Structural composition and differential anticoagulant activities of dermatan sulfates from the skin of four species of rays, Dasyatis americana, Dasyatis gutatta, Aetobatus narinari and Potamotrygon motoro

We compared the disaccharide composition of dermatan sulfate (DS) purified from the ventral skin of three species of rays from the Brazilian seacoast, Dasyatis americana, Dasyatis gutatta, Aetobatus narinari and of Potamotrygon motoro, a fresh water species that habits the Amazon River. DS obtained from the four species were composed of non-sulfated, mono-sulfated disaccharides bearing esterified sulfate groups at positions C-4 or C-6 of N-acetyl galactosamine (GalNAc), and disulfated disaccharides bearing esterified sulfate groups at positions C-2 of the uronic acid and at position C-4 or C-6 of GalNAc. However, DS from the skin of P. motoro presented a very low content of the disulfated disaccharides. The anticoagulant actions of ray skin DS, measured by both APTT clotting and HCII-mediated inhibition of thrombin assays, were compared to that of mammalian DS. DS from D. americana had both high APTT and HCII activities, whereas DS from D. gutatta showed activity profiles similar to those of mammalian DS. In contrast, DS from both A. narinari and P. motoro had no measurable activity in the APTT assay. Thus, the anticoagulant activity of ray skin DS is not merely a consequence of their charge density. We speculate that the differences among the anticoagulant activities of these three DS may be related to both different composition and arrangements of the disulfated disaccharide units within their polysaccharide chains.     

Purification and characterization of dermatan sulfate from the skin of the eel,Anguilla japonica

Glycosaminoglycans were isolated from the eel skin (Anguilla japonica) by actinase and endonuclease digestions, followed by a beta-elimination reaction and DEAE-Sephacel chromatography. Dermatan sulfate was the major glycosaminoglycan in the eel skin with 88% of the total uronic acid. The content of the IdoA2Salpha1-->4GalNAc4S sequence in eel skin, which shows anticoagulant activity through binding to heparin cofactor II, was two times higher than that of dermatan sulfate from porcine skin. The anti-IIa activity of eel skin dermatan sulfate was determined to be 2.4 units/mg, whereas dermatan sulfate from porcine skin shows 23.2 units/mg. The average molecular weight of dermatan sulfate was determined by gel chromatography on a TSKgel G3000SWXL column as 14 kDa. Based on 1H NMR spectroscopy, the presence of 3-sulfated and/or 2,3-sulfated IdoA residues was suggested. The reason why highly sulfated dermatan sulfate does not show anticoagulant activity is discussed. In addition to dermatan sulfate, the eel skin contained a small amount of keratan sulfate, which was identified by keratanase treatment.     

N-Acetylgalactosamine 4,6-O-sulfate residues mediate binding and activation of heparin cofactor II by porcine mucosal dermatan sulfate

Dermatan sulfate (DS) accelerates the inhibition of thrombin by heparin cofactor II (HCII). A hexasaccharide consisting of three l-iduronic acid 2-O-sulfate (IdoA2SO3)-->N-acetyl-D-galactosamine 4-O-sulfate (GalNAc4SO3) subunits was previously isolated from porcine skin DS and shown to bind HCII with high affinity. DS from porcine intestinal mucosa has a much lower content of this disaccharide but activates HCII with potency similar to that of porcine skin DS. Therefore, we sought to characterize oligosaccharides from porcine mucosal DS that interact with HCII. DS was partially depolymerized with chondroitinase ABC, and oligosaccharides containing 2-12 monosaccharide units were isolated. The oligosaccharides were then fractionated by anion-exchange and affinity chromatography on HCII-Sepharose, and the disaccharide compositions of selected fractions were determined. We found that the smallest oligosaccharides able to bind HCII were hexasaccharides. Oligosaccharides 6-12 units long that lacked uronic acid (UA)2SO3 but contained one or two GalNAc4,6SO3 residues bound, and binding was proportional to both oligosaccharide size and number of GalNAc4,6SO3 residues. Intact DS and bound dodecasaccharides contained predominantly IdoA but little D-glucuronic acid. Decasaccharides and dodecasaccharides containing one or two GalNAc4,6SO3 residues stimulated thrombin inhibition by HCII and prolonged the clotting time of normal but not HCII-depleted human plasma. These data support the hypothesis that modification of IdoA-->GalNAc4SO3 subunits in the DS polymer by either 2-O-sulfation of IdoA or 6-O-sulfation of GalNAc can generate molecules with HCII-binding sites and anticoagulant activity.     

Structure of a dermatan sulfate hexasaccharide that binds to heparin cofactor II with high affinity

Dermatan sulfate increases the rate of inhibition of thrombin by heparin cofactor II (HCII) approximately 1000-fold by providing a catalytic template to which both the inhibitor and the protease bind. Dermatan sulfate is a linear polymer of D-glucuronic acid (GlcA) or L-iduronic acid (IdoA) alternating with N-acetyl-D-galactosamine (GalNAc) residues. Heterogeneity in dermatan sulfate results from varying degrees of O-sulfation and from the presence of the two types of uronic acid residues. To characterize the HCII-binding site in dermatan sulfate, we isolated the smallest fragment of dermatan sulfate that bound to HCII with high affinity. Dermatan sulfate was partially N-deacetylated by hydrazinolysis, cleaved with nitrous acid at pH 4, and reduced with [3H]NaBH4. The resulting fragments, containing an even number of monosaccharide units with the reducing terminal GalNAc converted to [3H]2,5-anhydro-D-talitol (ATalR), were size-fractionated and then chromatographed on an HCII-Sepharose column. The smallest HCII-binding fragments were hexasaccharides, of which approximately 6% bound. Based on ion-exchange chromatography, the bound material appeared to comprise a heterogeneous mixture of molecules possessing four, five, or six sulfate groups per hexasaccharide. Subsequently, hexasaccharides with the highest affinity for HCII were isolated by overloading the HCII-Sepharose column. The high-affinity hexasaccharides were fractionated by strong anion-exchange chromatography, and one major peak representing approximately 2% of the starting hexasaccharides was isolated. The high-affinity hexasaccharide was cleaved to disaccharides that were analyzed by anion-exchange chromatography, paper electrophoresis, and paper chromatography. A single disulfated disaccharide, IdoA(2-SO4)----ATalR(4-SO4) was observed, indicating that the hexasaccharide has the following structure: IdoA(2-SO4)----GalNAc(4-SO4)----IdoA(2-SO4)---- GalNAc(4-SO4)----IdoA(2-SO4)----ATalR(4-SO4). Since IdoA(2-SO4)----GalNAc(4-SO4) comprises only approximately 5% of the disaccharides present in intact dermatan sulfate, clustering of these disaccharides must occur during biosynthesis to form the high-affinity binding site for HCII.     

High-performance liquid chromatography and on-line mass spectrometry detection for the analysis of chondroitin sulfates/hyaluronan disaccharides derivatized with 2-aminoacridone

In this study, we developed an on-line reverse-phase high-performance liquid chromatography-electrospray ionization-mass spectrometry (RP-HPLC-ESI-MS) separation and structural characterization of hyaluronan (HA)/chondroitin sulfate (CS)/dermatan sulfate (DS) disaccharides released by enzymatic treatment and derivatized with 2-aminoacridone (AMAC), providing a high-resolution system also applicable by using a further fluorimetric detector (Fp) before ESI-MS spectral acquisition. Isomeric nonsulfated HA and CS/DS disaccharides, isomeric monosulfated and isomeric disulfated CS/DS disaccharides, and the trisulfated species were distinctly separated and unambiguously identified by their retention times and mass spectra in negative ionization mode. In general, no multiply charged ions were detected even for highly charged disaccharides, but the presence of desulfonated products for highly sulfated species due to the relative instability of sulfo groups was observed. RP-HPLC-ESI-MS of each AMAC disaccharide was found to be linear from 3 to 500 ng with very high coefficient of correlation values due to the high efficiency of separation and the sharp outline of the peaks. Various CS/DS samples were characterized for disaccharide composition, and minor oligomer species identified as GalNAcSO₄ at the nonreducing end of chains was observed as a common component of these macromolecules. Furthermore, purified endogenous normal human plasma CS disaccharides were also evaluated by means of RP-HPLC-(Fp)-ESI-MS.     

Conformational studies on five octasaccharides isolated from chondroitin sulfate using NMR spectroscopy and molecular modeling

Chondroitin sulfate proteoglycans (CS-PG) are involved in the regulation of the central nervous system in vertebrates due to their presence on cell surfaces and in the extracellular matrix of tissues. The CS moieties are built up from repeating -4)GlcA(beta1-3)GalNAc(beta1- disaccharide units, partly O-sulfated at different positions. The presence of the disulfated disaccharide D-unit, GlcA2S(beta1-3)GalNAc6S, in the CS moiety of the proteoglycan DSD-1-PG/phosphacan, correlates with neurite outgrowth promotion. The binding of monoclonal antibody (mAb) 473HD to DSD-1-PG, reducing neuronal stimulation, is inhibited by shark cartilage CS-D. CS-D is also recognized by two other mAbs, MO-225 and CS-56. Conformational studies were performed using NMR spectroscopy and molecular modeling on five octasaccharides isolated from shark cartilage CS-D. These octasaccharides present different binding properties toward the three mAbs. The combination of the experimental and theoretical approaches revealed that the sulfate group at position 2 of GlcA in disaccharide D and the presence of an exocyclic negative tail in disaccharides C [GlcA(beta1-3)GalNAc6S] and DeltaC [Delta4,5HexA(alpha1-3)GalNAc6S] are important for antibody recognition.     

Structural determination of novel tetra- and hexasaccharide sequences isolated from chondroitin sulfate H (oversulfated dermatan sulfate) of hagfish notochord

Oversulfated chondroitin sulfate H (CS-H) isolated from hagfish notochord is a unique dermatan sulfate consisting mainly of IdoAalpha1-3GalNAc(4S,6S), where IdoA, GalNAc, 4S and 6S represent L-iduronic acid, Nacetyl-D-galactosamine, 4-O-sulfate and 6-O-sulfate, respectively. Several tetra- and hexasccharide fractions were isolated from CS-H after partial digestion with bacterial chondroitinase B to investigate the sequential arrangement of the IdoAalpha1-3GalNAc(4S,6S) unit in the CS-H polysaccharide. A structural analysis of the isolated oligosaccharides by enzymatic digestions, mass spectrometry and 1H NMR spectroscopy demonstrated that the major tetrasaccharides shared the common disulfated core structure delta4,5HexAalpha1-3GalNAc(4S)beta1-4IdoAalpha1-3 GalNAc (4S) with 0 approximately 3 additional O-sulfate groups, where delta4,5HexA represents 4-deoxy-alpha-L-threo-hex-4-enepyranosyluronic acid. The major hexasaccharides shared the common trisulfated core structure delta4,5HexAalpha1-3 GalNAc(4S)beta1-4 IdoAalpha1-3 GalNAc(4S)beta1-4IdoAalpha1-3 GalNAc(4S) with 1 approximately 4 additional O-sulfate groups. Some extra sulfate groups in both tetra- and hexasaccharides were located at the C-2 position of a delta4,5HexA or an internal IdoA residue, or C-6 position of 4-O-sulfated GalNAc residues, forming the unique disulfated or trisulfated disaccharide units, IdoA (2S)-GalNAc(4S), IdoA-GalNAc(4S,6S) and IdoA (2S)-GalNAc(4S,6S), where 2S represents 2-O-sulfate. Of the demonstrated sequences, five tetra- and four hexasaccharide sequences containing these units were novel.     

Oversulfated dermatan sulfate exhibits neurite outgrowth-promoting activity toward embryonic mouse hippocampal neurons: implications of dermatan sulfate in neuritogenesis in the brain

Brain-specific chondroitin sulfate (CS) proteoglycan (PG) DSD-1-PG/6B4-PG/phosphacan isolated from neonatal mouse brains exhibits neurite outgrowth-promoting activity toward embryonic rat and mouse hippocampal neurons in vitro through the so-called DSD-1 epitope embedded in its glycosaminoglycan side chains. Oversulfated CS variants, CS-D from shark cartilage and CS-E from squid cartilage, also possess similar activities. We have proposed that the neuritogenic property of the DSD-1 epitope may be attributable to a distinct CS structure characterized by the disulfated D disaccharide unit [GlcUA(2S)-GalNAc(6S)]. In this study, we assessed neuritogenic potencies of various oversulfated dermatan sulfate (DS) preparations purified from hagfish notochord, the bodies of two kinds of ascidians and embryonic sea urchin, which are characterized by the predominant disulfated disaccharide units of [IdoUA-GalNAc(4S,6S)] (68%), [IdoUA(2S)-GalNAc(4S)] (66%) plus [IdoUA(2S)-GalNAc(6S)] (5%), [IdoUA(2S)-GalNAc (6S)] (>90%), and [IdoUA-GalNAc(4S,6S)] (74%), respectively. They exerted marked neurite outgrowth-promoting activities, resulting in distinct morphological features depending on the individual structural features. Such activities were not observed for a less sulfated DS preparation derived from porcine skin, which has a monosulfated disaccharide unit [IdoUA-Gal-NAc(4S)] as a predominant unit. The neurite outgrowth-promoting activities of these oversulfated DS preparations and DSD-1-PG were eliminated by the specific enzymatic cleavage of GalNAc-IdoUA linkages characteristic of DS using chondroitinase B. In addition, chemical analysis of the glycosaminoglycan side chains of DSD-1-PG revealed the DS-type structures. These observations suggest potential novel neurobiological functions of oversulfated DS structures and may reflect the physiological neuritogenesis during brain development by mammalian oversulfated DS structures exemplified by the DSD-1 epitope.     

The disaccharide repeating-units of heparan sulfate

Five disaccharides have been isolated after degradation of heparan sulfate by heparinase (heparin lyase) and heparitinase (heparan sulfate lyase) and are suggested to represent the repeating units of the polysaccharide. They all contain a 4,5-unsaturated uronic acid residue and are: (a) A trisulfated disaccharide that is apparently identical to a disaccharide repeating-unit of heparin; (b) a disulfated disaccharide that seems unique for heparan sulfate and contains 2-deoxy-2-sulfamidoglucose and uronic acid sulfate residues; (c) a nonsulfated disaccharide containing a 2-acetamido-2-deoxyglucose residue; (d) a monosulfated disaccharide containing a 2-acetamido-2-deoxyglucose sulfate residue; and (e) a monosulfated disaccharide containing a 2-deoxy-2-sulfamidoglucose residue. Yields of these disaccharides from different heparan sulfate fractions are discussed in relation to possible arrangements of these units in the intact polymer.     

Heterogeneity of rat rib chondroitin sulfate and susceptibility to rat gastric chondrosulfatase

Cartilage chondroitin sulfate isolated directly from rat rib or from in vitro culture of rat rib constitutes a population of glycosaminoglycans which is heterogeneous with respect to size, degree of sulfation and content of N-acetylgalactosamine 4-sulfate. Fractions elute from Dowex-1 in order of increasing molecular size and degree of sulfation up to a certain limit. Unsulfated disaccharides and disulfated disaccharides are present in both the undersulfated chondroitin sulfate fractions and in the average or more representative chondroitin sulfate. A small content of disaccharide 6-sulfate is present in all fractions and appears to be an integral part of the chondroitin 4-sulfate molecules. Rat gastric chondrosulfatase hydrolyzes sulfate preferentially from the larger chondroitin 4-sulfate molecules, and the sulfate is removed primarily from the disaccharide 4-sulfate units.     

Structural requirements of heparin disaccharides responsible for hemorrhage: reversion of the antihemostatic effect by ATP

Topically applied heparin and heparan sulfate disaccharides, with the basic structure delta-4,5 uronyl-(1----4)-glucosamine and bearing a sulfate at the C-6 position of the glucosamine residue, are antihemostatics as potent as heparin, producing uncontrollable hemorrhage from small blood vessels. The finding that other sulfated disaccharides with the same sulfate:hexosamine:uronic acid ratios but with the sulfate at a different position (C-2), or with different glycosidic linkage (1----3), were inactive as inhibitors of hemostasis indicates that a specific structure is needed to produce the effect. The inhibitory activity of the normal hemostatic process could be reversed by ATP. Molecular models show that part of the disaccharide inhibitors and ATP hold a similar structural conformation.     

Structural studies on sulfated oligosaccharides derived from the carbohydrate-protein linkage region of chondroitin 6-sulfate proteoglycans of shark cartilage. II. Seven compounds containing 2 or 3 sulfate residues.

Shark cartilage proteoglycans bear predominantly chondroitin 6-sulfate. After exhaustive protease digestion, reductive beta-elimination and subsequent chondroitinase ABC digestion, 13 hexasaccharide alditols were obtained from the carbohydrate-protein linkage region and six of them contain 0 or 1 sulfate and/or 1 phosphate residue (Sugahara, K., Ohi, Y., Harada, T., de Waard, P., and Vliegenthart, J. F. G. (1992) J. Biol. Chem. 267, 6027-6035). The other seven compounds, which represent approximately 60% of the isolated linkage hexasaccharides, were analyzed by chondroitinase ACII digestion in conjunction with high performance liquid chromatography and by 500-MHz one- and two dimensional 1H NMR spectroscopy. All seven compounds have the following conventional structure in common. [formula: see text] Two disulfated compounds have an O-sulfate on C-6 of the Gal-2 residue attached to xylitol in combination with an O-sulfate on C-4 or on C-6 of the GalNAc residue. The third disulfated compound has O-sulfate on C-6 of Gal-2, and also on C-6 of Gal-3. Two of the trisulfated compounds also have O-sulfate on C-6 of both Gal-2 and Gal-3 with in addition sulfate on C-6 or C-4 of GalNAc. The other two trisulfated compounds have O-sulfate on C-6 of Gal-2 and on C-4 of Gal-3 in conjunction with sulfate on C-6 or C-4 of GalNAc.     

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.     

Structural studies on sulfated glycopeptides from the carbohydrate-protein linkage region of chondroitin 4-sulfate proteoglycans of swarm rat chondrosarcoma. Demonstration of the structure Gal(4-O-sulfate)beta 1-3Gal beta 1-4XYL beta 1-O-Ser

Nonsulfated, monosulfated, and disulfated glycopeptides containing the entire carbohydrate sequence of the glycosaminoglycan-specific linkage region were isolated after exhaustive enzymatic digestions of Swarm rat chondrosarcoma proteoglycans with chondroitinase ABC, papain, and Pronase. Their structures were examined by 500 MHz 1H NMR spectroscopy. The nonsulfated compound has the following structure with trace amounts of a few additional amino acids: delta 4,5-GlcA beta 1-3GalNAc beta 1-4GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser. The monosulfated compound has an ester sulfate on C-4 of the GalNAc residue and the disulfated compound has an additional hitherto unrecognized ester sulfate on C-4 of the second galactose residue which is remote from the innermost xylose. This new structure was confirmed by two-dimensional homonuclear Hartmann-Hahn spectroscopy. The molar ratio of the isolated nonsulfated, monosulfated, and disulfated compounds was 53:37:10 based on the serine contents. Biological significance of the newly found sulfated linkage structure is discussed.     

Purification and substrate specificity of heparitinase I and heparitinase II from Flavobacterium heparinum. Analyses of the heparin and heparan sulfate degradation products by 13C NMR spectroscopy

The purification of two heparitinases and a heparinase, in high yields from Flavobacterium heparinum was achieved by a combination of molecular sieving and cation-exchange chromatography. Heparinase acts upon N-sulfated glucosaminido-L-iduronic acid linkages of heparin. Substitution of N-sulfate by N-acetyl groups renders the heparin molecule resistant to degradation by the enzyme. Heparitinase I acts on N-acetylated or N-sulfated glucosaminido-glucuronic acid linkages of the heparan sulfate. Sulfate groups at the 6-position of the glucosamine moiety of the heparan sulfate chains seem to be impeditive for heparitinase I action. Heparitinase II acts upon heparan sulfate producing disulfated, N-sulfated and N-acetylated-6-sulfated disaccharides, and small amounts of N-acetylated disaccharide. These and other results suggest that heparitinase II acts preferentially upon N,6-sulfated glucosaminido-glucuronic acid linkages. The total degradation of heparan sulfate is only achieved by the combined action of both heparitinases. The 13C NMR spectra of the disaccharides formed from heparan sulfate and a heparin oligosaccharide formed by the action of the heparitinases are in accordance to the proposed mode of action of the enzymes. Comparative studies of the enzymes with the commercially available heparinase and heparitinase are described.     

An enhanced humoral immune response against the swimbladder nematode, Anguillicola crassus, in the Japanese eel, Anguilla japonica, compared with the European eel, A. anguilla.

The humoral immune response in the two eel species, Anguilla japonica and Anguilla anguilla against two fractions of antigens in Anguillicola crassus were studied. Within species, both eel species showed significantly elevated titres compared with controls when immunized with antigens from Anguillicola crassus. In interspecific comparison, Anguilla japonica showed significantly elevated titres in comparison with Anguilla anguilla. Immunization of Anguilla anguilla caused a significantly decrease in the plasma levels of protein in comparison with control fish and all groups of Anguilla japonica. In contrast, Anguilla japonica showed significantly lower plasma levels of Ig in all groups compared with Anguilla anguilla. The different susceptibilities to Anguillicola crassus between the natural host, Anguilla japonica, and the naive, Anguilla anguilla, is partly due to differences in the ability of the two eel species to mount a humoral immune response.     

Isolation of dermatan sulfate with high heparin cofactor II-mediated thrombin-inhibitory activity from porcine spleen

We prepared dermatan sulfate specimens from various porcine tissues, and compared their heparin cofactor II-mediated thrombin-inhibitory activities and chemical natures, including disaccharide composition. Electrophoresis of the specimens on cellulose acetate membrane indicated that spleen dermatan sulfate was the most acidic of the dermatan sulfates prepared from the various porcine tissues. Analysis of the disaccharide units of the dermatan sulfate specimens by high-performance liquid chromatography revealed that spleen dermatan sulfate was rich in 4,6-di-O-sulfated N-acetylgalactosamine residues as compared with those of the other tissues. Spleen dermatan sulfate exhibited the highest thrombin-inhibitory activity, which may be related to its high content of the disulfated N-acetylgalactosamine residue.     

Two squid skin proteoglycans each containing chondroitin sulfates with different sulfation patterns.

Two chondroitin sulfate containing proteoglycans, amounting to approximately 6% of the tissue proteoglycans, were isolated from the skin of the squid. They were almost completely extracted by 4 M guanidine hydrochloride in the presence of proteinase inhibitors, and then they were separated by DEAE-Sephacel chromatography and isolated by further chromatography on Sepharose CL-4B. Each proteoglycan contained two types of chondroitin sulfates that differed in their sulfation patterns. One proteoglycan (molecular mass (M(r)) 5.6 x 10(5)) contained, on the average, four chondroitins (M(r) 8.4 x 10(4)) and five chondroitin sulfates (M(r) 3.4 x 10(4)), whereas the other proteoglycan (M(r) 5.2 x 10(5)) contained three chondroitin sulfates (M(r) 1.1 x 10(5)) and five oversulfated chondroitin sulfates (M(r) 4.3 x 10(4)). The glycosaminoglycans were released from the proteoglycans by treatment with alkaline borohydride, separated from the oligosaccharides by chromatography on Bio-Gel P-30, and isolated by chromatography on DEAE-Sephacel and Sepharose CL-6B. Chondroitin sulfates were degraded by chondroitinase AC to an extent of 70% and consisted of significant amounts of disaccharides sulfated at C-4 of the galactosamine, disulfated disaccharides, and small amounts of nonsulfated disaccharides, as well as disaccharides that bore sulfates at C-6. Oversulfated chondroitin sulfate was degraded by chondroitinase AC to only 40% and contained appreciable amounts of disulfated and trisulfated disaccharides. The glycosaminoglycans also contained neutral monosaccharides; glucose was the predominant neutral sugar. A part of the oligosaccharides of both proteoglycans was of identical structure to that of chondroitin sulfate.     

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.     

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.