Expanding the chondroitin glycoproteome of Caenorhabditis elegans.

Chondroitin sulfate proteoglycans (CSPGs) are important structural components of connective tissues in essentially all metazoan organisms. In vertebrates, CSPGs are involved also in more specialized processes such as neurogenesis and growth factor signaling. In invertebrates, however, knowledge of CSPGs core proteins and proteoglycan-related functions is relatively limited, even for Caenorhabditis elegans. This nematode produces large amounts of non-sulfated chondroitin in addition to low-sulfated chondroitin sulfate chains. So far, only nine core proteins (CPGs) have been identified, some of which have been shown to be involved in extracellular matrix formation. We recently introduced a protocol to characterize proteoglycan core proteins by identifying CS-glycopeptides with a combination of biochemical enrichment, enzymatic digestion, and nano-scale liquid chromatography MS/MS analysis. Here, we have used this protocol to map the chondroitin glycoproteome in C. elegans, resulting in the identification of 15 novel CPG proteins in addition to the nine previously established. Three of the newly identified CPGs displayed homology to vertebrate proteins. Bioinformatics analysis of the primary protein sequences revealed that the CPG proteins altogether contained 19 unique functional domains, including Kunitz and endostatin domains, suggesting direct involvement in protease inhibition and axonal migration, respectively. The analysis of the core protein domain organization revealed that all chondroitin attachment sites are located in unstructured regions. Our results suggest that CPGs display a much greater functional and structural heterogeneity than previously appreciated and indicate that specialized proteoglycan-mediated functions evolved early in metazoan evolution.

Major chondroitin sulfate proteoglycans identified in L6J1 myoblast culture.

The major proteoglycans from L6J1 rat myoblast culture were identified. The proteoglycans were isolated from different constituents of cell culture: culture medium, extracellular matrix (ECM), and myoblasts. To identify their core proteins, the proteoglycans were treated with enzymes specifically digesting chondroitin/dermatan sulfates or chondroitin sulfates. Subsequent electrophoresis and mass spectrometry revealed versican, collagen XII, and inter-α-trypsin inhibitor classified as chondroitin sulfate proteoglycans and biglycan known to be chondroitin/dermatan sulfate proteoglycan. Versican was identified in ECM and the other proteoglycans in the culture medium. Such difference in localization is likely to be a consequence of different biological functions. Versican, collagen XII, and biglycan are synthesized by myoblasts and inter-α-trypsin inhibitor originates from fetal bovine serum (a culture medium component).

A positively charged channel within the Smc1/Smc3 hinge required for sister chromatid cohesion.

Cohesin's structural maintenance of chromosome 1 (Smc1) and Smc3 are rod-shaped proteins with 50-nm long intra-molecular coiled-coil arms with a heterodimerization domain at one end and an ABC-like nucleotide-binding domain (NBD) at the other. Heterodimerization creates V-shaped molecules with a hinge at their centre. Inter-connection of NBDs by Scc1 creates a tripartite ring within which, it is proposed, sister DNAs are entrapped. To investigate whether cohesin's hinge functions as a possible DNA entry gate, we solved the crystal structure of the hinge from Mus musculus, which like its bacterial counterpart is characterized by a pseudo symmetric heterodimeric torus containing a small channel that is positively charged. Mutations in yeast Smc1 and Smc3 that together neutralize the channel's charge have little effect on dimerization or association with chromosomes, but are nevertheless lethal. Our finding that neutralization reduces acetylation of Smc3, which normally occurs during replication and is essential for cohesion, suggests that the positively charged channel is involved in a major conformational change during S phase.

Plasmodium falciparum: Assessment of parasite-infected red blood cell binding to placental chondroitin proteoglycan and bovine tracheal chondroitin sulfate A.

In pregnant women infected with Plasmodium falciparum, the infected red blood cells (IRBCs) sequester in placenta by binding to the chondroitin 4-sulfate (C4S) chains of low sulfated chondroitin sulfate proteoglycan (CSPG). Placental CSPG, the natural receptor for IRBC adherence in the placenta, is the ideal molecule for studying structural interactions in IRBC adhesion to C4S, adhesion inhibitory antibody responses, and identification of parasite adhesive protein(s). However, because of difficulty involved in purifying placental CSPG, the commercially available bovine tracheal chondroitin sulfate A (bCSA), a copolymer having structural features of both C4S and C6S, has been widely used. To determine the validity of bCSA for C4S-IRBC interaction studies, we comparatively evaluated the characteristics of IRBC binding to placental CSPG and bCSA using three commonly used parasite strains. The results indicate that, in all three parasites studied, the characteristics of IRBC binding to placental CSPG and bCSA are qualitatively similar, but the binding capacity with respect to both the number of IRBCs bound per unit area of coated surface and binding strength is significantly higher for CSPG than bCSA regardless of whether parasites were selected on CSPG or bCSA. These results demonstrate that placental CSPG is best suited for studying interactions between parasite adhesive protein(s) and C4S, and have implications in understanding C4S-IRBC structural interactions.

Structural interactions in chondroitin 4-sulfate mediated adherence of Plasmodium falciparum infected erythrocytes in human placenta during pregnancy-associated malaria.

Infection with Plasmodium falciparum during pregnancy results in the adherence of infected red blood cells (IRBCs) in placenta, causing pregnancy-associated malaria with severe health complications in mothers and fetuses. The chondroitin 4-sulfate (C4S) chains of very low sulfated chondroitin sulfate proteoglycans (CSPGs) in placenta mediate the IRBC adherence. While it is known that partially sulfated but not fully sulfated C4S effectively binds IRBCs, structural interactions involved remain unclear and are incompletely understood. In this study, structurally defined C4S oligosaccharides of varying sulfate contents and sizes were evaluated for their ability to inhibit the binding of IRBCs from different P. falciparum strains to CSPG purified from placenta. The results clearly show that, with all parasite strains studied, dodecasaccharide is the minimal chain length required for the efficient adherence of IRBCs to CSPG and two 4-sulfated disaccharides within this minimal structural motif are sufficient for maximal binding. Together, these data demonstrate for the first time that the C4S structural requirement for IRBC adherence is parasite strain-independent. We also show that the carboxyl group on nonreducing end glucuronic acid in dodecasaccharide motif is important for IRBC binding. Thus, in oligosaccharides containing terminal 4,5-unsaturated glucuronic acid, the nonreducing end disaccharide moiety does not interact with IRBCs due to the altered spatial orientation of carboxyl group. In such C4S oligosaccharides, 14-mer but not 12-mer constitutes the minimal motif for inhibition of IRBC binding to placental CSPG. These data have important implications for the development and evaluation of therapeutics and vaccine for placental malaria.

[Proteoglycans of L6J1 myoblast extracellular matrix. Characteristics and effect on myoblast adhesion].

Proteoglycans were isolated from extracellular matrix of L6J1 rat myoblasts and their influence on myoblast adhesion was studied. Proteoglycan digestion with chondroitinase AC and heparinase III degrading the polysaccharide moieties revealed that chondroitin sulfate proteoglycans are the main class of myoblast extracellular matrix proteoglycans. Electrophoresis of enzymatically processed proteoglycans was used to examine their core proteins. Myoblast adhesion was suppressed by proteoglycans or the mixture of proteoglycans and fibronectin/extracellular matrix. When being processed with chondroitinase AC the combined substrate of fibronectin and proteoglycans lost the capability of myoblast adhesion suppression. Thus, as a result of presented work the proteoglycans of L6J1 rat myoblast extracellular matrix were isolated and purified. The main class of proteoglycans was chondroitin sulphate proteoglycans. Isolated proteoglycans suppressed myoblast adhesion and this effect was mediated by polysaccharide moieties of proteoglycans.

Cohesin's ATPase activity is stimulated by the C-terminal Winged-Helix domain of its kleisin subunit.

BACKGROUND: Cohesin, a multisubunit protein complex conserved from yeast to humans, holds sister chromatids together from the onset of replication to their separation during anaphase. Cohesin consists of four core subunits, namely Smc1, Smc3, Scc1, and Scc3. Smc1 and Smc3 proteins are characterized by 50-nm-long anti-parallel coiled coils flanked by a globular hinge domain and an ABC-like ATPase head domain. Whereas Smc1 and Smc3 heterodimerize via their hinge domains, the kleisin subunit Scc1 connects their ATPase heads, and this results in the formation of a large ring. Biochemical studies suggest that cohesin might trap sister chromatids within its ring, and genetic evidence suggests that ATP hydrolysis is required for the stable association of cohesin with chromosomes. However, the precise role of the ATPase domains remains enigmatic. RESULTS: Characterization of cohesin's ATPase activity suggests that hydrolysis depends on the binding of ATP to both Smc1 and Smc3 heads. However, ATP hydrolysis at the two active sites is not per se cooperative. We show that the C-terminal winged-helix domain of Scc1 stimulates the ATPase activity of the Smc1/Smc3 heterodimer by promoting ATP binding to Smc1's head. In contrast, we do not detect any effect of Scc1's N-terminal domain on Smc1/Smc3 ATPase activity. CONCLUSIONS: Our studies reveal that Scc1 not only connects the Smc1 and Smc3 ATPase heads but also regulates their ATPase activity.

Human intervertebral disc cells promote nerve growth over substrata of human intervertebral disc aggrecan.

STUDY DESIGN: Coculture assays of the migration and interaction of human intervertebral disc cells and chick sensory nerves on alternate substrata of collagen and aggrecan. OBJECTIVE: To examine the effects of aggrecan on disc cell migration, how disc cells and sensory nerves interact, and whether disc cells affect previously reported inhibitory effects of aggrecan on sensory nerve growth. SUMMARY OF BACKGROUND DATA: Human intervertebral disc aggrecan is inhibitory to sensory nerve growth in vitro, suggesting that a loss of aggrecan from the disc may have a role in the increased innervation seen in disc degeneration. Endothelial cells that appear to co-migrate with nerves into degenerated intervertebral disc express neurotrophic factors, but the effects of disc cells on nerve growth are not known. METHODS: Human disc cells were seeded onto tissue culture plates that had been coated with type I collagen and human intervertebral disc aggrecan. Explants of chick dorsal root ganglions (DRGs) were subsequently added to the plates and sensory neurite outgrowth stimulated by the addition of nerve growth factor. Time-lapse video and fluorescence microscopy were used to examine the migration and interaction of the disc cells and sensory neurites, in the context of the different matrix substrata. The effects of disc cell conditioned medium on nerve growth were also examined. RESULTS: Disc cells spread and migrated on collagen until they encountered the aggrecan substrata, where some cells, but not all, were repelled. In coculture, DRG neurites extended onto the collagen/disc cells until they encountered the aggrecan, where, like the disc cells, many were repelled. However, in the presence of disc cells, some neurites were able to cross onto this normally inhibitory substratum. The number of neurite crossings onto aggrecan correlated significantly with the number of disc cells present on the aggrecan. In control experiments using DRG alone, all extending neurites were repelled at the collagen/aggrecan border. Conditioned medium from disc cell cultures stimulated DRG neurite outgrowth on collagen but did not increase neurite crossing onto aggrecan substrata. CONCLUSIONS: Human disc cells migrate across aggrecan substrata that are repellent to sensory DRG neurites. Disc cells synthesize neurotrophic factors in vitro that promote neurite outgrowth. Furthermore, the presence of disc cells in coculture with DRG partially abrogates the inhibitory effects of aggrecan on nerve growth. These findings have important implications for the regulation of nerve growth into the intervertebral disc, but whether disc cells promote nerve growth in vivo remains to be determined.

Composition of perineuronal net extracellular matrix in rat brain: a different disaccharide composition for the net-associated proteoglycans.

We developed a method to extract differentially chondroitin sulfate proteoglycans (CSPGs) that are diffusely present in the central nervous system (CNS) matrix and CSPGs that are present in the condensed matrix of perineuronal nets (PNNs). Adult rat brain was sequentially extracted with Tris-buffered saline (TBS), TBS-containing detergent, 1 m NaCl, and 6 m urea. Extracting tissue sections with these buffers showed that the diffuse and membrane-bound CSPGs were extracted in the first three buffers, but PNN-associated CSPGs remained and were only removed by 6 m urea. Most of the CSPGs were extracted to some degree with all the buffers, with neurocan, brevican, aggrecan, and versican particularly associated with the stable urea-extractable PNNs. The CSPGs in stable complexes only extractable in urea buffer are found from postnatal day 7-14 coinciding with PNN formation. Disaccharide composition analysis indicated a different glycosaminoglycan (GAG) composition for PGs strongly associated with extracellular matrix (ECM). For CS/dermatan sulfate (DS)-GAG the content of nonsulfated, 6-O-sulfated, 2,6-O-disulfated, and 4,6-O-disulfated disaccharides were higher and for heparan sulfate (HS)-GAG, the content of 6-O-sulfated, 2-N-, 6-O-disulfated, 2-O-, 2-N-disulfated, and 2-O-, 2-N-, 6-O-trisulfated disaccharides were higher in urea extract compared with other buffer extracts. Digestions with chondroitinase ABC and hyaluronidase indicated that aggrecan, versican, neurocan, brevican, and phosphacan are retained in PNNs through binding to hyaluronan (HA). A comparison of the brain and spinal cord ECM with respect to CSPGs indicated that the PNNs in both parts of the CNS have the same composition.

A sulfated disaccharide derived from chondroitin sulfate proteoglycan protects against inflammation-associated neurodegeneration.

Chondroitin sulfate proteoglycan (CSPG), a matrix protein that occurs naturally in the central nervous system (CNS), is considered to be a major inhibitor of axonal regeneration and is known to participate in activation of the inflammatory response. The degradation of CSPG by a specific enzyme, chondroitinase ABC, promotes repair. We postulated that a disaccharidic degradation product of this glycoprotein (CSPG-DS), generated following such degradation, participates in the modulation of the inflammatory responses and can, therefore, promote recovery in immune-induced neuropathologies of the CNS, such as experimental autoimmune encephalomyelitis (EAE) and experimental autoimmune uveitis (EAU). In these pathologies, the dramatic increase in T cells infiltrating the CNS is far in excess of the numbers needed for regular maintenance. Here, we show that CSPG-DS markedly alleviated the clinical symptoms of EAE and protected against the neuronal loss in EAU. The last effect was associated with a reduction in the numbers of infiltrating T cells and marked microglia activation. This is further supported by our in vitro results indicating that CSPG-DS attenuated T cell motility and decreased secretion of the cytokines interferon-gamma and tumor necrosis factor-alpha. Mechanistically, these effects are associated with an increase in SOCS-3 levels and a decrease in NF-kappaB. Our results point to a potential therapeutic modality, in which a compound derived from an endogenous CNS-resident molecule, known for its destructive role in CNS recovery, might be helpful in overcoming inflammation-induced neurodegenerative conditions.

Chondroitin sulfate proteoglycans are structural renewable constituents of the rabbit vitreous body.

PURPOSE: To characterize the vitreous intrinsic proteoglycans, investigate their dynamics, and examine their role in the supramolecular organization of the vitreous. METHODS: Vitreous from normal rabbits was collected and processed for observation with the transmission electron microscope after treatment with glycosidases. Also, rabbits were injected intravitreally with [35S]-sodium sulfate and sacrificed at several time intervals after the injection. Proteoglycans (PGs) were assayed in the vitreous supernatant or in whole samples extracted with guanidine hydrochloride by polyacrylamide or agarose gel electrophoresis, followed respectively by fluorography or autoradiography, and ion-exchange chromatography and gel-filtration chromatography, combined with glycolytic treatment of the samples. The sulfated glycosaminoglycans (GAGs) were characterized by agarose gel electrophoresis after treating vitreous samples with protease and specific glycosidases. RESULTS: The electron microscopic study revealed a network with hyaluronic acid (HA) as thin threads coating and connecting collagen fibrils. The elimination of the HA coat showed chondroitin sulfate granules (8-25 nm) arranged at regular intervals on the fibril surface. The chondroitinase ABC digestion, besides removing the granules, also caused the formation of thicker bundles of the collagen fibrils. The PG and GAG analysis indicated that there are three renewable PGs in the vitreous (e.g., one heparan- and two chondroitin-sulfate ones). CONCLUSIONS: At least one of the chondroitin sulfate PGs is involved in the interactions that occur in the vitreous structure, mainly by providing adequate spacing between the collagen fibrils, a condition that is probably required for the transparency of the vitreous.

Identification of versican as an isolectin B4-binding glycoprotein from mammalian spinal cord tissue.

Nociceptors are specialized nerve fibers that transmit noxious pain stimuli to the dorsal horn of the spinal cord. A subset of nociceptors, the nonpeptidergic C-fibers, is characterized by its reactivity for the plant isolectin B4 (IB4) from Griffonia simplicifolia. The molecular nature of the IB4-reactive glycoconjugate, although used as a neuroanatomical marker for more than a decade, has remained unknown. We here present data which strongly suggest that a splice variant of the extracellular matrix proteoglycan versican is the IB4-reactive glycoconjugate associated with these nociceptors. We isolated (by subcellular fractionation and IB4 affinity chromatography) a glycoconjugate from porcine spinal cord tissue that migrated in SDS/PAGE as a single distinct protein band at an apparent molecular mass of > 250 kDa. By using MALDI-TOF/TOF MS, we identified this glycoconjugate unambiguously as a V2-like variant of versican. Moreover, we demonstrate that the IB4-reactive glycoconjugate and the versican variant can be co-released from spinal cord membranes by hyaluronidase, and that the IB4-reactive glycoconjugate and the versican variant can be co-precipitated by an anti-versican immunoglobulin and perfectly co-migrate in SDS/PAGE. Our findings shed new light on the role of the extracellular matrix, which is thought to be involved in plastic changes underlying pain-related phenomena such as hyperalgesia and allodynia.

Soluble lumican glycoprotein purified from human amniotic membrane promotes corneal epithelial wound healing.

PURPOSE: To purify and characterize the glycoprotein lumican, isolated from human amniotic membrane (AM), and to examine its efficacy in treating corneal epithelium debridement. METHODS: An affinity-purified, anti-human lumican antibody-conjugated protein A Sepharose column was used to purify soluble lumican protein from human AM. The purified AM lumican was characterized by two-dimensional and SDS gel electrophoresis, plus Western blot analysis with anti-lumican antibody. The effects of lumican on corneal epithelial wound healing were examined in an organ culture mouse eye model. RESULTS: Lumican was found to be abundantly present in the stroma of human AM. It was extracted from the AM by isotonic, 1 M NaCl, and 4 M guanidine HCl solutions, suggesting that it is present in both the soluble and matrix-bound states. In two-dimensional gel electrophoresis, the 50-kDa human amniotic lumican purified by antibody-conjugated affinity chromatography migrated in a smear between pH 3.0 and 6.0. After endo-beta-galactosidase digestion, it existed as a single core protein at pH 6.0, suggesting that native human amniotic lumican is a glycoprotein with short sugar moiety. Addition of purified human AM lumican to cultured medium promoted re-epithelialization and enhanced cell proliferation of wild-type mouse corneal epithelial cells in an organ culture. In lumican-knockout (lum(-/-)) mice, the effect of human lumican on promoting corneal epithelial wound healing was even more dramatic than in wild-type mice. CONCLUSIONS: The diversified functions of lumican include modulation of epithelial cells in wound healing and serving as an extracellular matrix component. Administration of lumican may be beneficial for treating epithelial defects in the cornea and other tissues.

Expression and purification of functionally active hyaluronan-binding domains from human cartilage link protein, aggrecan and versican: formation of ternary complexes with defined hyaluronan oligosaccharides.

The chondroitin sulfate proteoglycan aggrecan forms link protein-stabilized complexes with hyaluronan (HA), via its N-terminal G1-domain, that provide cartilage with its load bearing properties. Similar aggregates (potentially containing new members of the link protein family), in which other chondroitin sulfate proteoglycans (i.e. versican, brevican, and neurocan) substitute for aggrecan, may contribute to the structural integrity of many other tissues including skin and brain. In this study, cartilage link protein (cLP) and the G1-domains of aggrecan (AG1) and versican (VG1) were expressed in Drosophila S2 cells. The recombinant human proteins were found to have properties similar to those described for the native molecules (e.g. cLP was able to form oligomers, and HA decasaccharides were the minimum size that could compete effectively for their binding to polymeric HA). Gel filtration and protein cross-linking/matrix-assisted laser desorption ionization time-of-flight peptide fingerprinting showed that cLP and AG1 interact in the absence or presence of HA. Conversely, cLP and VG1 did not bind directly to each other in solution yet formed ternary complexes with HA24. N-linked glycosylation of AG1 and VG1 was demonstrated to be unnecessary for either HA binding or the formation of ternary complexes. Surprisingly, the length of HA required to accommodate two G1-domains was found to be significantly larger for aggrecan than versican, which may reflect differences in the conformation of HA stabilized on binding these proteins.

Interaction of lumican with aggrecan in the aging human sclera.

PURPOSE: Lumican is a keratan sulfate proteoglycan originally identified in cornea, but present in a variety of connective tissues where it presumably regulates collagen fibril formation and organization. The present study was designed to describe the chemical nature of lumican core protein in the aging human sclera. METHODS: Western blot analyses, immunohistochemistry, and immunoaffinity chromatography were used to detect and purify the lumican core protein from tissue extracts from human donors 6 to 89 years of age. Treatment of lumican with chondroitinase ABC, keratanase-I and -II, and/or endo-beta-galactosidase was used to determine the degree of glycosylation of the lumican core protein. RESULTS: Lumican was present in the human sclera as a 70- to 80-kDa core protein with short unsulfated lactosaminoglycan side chains. In addition, on Western blots, a larger >200-kDa species was apparent that was immunologically related to lumican. This high-molecular-weight material increased in scleral extracts with increasing age. The complex was most abundant in unreduced samples, and approximately two thirds of the 70- to 80-kDa lumican core protein was released from the complex on reduction of the scleral extract. Further characterization of the >200-kDa lumican-immunopurified complex indicated that aggrecan (the cartilage proteoglycan) was covalently associated with lumican. CONCLUSIONS: Reducible and nonreducible lumican-aggrecan interactions occur in the scleral extracellular matrix and result in the formation of high-molecular-weight complexes that increase with age. These results represent the first report demonstrating lumican-aggrecan interactions and suggest they may play a role in age-related scleral extracellular matrix changes.

Chondroitin sulfate proteoglycans of bovine cornea: structural characterization and assessment for the adherence of Plasmodium falciparum-infected erythrocytes.

The structures of the bovine corneal chondroitin sulfate (CS) chains and the nature of core proteins to which these chains are attached have not been studied in detail. In this study, we show that structurally diverse CS chains are present in bovine cornea and that they are mainly linked to decorin core protein. DEAE-Sephacel chromatography fractionated the corneal chondroitin sulfate proteoglycans (CSPGs) into three distinct fractions, CSPG-I, CSPG-II, and CSPG-III. These CSPGs markedly differ in their CS and dermatan sulfate (DS) contents, and in particular the CS structure-the overall sulfate content and 4- to 6-sulfate ratio. In general, the CS chains of the corneal CSPGs have low to moderate levels (15-64%) of sulfated disaccharides and 0-30% DS content. Structural analysis indicated that the DS disaccharide units in the CS chains are segregated as large blocks. We have also assessed the suitability of the corneal CSPGs as an alternative to placental CSPG or the widely used bovine tracheal chondroitin sulfate A (CSA) for studying the structural interactions involved in the adherence of Plasmodium falciparum-infected red blood cells (IRBCs) to chondroitin 4-sulfate. The data demonstrate that the corneal CSPGs efficiently bind IRBCs, and that the binding strength is either comparable or significantly higher than the placental CSPG. In contrast, the IRBC binding strength of bovine tracheal CSA is markedly lower than the human placental and bovine corneal CSPGs. Thus, our data demonstrate that the bovine corneal CSPG but not tracheal CSA is suitable for studying structural interactions involved in IRBC-C4S binding.

Characterization of a chondroitin sultate proteoglycan associated with regeneration in goldfish optic tract.

Regeneration of goldfish optic nerve axons is accompanied by a major increase in axonally transported proteoglycans (21). To identify specific proteoglycans increased during regeneration, we have used proteoglycan preparations from regenerating goldfish optic tracts to produce monoclonal antibodies. Western blot analysis shows a 28-kD antigen reacting with our 1G4/G5 antibody is present in optic tract 21 days after nerve crush, but absent in nonregenerating tract. Treatment with chondroitinase AC removes IG4/G5 immunostaining of the 28-kD molecule. An anti-CS antibody further confirmed this molecule as a chondroitin sulfate proteoglycan. A slightly smaller core protein following chondroitinase AC treatment indicates a low level of glycosylation. The N-terminal amino acid sequence of its core protein is not similar to any known proteoglycans. The CSPG sediments through 1.4 M sucrose, indicative of an extracellular matrix localization. It is expressed during the outgrowth of regenerating axons. Cut nerve and retinal explant studies demonstrate that the 1G4/G5 CSPG is not axonal, suggesting a glial localization.

Structural characterization of the bovine tracheal chondroitin sulfate chains and binding of Plasmodium falciparum-infected erythrocytes.

Sequestration of Plasmodium falciparum-infected red blood cells (IRBCs) in the human placenta is mediated by chondroitin 4-sulfate (C4S). A cytoadherence assay using chondroitin sulfate proteoglycans (CSPGs) is widely used for studying C4S-IRBC interactions. Bovine tracheal chondroitin sulfate A (CSA) preparation lacking a major portion of core protein has been frequently used for the assay. Here the CSPG purified from bovine trachea and CSA were assessed for IRBC binding and the CS chains studied in detail for structure-activity relationship. The IRBCs bound at significantly higher density to the CSPG than CSA. The CS chains of CSPG/CSA are heterogeneous with varying levels of 4- and 6-sulfates, which are distributed such that approximately 80% of the 4-sulfated disaccharides are present as single and blocks of two or three separated by one to three 6-sulfated disaccharides. The remainder of the 4-sulfated disaccharides is present in blocks composed of 4-12 units, separated by 6-sulfated disaccharides. In the IRBC adherence inhibition analysis, CSA fragments with 88%-92% 4-sulfate were significantly less inhibitory than the intact CSA, indicating that the regions consisting of shorter 4-sulfated blocks efficiently bind IRBCs despite the presence of relatively high levels of 6-sulfate. This is because the 6-sulfated disaccharides have unsubstituted C-4 hydroxyls that are crucial for IRBC binding. The presence of high levels of 6-sulfate, however, significantly interfere with the IRBC binding activity of CSA, which otherwise would more efficiently bind IRBCs. Thus our study revealed the distribution pattern of 4- and 6-sulfate in bovine tracheal CSA and structural basis for IRBC binding.

Chondroitin sulfate proteoglycans of the endothelia of human umbilical vein and arteries and assessment for the adherence of Plasmodium falciparum-infected erythrocytes.

Infection with Plasmodium falciparum during pregnancy leads to chondroitin 4-sulfate-mediated adhesion of the infected red blood cells (IRBCs) in the placenta, causing severe health complications to fetus and the mother. The IRBCs are also frequently found in low density in the umbilical cord of infected placentas. In this study, the CSPGs of umbilical vein and arteries were purified, characterized, and their localization and IRBC-binding abilities were studied. While a versican type CSPG was found both in the vein and arteries, a serglycin type CSPG was present exclusively in the vein. The CSPGs were present at significant level on the endothelial surface of the umbilical vein but not on that of arteries. Although the purified versican and serglycin type CSPGs could bind IRBCs, their binding abilities were significantly less compared to the low sulfated CSPGs of the placenta because of the predominance of 6-sulfated disaccharide moieties in the CS chains. Therefore, IRBCs were unable to bind efficiently onto the umbilical cord endothelial surface. Unexpectedly, however, the IRBCs adhered densely in the blood vessels of fetal villi in the placental tissue sections and sparingly in the blood spaces of the umbilical cord vein, presumably because the CSPG that can efficiently bind IRBCs is present at high levels in the fetal blood vessels and at very low levels in the umbilical cord blood vessels. Since the C4S-adherent IRBCs that enter the fetal blood vessels cannot adhere to the cord endothelial surface and parasites cannot efficiently grow due to fetal hemoglobin toxicity and protection by maternal antibodies, transplacental infection may be quickly cleared without clinical episodes.