation analysis. The enzyme has been named GDP-l-fucose: 2-acetamido-2-deoxy-β-d-glucoside (Fuc→Asn-linked GlcNAc) 6-α-l-fucosyltransferase, because the substrate requires a terminal β-(1→2)-linked GlcNAc residue on the α-Man (1→3) arm of the core. Glycopeptides with this residue were shown to be acceptors whether they contained 3 or 5 Man residues. Substrate-specificity studies have shown that diantennary glycopeptides with two terminal β-(1→2)-linked GlcNAc residues and glycopeptides with more than two terminal GlcNAc residues are also excellent acceptors for the fucosyltransferase. An examination of four pairs of glycopeptides differing only by the absence or presence of a bisecting GlcNAc residue in β-(1→4) linkage to the β-linked Man residue of the core showed that the bisecting GlcNAc prevented 6-α-l-fucosyltransferase action. These findings probably explain why the oligosaccharides with a high content of mannose and the hybrid oligosaccharides with a bisecting GlcNAc residue that have been isolated to date do not contain a core l-fucosyl residue.     

Studies on (β,1→5) and (β,1→6) linked octyl Galf disaccharides as substrates for mycobacterial galactosyltransferase activity

The emergence of multi-drug resistant (MDR) strains of Mycobacterium tuberculosis (MTB) and the continuing pandemic of tuberculosis emphasizes the urgent need for the development of new anti-tubercular agents with novel drug targets. The recent structural elucidation of the mycobacterial cell wall highlights a large variety of structurally unique components that may be a basis for new drug development. This publication describes the synthesis, characterization, and screening of several octyl Galf(β,1→5)Galf and octyl Galf(β,1→6)Galf derivatives. A cell-free assay system has been utilized for galactosyltransferase activity using UDP[¹⁴C]Galf as the glycosyl donor, and in vitro inhibitory activity has been determined in a colorimetric broth microdilution assay system against MTB H37Ra and three clinical isolates of Mycobacterium avium complex (MAC). Certain derivatives showed moderate activities against MTB and MAC. The biological evaluation of these disaccharides suggests that more hydrophobic analogues with a blocked reducing end showed better activity as compared to totally deprotected disaccharides that more closely resemble the natural substrates in cell wall biosynthesis.     

Organization of the inter-alpha-inhibitor heavy chains on the chondroitin sulfate originating from Ser(10) of bikunin: posttranslational modification of IalphaI-derived bikunin.

Inter-alpha-inhibitor-derived bikunin was purified and the molecular mass was determined to be approximately 8.7 kDa higher than the prediction based on the protein sequence, suggesting extensive posttranslational modifications. These modifications were identified and characterized by a combination of protein and carbohydrate analytical techniques. Three modifications were identified: (i) glycosylation of Ser(10), (ii) glycosylation of Asn(45), and (iii) a heterogeneous truncation of the C-terminus. The Asn(45) associated glycan was shown to be a homogenous "complex type" biantennary structure. The chondroitin-4-sulfate (CS) chain attached to Ser(10) was analyzed by both matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and acrylamide gel electrophoresis after partial chondroitin ABC lyase digestion. The analyses showed that the CS chains were composed of 15 +/- 3 [GlcUA-GalNAc] disaccharide units. On average, every forth disaccharide was sulfated, and these sulfated disaccharides appeared to be more common near the reducing end. Anion exchange chromatography at pH 3. 4 of intact bikunin resulted in the isolation of four isotypes shown to differ only in the amount of sulfation. Heavy chain 1 (HC1) and heavy chain 2 (HC2) are attached to the CS by a novel cross-link [Enghild, J. J., Salvesen, G., Hefta, S. A., Thogersen, I. B., Rutherfurd, S., and Pizzo, S. V. (1991) J. Biol. Chem. 266, 747-751], and the order in which the two heavy chains are positioned on the CS was examined. The results indicate that HC1 is in close proximity to HC2 and both are near the less sulfated nonreducing end of the CS. Taken together, the data show the following organization of the IalphaI molecule: [GlcUA-GalNAc](a)-HC1-[GlcUA-GalNAc](b)-HC2-[GlcUA-GalNAc](c)-Gal -Gal-Xyl-Ser(10)-bikunin, (a + b + c = 12-18 disaccharides).