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 functional dermatan sulfate epitope containing iduronate(2-O-sulfate)alpha1-3GalNAc(6-O-sulfate) disaccharide in the mouse brain: demonstration using a novel monoclonal antibody raised against dermatan sulfate of ascidian Ascidia nigra

Oversulfated chondroitin sulfate (CS), dermatan sulfate (DS), and CS/DS hybrid structures bind growth factors, promote the neurite outgrowth of hippocampal neurons in vitro, and have been implicated in the development of the brain. To investigate the expression of functional oversulfated DS structures in the brain, a novel monoclonal antibody (mAb), 2A12, was generated against DS (An-DS) from ascidian Ascidia nigra, which contains a unique iD disaccharide unit, iduronic acid (2-O-sulfate)alpha1-->3GalNAc(6-O-sulfate), as a predominant disaccharide. mAb 2A12 specifically reacted with the immunogen, and recognized iD-enriched decasaccharides as minimal structures. The 2A12 epitope was specifically observed in the hippocampus and cerebellum of the mouse brain on postnatal day 7, and the expression in the cerebellum disappeared in the adult brain, suggesting a spatiotemporally regulated expression of this epitope. Embryonic hippocampal neurons were immunopositive for 2A12, and the addition of the antibody to the culture medium significantly reduced the neurite growth of hippocampal neurons. In addition, two minimum 2A12-reactive decasaccharide sequences with multiple consecutive iD units were isolated from the An-DS chains, which exhibited stronger inhibitory activity against the binding of various growth factors and neurotrophic factors to immobilized embryonic pig brain CS/DS chains (E-CS/DS) than the intact E-CS/DS, suggesting that the 2A12 epitope at the neuronal surface acts as a receptor or co-receptor for these molecules. Thus, we have selected a unique antibody that recognizes iD-enriched oversulfated DS structures, which are implicated in the development of the hippocampus and cerebellum in the central nervous system. The antibody will also be applicable for investigating structural alterations in CS/DS in aging and pathological conditions.     

Heparin-binding growth factor, pleiotrophin, mediates neuritogenic activity of embryonic pig brain-derived chondroitin sulfate/dermatan sulfate hybrid chains

Chondroitin sulfate (CS) and dermatan sulfate (DS) chains play roles in the central nervous system. Most notably, CS/DS hybrid chains (E-CS/DS) purified from embryonic pig brains bind growth factors and promote neurite outgrowth toward embryonic mouse hippocampal neurons in culture. However, the neuritogenic mechanism is not well understood. Here we showed that pleiotrophin (PTN), a heparin-binding growth factor, produced mainly by glia cells, was the predominant binding partner for E-CS/DS in the membrane-associated protein fraction of neonatal rat brain. The CS/DS chains were separated on a PTN column into unbound, low affinity, and high affinity fractions. The latter two fractions promoted outgrowth of dendrite- and axon-like neurites, respectively, whereas the unbound fraction showed no such activity. The activity of the low affinity fraction was abolished by an anti-PTN antibody or when glia cells were removed from the culture. In contrast, the high affinity fraction displayed activity under both these conditions. Hence, PTN mainly from glia cells mediated the activity of the low affinity but not the high affinity fraction. The anti-CS antibody 473HD neutralized the neuritogenic activities of both fractions. Interaction analysis indicated that the 473HD epitope and PTN-binding domains in the E-CS/DS chains largely overlap. The three affinity subfractions differed in disaccharide composition and the distribution of l-iduronic acid-containing disaccharides along the chains. Oversulfated disaccharides and nonconsecutive iduronic acid-containing units were the requirements for the E-CS/DS chains to bind PTN and to exhibit the neuritogenic activities. Thus, CS subpopulations with distinct structures in the mammalian brain play different roles in neuritogenesis through distinct molecular mechanisms, at least in part by regulating the functions of growth factors.     

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.     

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.     

Appican, the proteoglycan form of the amyloid precursor protein, contains chondroitin sulfate E in the repeating disaccharide region and 4-O-sulfated galactose in the linkage region

Chondroitin sulfate (CS)-D and CS-E, which are characterized by oversulfated disaccharide units, have been shown to regulate neuronal adhesion, cell migration, and neurite outgrowth. CS proteoglycans (CSPGs) consist of a core protein to which one or more CS chains are attached via a serine residue. Although several brain CSPGs, including mouse DSD-1-PG/phosphacan, have been found to contain the oversulfated D disaccharide motif, no brain CSPG has been reported to contain the oversulfated E motif. Here we analyzed the CS chain of appican, the CSPG form of the Alzheimer's amyloid precursor protein. Appican is expressed almost exclusively by astrocytes and has been reported to have brain- and astrocyte-specific functions including stimulation of both neural cell adhesion and neurite outgrowth. The present findings show that the CS chain of appican has a molecular mass of 25-50 kDa. This chain contains a significant fraction (14.3%) of the oversulfated E motif GlcUA beta 1-3GalNAc(4,6-O-disulfate). The rest of the chain consists of GlcUA beta 1-3GalNAc(4-O-sulfate) (81.2%) and minor fractions of GlcUA beta 1-3GalNAc and GlcUA beta 1-3GalNAc(6-O-sulfate). We also show that the CS chain of appican contains in its linkage region the 4-O-sulfated Gal structure. Thus, appican is the first example of a specific brain CSPG that contains the E disaccharide unit in its sugar backbone and the 4-O-sulfated Gal residue in its linkage region. The presence of the E unit is consistent with and may explain the neurotrophic activities of appican.     

Oversulfated chondroitin sulfate plays critical roles in the neuronal migration in the cerebral cortex

Chondroitin sulfate (CS) proteoglycans bind with various proteins through CS chains in a CS structure-dependent manner, in which oversulfated structures, such as iB (IdoA(2-O-sulfate)alpha1-3GalNAc(4-O-sulfate)), D (GlcA(2-O-sulfate)beta1-3GalNAc(6-O-sulfate)), and E (GlcAbeta1-3GalNAc(4,6-O-disulfate)) units constitute the critical functional module. In this study, we examined the expression and function of three CS sulfotransferases in the developing neocortex: uronyl 2-O-sulfotransferase (UST), N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (4,6-ST) and dermatan 4-O-sulfotransferase-1 (D4-ST), which are responsible for the synthesis of oversulfated structures. The CS chains of the neocortex of mouse embryos contained significant amounts of D and E units that are generated by UST and 4,6-ST, respectively. UST and 4,6-ST mRNAs were expressed in the ventricular and subventricular zones, and their expression increased during late embryonic development. In utero electroporation experiments indicated that knockdown of UST and 4,6-ST resulted in the disturbed migration of cortical neurons. The neurons electroporated with the short hairpin RNA constructs of UST and 4,6-ST accumulated in the lower intermediate zone and in the subventricular zone, showing a multipolar morphology. The cDNA constructs of UST and 4,6-ST rescued the defects caused by the RNA interference, and the neurons were able to migrate radially. On the other hand, knockdown of D4-ST, which is involved in the biosynthesis of the iB unit, caused no migratory defects. These results revealed that specific oversulfated structures in CS chains play critical roles in the migration of neuronal precursors during cortical development.     

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.     

Two related but distinct chondroitin sulfate mimetope octasaccharide sequences recognized by monoclonal antibody WF6

Chondroitin sulfate (CS) proteoglycans are major components of cartilage and other connective tissues. The monoclonal antibody WF6, developed against embryonic shark cartilage CS, recognizes an epitope in CS chains, which is expressed in ovarian cancer and variably in joint diseases. To elucidate the structure of the epitope, we isolated oligosaccharide fractions from a partial chondroitinase ABC digest of shark cartilage CS-C and established their chain length, disaccharide composition, sulfate content, and sulfation pattern. These structurally defined oligosaccharide fractions were characterized for binding to WF6 by enzyme-linked immunosorbent assay using an oligosaccharide microarray prepared with CS oligosaccharides derivatized with a fluorescent aminolipid. The lowest molecular weight fraction recognized by WF6 contained octasaccharides, which were split into five subfractions. The most reactive subfraction contained several distinct octasaccharide sequences. Two octasaccharides, DeltaD-C-C-C and DeltaC-C-A-D (where A represents GlcUAbeta1-3GalNAc(4-O-sulfate), C is GlcUAbeta1-3Gal-NAc(6-O-sulfate), D is GlcUA(2-O-sulfate)beta1-3GalNAc(6-O-sulfate), DeltaCis Delta(4,5)HexUAalpha1-3GalNAc(6-O-sulfate), and DeltaDis Delta(4,5)HexUA(2-O-sulfate)alpha1-3GalNAc(6-O-sulfate)), were recognized by WF6, but other related octasaccharides, DeltaC-A-D-C and DeltaC-C-C-C, were not. The structure and sequences of both the binding and nonbinding octasaccharides were compared by computer modeling, which revealed a remarkable similarity between the shape and distribution of the electrostatic potential in the two different octasaccharide sequences that bound to WF6 and that differed from the nonbinding octasaccharides. The strong similarity in structure predicted for the two binding CS octasaccharides (DeltaD-C-C-C and DeltaC-C-A-D) provided a possible explanation for their similar affinity for WF6, although they differed in sequence and thus form two specific mimetopes for the antibody.     

Chondroitin sulfate/dermatan sulfate hybrid chains from embryonic pig brain, which contain a higher proportion of L-iduronic acid than those from adult pig brain, exhibit neuritogenic and growth factor binding activities

We have shown that over-sulfated chondroitin sulfate/dermatan sulfate (CS/DS) chains from various marine organisms exhibit growth factor binding activities and neurite outgrowth-promoting activities in embryonic mouse hippocampal neurons in vitro. In this study we demonstrated that CS/DS hybrid chains purified from embryonic pig brain displayed marked neuritogenic activity and growth factor binding activities toward fibroblast growth factor 2 (FGF2), FGF10, FGF18, pleiotrophin, and midkine, all of which exhibit neuroregulatory activities in the brain. In contrast, the CS/DS preparation from adult pig brain showed considerably less activity to bind these growth factors and no neuritogenic activity. Structural analysis indicated that the average size of the CS/DS chains was similar (40 kDa) between these two preparations, but the disaccharide compositions differed considerably, with a significant proportion of l-iduronic acid (IdoUA)-containing disaccharides (8 approximately 9%) in the CS/DS chains from embryos but not in those from adults (<1%). Interestingly, both neurite outgrowth-promoting activity and growth factor binding activities of the CS/DS chains from embryos were abolished by digestion not only with chondroitinase ABC but also with chondroitinase B, suggesting that the IdoUA-containing motifs are essential for these activities. These findings imply that the temporal expression of CS/DS hybrid structures containing both GlcUA and IdoUA and binding activities toward various growth factors play important roles in neurogenesis in the early stages of the development of the brain.     

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.     

Specificities of three distinct human chondroitin/dermatan N-acetylgalactosamine 4-O-sulfotransferases demonstrated using partially desulfated dermatan sulfate as an acceptor: implication of differential roles in dermatan sulfate biosynthesis

4-O-Sulfation of GalNAc is a high frequency modification of chondroitin sulfate and dermatan sulfate (DS), and three major GalNAc 4-O-sulfotransferases including dermatan 4-O-sulfotransferase-1 (D4ST-1) and chondroitin 4-O-sulfotransferases-1 and -2 (C4ST-1 and -2) have been identified. 4-O-Sulfation of GalNAc during DS biosynthesis had long been postulated to be a prerequisite for iduronic acid (IdoUA) formation by C5-epimerization of GlcUA. This hypothesis has recently been argued based on enzymological studies using microsomes that C5-epimerization precedes 4-O-sulfation, which was further supported by the specificity of the cloned D4ST-1 with predominant preference for IdoUA-GalNAc flanked by GlcUA-GalNAc over IdoUA-GalNAc flanked by IdoUA-GalNAc in exhaustively desulfated dermatan. Whereas the counterproposal explains the initial reactions, apparently it cannot rationalize the synthetic mechanism of IdoUA-GalNAc(4-O-sulfate)-rich clusters typical of mature DS chains. In this study, we examined detailed specificities of the three recombinant human 4-O-sulfotransferases using partially desulfated DS as an acceptor. Enzymatic analysis of the transferase reaction products showed that D4ST-1 far more efficiently transferred sulfate to GalNAc residues in -IdoUA-Gal-NAc-IdoUA-than in -GlcUA-GalNAc-GlcUA-sequences. In contrast, C4ST-1 showed the opposite preference, and C4ST-2 used GalNAc residues in both sequences to comparable degrees, being consistent with its phylogenetic relations to D4ST-1 and C4ST-1. Structural analysis of the oligosaccharides, which were isolated after chondroitinase AC-I digestion of the 35S-labeled transferase reaction products, revealed for the first time that D4ST-1, as compared with C4ST-1 and C4ST-2, most efficiently utilized GalNAc residues located not only in the sequence -IdoUA-GalNAc-IdoUA- but also in -GlcUA-Gal-NAc-IdoUA- and -IdoUA-GalNAc-GlcUA-. The isolated oligosaccharide structures also suggest that 4-O-sulfation promotes subsequent 4-O-sulfation of GalNAc in the neighboring disaccharide unit.     

Dermatan sulfate epimerase 2 is the predominant isozyme in the formation of the chondroitin sulfate/dermatan sulfate hybrid structure in postnatal developing mouse brain

Chondroitin sulfate (CS) and dermatan sulfate (DS) are expressed in significant amounts in the brain and play important roles in the development of the central nervous system in mammals. CS and DS structures are often found in a single CS/DS hybrid chain. The l-iduronic acid (IdoA)-containing domain, which defines a DS-type domain, appears key to the biological functions of the CS/DS hybrid chain. In this study, to clarify the distribution of the DS-type structure in the brain during development, the expression patterns of DS epimerase 1 (DS-epi1) and DS-epi2, both of which convert d-glucuronic acid into IdoA, were investigated by in situ hybridization. DS-epi2 was ubiquitously expressed in the developing brain after birth, whereas the expression of DS-epi1 was faint and obscure at all developmental stages. Quantitative real-time polymerase chain reaction revealed the expression of DS-epi2 to be higher than that of DS-epi1 throughout development, suggesting that DS-epi2 but not DS-epi1 is mostly expressed in the brain and plays key roles in the epimerization of CS/DS during its biosynthesis. Moreover, an analysis of the disaccharides of CS/DS demonstrated significant amounts of IdoA-containing iD units [IdoA(2S)-GalNAc(6S)] and iB units [IdoA(2S)-GalNAc(4S)], where 2S, 4S and 6S stand for 2-O-, 4-O- and 6-O-sulfate, respectively, in every region of the brain examined. The proportion of these units in cerebellar CS/DS was greatly altered during postnatal development. These results suggest that the IdoA-containing structures in the developing brain are mainly produced by the actions of DS-epi2 and play crucial roles in postnatal development.     

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.     

Structural determination of five novel tetrasaccharides containing 3-O-sulfated D-glucuronic acid and two rare oligosaccharides containing a beta-D-glucose branch isolated from squid cartilage chondroitin sulfate E

Oversulfated chondroitin sulfate E (CS-E) derived from squid cartilage exhibits intriguing biological activities, which appear to reflect the biological activities of mammalian CS chains containing the so-called E disaccharide unit [GlcAbeta1-3GalNAc(4,6-O-disulfate)]. Previously, we isolated novel tetra- and hexasaccharides containing a rare GlcA(3-O-sulfate) at the nonreducing end after digestion of squid cartilage CS-E with testicular hyaluronidase. In this study, squid cartilage CS-E was extensively digested with chondroitinase AC-II, which yielded five highly sulfated novel tetrasaccharides and two odd-numbered oligosaccharides (tri- and pentasaccharides) containing D-Glc. Their structures were determined by fast atom bombardment mass spectrometry and (1)H NMR spectroscopy. The results revealed an internal GlcA(3-O-sulfate) residue for all the novel tetrasaccharide sequences, which rendered the oligosaccharides resistant to the enzyme. The results suggest that GlcA(3-O-sulfate) units are not clustered but rather interspersed in the CS-E polysaccahride chains, being preferentially located in the highly sulfated sequences. The predominant structure on the nearest nonreducing side of a GlcA(3-O-sulfate) residue was GalNAc(4-O-sulfate) (80%), whereas that on the reducing side was GalNAc(4,6-O-disulfate) (59%). The structural variety in the vicinity of the GlcA(3-O-sulfate) residue might represent the substrate specificity of the unidentified chondroitin GlcA 3-O-sulfotransferase. The results also revealed a trisaccharide and a pentasaccahride sequence, both of which contained a beta-d-Glc branch at the C6 position of the constituent GalNAc residue. Approximately 5 mol % of all disaccharide units were substituted by Glc in the CS-E preparation used.     

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.     

Structural determination of novel sulfated octasaccharides isolated from chondroitin sulfate of shark cartilage and their application for characterizing monoclonal antibody epitopes

Twelve octasaccharide fractions were obtained from chondroitin sulfate C derived from shark cartilage after hyaluronidase digestion. Their sugar and sulfate composition was assigned by matrix-assisted laser desorption ionization time of flight mass spectrometry. The sequences were determined at low picomole amounts by a combination of enzymatic digestions with high-performance liquid chromatography, and were composed of disaccharide building units including O [GlcUAbeta1-3GalNAc], C [GlcUAbeta1-3GalNAc(6S)], A [GlcUAbeta1-3GalNAc(4S)], and/or D [GlcUA(2S)beta1-3GalNAc(6S)], where 2S, 4S, and 6S represent 2-O-, 4-O-, and 6-O-sulfate, respectively. As many as 24 different sequences including minor ones were revealed, exhibiting a high degree of structural diversity reflecting the enormous heterogeneity of the parent polysaccharides. Nineteen of them were novel, with the other four reported previously as unsaturated counterparts obtained after digestion with chondroitinase. Microarrays of these structurally defined octasaccharide fractions were prepared using low picomole amounts of their lipid-derivatives to investigate the binding specificity of four commercial anti-chondroitin sulfate antibodies CS-56, MO-225, 2H6, and LY111. The results revealed that multiple unique sequences were recognized by each antibody, which implies that the common conformation shared by the multiple primary sequences in the intact chondroitin sulfate chains is important as an epitope for each monoclonal antibody. Comparison of the specificity of the tested antibodies indicates that CS-56 and MO-225 specifically recognize octasaccharides containing an A-D tetrasaccharide sequence, whereas 2H6 and LY111 require a hexasaccharide as a minimum size for their binding, and prefer sequences with A- and C-units such as C-C-A-C (2H6) or C-C-A-O, C-C-A-A, and C-C-A-C (LY111) for strong binding but require no D-unit.