We previously reported the cloning and sequencing of a 1.5-kilobase cDNA which encoded a portion of the chondroitin sulfate domain from the chick cartilage proteoglycan core protein and the localization of a species-specific monoclonal antibody epitope. Using polymerase chain reaction amplification and primer extension, cDNA clones which code for the entire proteoglycan core protein have now been obtained from a 10-day chick embryo cDNA library. The composite sequence is 6464 nucleotides long, coding for a protein of 2109 amino acid residues with a calculated M(r) = 223,500. The overall arrangement of globular and carbohydrate-attachment domains is similar to human and rat chondrosarcoma aggrecan, but there are significant differences in detailed homology between chick and mammalian core proteins. Most significantly a highly repetitive region (19 repeat units of 20 residues each), not found in either human or rat, enlarges one of the characteristic serine-glycine containing regions (designated CS-2) while the other serine-glycine containing domain (designated CS-1) is approximately one-fourth the length of the mammalian CS-1. Analysis of a polymerase chain reaction-amplified fragment encoding the chick-specific repeat region revealed a single base mutation at position 4553 (G to T transversion) that converted the codon GAA for glutamate at amino acid 1513 to TAA, a stop codon, in nanomelic chondrocytes. Genomic DNA from nanomelic liver was also digested with restriction enzyme BsaBI to verify the G to T transversion. This single mutation leads to a shortened core protein precursor with a calculated M(r) = 158,300. The resulting phenotype, nanomelia, arises because the truncated core protein is neither processed to a mature proteoglycan, nor secreted from the chondrocyte.

Emerging studies have revealed new roles for the neural extracellular matrix in neuropathologies. The structure of this matrix is unusual and uniquely enriched in chondroitin sulfate proteoglycans, particularly those of the lectican family. Historically, lecticans have attracted considerable interest in the normal and injured brain for their prominent roles as inhibitors of cellular motility, neurite extension and synaptic plasticity. However, these molecules are structurally heterogeneous, have distinct expression patterns and mediate unique interactions, suggesting that they might have other functions in addition to their traditional role as chemorepulsants. Here, we review recent work demonstrating unique modifications and structural microheterogeneity of the lecticans in the diseased CNS, which might relate to novel roles of these molecules in neuropathologies.

Binding of immunoglobulin M (IgM) antibodies from normal human serum to the surface of Plasmodium falciparum-infected red blood cells (iRBC) has previously been demonstrated only in parasites that form rosettes with uninfected red cells. We show that natural, nonspecific IgM but not IgG, IgA, IgD, or IgE also binds to the surface of iRBC selected for adhesion to chondroitin sulfate A (CSA), a placental receptor for parasites associated with malaria in pregnancy. The protease sensitivity of IgM-binding appears to match that of CSA binding, suggesting that the two phenotypes may be mediated by the same parasite molecule. We also show that a wide range of mouse monoclonal antibodies of the IgM class bind nonspecifically to CSA-selected iRBC, an important consideration in the interpretation of immunological assays performed on these parasite lines.

Cell adhesion to thrombospondin-1 (TSP-1) correlates with assembly of cell-substratum contact structures that contain fascin microspikes. In this analysis, cell-matrix requirements for assembly of fascin microspikes were examined in detail. In six cell lines, cell spreading on a TSP-1 substratum correlated with expression of fascin protein and formation of fascin microspikes. Microspikes were not formed by H9c2 cells adherent on fibronectin, vitronectin, collagen IV, or platelet factor 4. However, both fascin microspikes and focal contacts were assembled by cells adherent on laminin-1. Using mixed substrata containing different proportions of TSP-1, and fibronectin, fascin microspike formation by H9c2 and C2C12 cells was found to be reduced on substrata containing 25% fibronectin and abolished on substrata containing 75% fibronectin. Adhesion to intermediate mixtures of TSP-1 and fibronectin resulted in coassembly of fascin microspikes and focal contacts, colocalization of fascin with actin stress fiber bundles and altered distributions of beta 1 integrins, cortical alpha-actinin, and tropomyosin. In cells adherent on 50% TSP-1:50% fibronectin, GRGDSP peptide treatment decreased focal contact assembly and altered cytoskeletal organization but did not inhibit microspike assembly. Treatment with chondroitin sulfate A or p-nitrophenol beta-D-xylopyranoside decreased microspike formation and modified cytoskeletal organization but did not inhibit focal contact formation. In polarized migratory and postmitotic C2C12 cells, fascin microspikes and ruffles were localized at leading edges and TSP matrix deposition was also concentrated in this region. Depletion of matrix TSP by heparin treatment correlated with decreased microspike formation and cell motility. Thus, the balance of adhesive receptors ligated at the cell surface during initial cell-matrix attachment serves to regulate the type of substratum adhesion contact assembled and subsequent cytoskeletal organization. A role for fascin microspikes in cell motile behavior is indicated.

A new method of identifying and quantifying the disaccharide building blocks of glycosaminoglycans is introduced. The polysaccharides are subjected to an enzymatic digestion that releases the sulfated disaccharides. The disaccharides are then identified using a combination of electrospray ionization mass spectrometry and tandem mass spectrometry. Quantification of the isomeric disaccharides is also achieved by tandem mass spectrometry, using a recently developed methodology which quantifies mixtures of isomers without the use of chromatography or prior separation. Using mass spectrometry to characterize the components of glycosaminoglycans significantly reduces both sample consumption and analysis time of traditional methods.

Oligodendrocyte precursor cells (OPCs) are a newly recognized glial component of the adult central nervous system of unknown function. Antibodies against the NG2 chondroitin sulfate proteoglycan have been useful tools to identify these cells in intact tissue. Here we review studies that show that OPCs react to several types of experimentally induced brain injury. Injury stimulates OPCs to re-enter the cell cycle, divide, and accumulate at the site of damage. OPCs, together with microglia and astrocytes, form the glial scar. Glial scars are thought to inhibit or prevent axonal regeneration and reactive OPCs contribute to this inhibition by producing growth-inhibiting chondroitin sulfate proteoglycans, particularly NG2. In developing animals, NG2 is found in areas, such as the perinotochordal mesenchyme, that are avoided by growing motor and sensory axons. Within the developing CNS, NG2-expressing cells surround the developing optic chiasm and tract and separate it from the overlying diencephalon. Thus, NG2-expressing cells are well positioned to inhibit axonal growth from developing as well as regenerating neurons.

Chondroitinase B from Pedobacter heparinus is the only known enzyme strictly specific for dermatan sulfate and is a widely used enzymatic tool for the structural characterization of glycosaminoglycans. This beta-helical polysaccharide lyase belongs to family PL-6 and cleaves the beta(1,4) linkage of dermatan sulfate in a random manner, yielding 4,5-unsaturated dermatan sulfate disaccharides as the product. The previously reported structure of its complex with a dermatan sulfate disaccharide product identified the -1 and -2 subsites of the catalytic groove. We present here the structure of chondroitinase B complexed with several dermatan sulfate and chondroitin sulfate oligosaccharides. In particular, the soaking of chondroitinase B crystals with a dermatan sulfate hexasaccharide results in a complex with two dermatan sulfate disaccharide reaction products, enabling the identification of the +2 and +1 subsites. Unexpectedly, this structure revealed the presence of a calcium ion coordinated by sequence-conserved acidic residues and by the carboxyl group of the l-iduronic acid at the +1 subsite. Kinetic and site-directed mutagenesis experiments have subsequently demonstrated that chondroitinase B absolutely requires calcium for its activity, indicating that the protein-Ca(2+)-oligosaccharide complex is functionally relevant. Modeling of an intact tetrasaccharide in the active site of chondroitinase B provided a better understanding of substrate specificity and the role of Ca(2+) in enzymatic activity. Given these results, we propose that the Ca(2+) ion neutralizes the carboxyl moiety of the l-iduronic acid at the cleavage site, whereas the conserved residues Lys-250 and Arg-271 act as Brønsted base and acid, respectively, in the lytic degradation of dermatan sulfate by chondroitinase B.

Costello syndrome is characterized by mental retardation, loose skin, coarse face, skeletal deformations, cardiomyopathy, and predisposition to numerous malignancies. The genetic origin of Costello syndrome has not yet been defined. Using immunohistochemistry and metabolic labeling with [3H]-valine, we have established that cultured skin fibroblasts obtained from patients with Costello syndrome did not assemble elastic fibers, despite an adequate synthesis of tropoelastin and normal deposition of the microfibrillar scaffold. We found that impaired production of elastic fibers by these fibroblasts is associated with a functional deficiency of the 67-kD elastin-binding protein (EBP), which is normally required to chaperone tropoelastin through the secretory pathways and to its extracellular assembly. Metabolic pulse labeling of the 67-kD EBP with radioactive serine and further chase of this tracer indicated that both normal fibroblasts and fibroblasts from patients with Costello syndrome initially synthesized comparable amounts of this protein; however, the fibroblasts from Costello syndrome patients quickly lost it into the conditioned media. Because the normal association between EBP and tropoelastin can be disrupted on contact with galactosugar-bearing moieties, and the fibroblasts from patients with Costello syndrome revealed an unusual accumulation of chondroitin sulfate-bearing proteoglycans (CD44 and biglycan), we postulate that a chondroitin sulfate may be responsible for shedding EBP from Costello cells and in turn for their impaired elastogenesis. This was further supported by the fact that exposure to chondroitinase ABC, an enzyme capable of chondroitin sulfate degradation, restored normal production of elastic fibers by fibroblasts from patients with Costello syndrome. We also present evidence that loss of EBP from fibroblasts of Costello syndrome patients is associated with an unusually high rate of cellular proliferation.

Glycosaminoglycans (GAGs) and glycoprotein-derived glycopeptide from mouse BALB/c3T3 and simian virus 40-transformed 3T3 whole cells or their adhesion sites, which are left bound to the serum-coated tissue culture substratum after detachment of cells mediated by [ethylenebis-(oxyethylenenitrilo]tetraacetic acid (EGTA), were analyzed for specific binding to Sepharose columns derivatized with cold-insoluble globulin (CIg). CIg is the serum-contained form of fibronectin and is required for the adhesion of these fibroblasts to the substratum. Of the various GAGs present in these fractions of either cell type, only the highly N-sulfated sequences of heparan sulfate and a small subset of dermatan sulfate bind to CIg-Sepharose. There was no detectable binding of glycopeptide, undersulfated heparan sulfate, the various chondroitin species, or hyaluronate. Adhesion sites from newly attaching cells were greatly enriched in CIg-binding heparan sulfate when compared to long-term-growth adhesion sites or EGTA-detached cells. Various properties of binding were determined. The reference standard standard GAGs heparin (or heparan sulfate) and dermatan sulfate were able to displace bound radiolabeled adhesion site GAG from the column, whereas the other GAGs had no effect. CIg has been shown to be the only adhesion-promoting activity in the serum layer of this culture system. Because these fibroblast adhesion sites do not contain collagen, which could potentially mediate adhesion to the substratum-bound CIg, these data support other evidence that multivalent heparan sulfate proteoglycans mediate substratum adhesion of these cells by coordinate binding to fibronectin on the cell surface and CIg on the substratum.

It has been postulated that lipoprotein lipase, an enzyme important in the uptake of fatty acids into tissues, is bound to the vascular endothelial cell surface and that this binding occurs through attachment to heparinlike glycosaminoglycans. Furthermore, it is thought that heparin releases the enzyme from its attachment to the endothelium into the circulation. These hypotheses have never been tested directly in cell systems in vitro. In the present study we have directly evaluated the interaction of lipoprotein lipase, purified from bovine skim milk with monolayer cultures of endothelial cells, isolated from bovine pulmonary artery. Endothelial cells in primary culture had no intrinsic lipoprotein lipase activity but were able to bind lipoprotein lipase quantitatively. The binding reached equilibrium and was saturable at 0.24 nmol of lipoprotein lipase/mg of cell protein. The concentration of lipoprotein lipase at half-maximal binding was 0.52 microM. Bound lipoprotein lipase could be detached from cultured cells by increasing concentrations of heparin, and at and above 0.6 microgram/ml of heparin, 90% of the cell-bound lipoprotein lipase activity was released. Heparan sulfate and dermatan sulfate released the enzyme to a lesser extent and chondroitin sulfate caused little, if any, release of lipoprotein lipase. The release of lipoprotein lipase with heparin was not associated with a release of [3S]glycosaminoglycans from 35S-prelabeled cells. Reductions of lipoprotein lipase binding to endothelial cells and of cell surface-associated [3S]glycosaminoglycans in 35S-prelabeled cells occurred in parallel both when cells were pretreated with crude Flavobacterium heparinum enzyme before lipoprotein lipase binding and when cells were treated with this enzyme after lipoprotein lipase binding. The removal of heparan sulfate from the cell surface by purified heparinase totally inhibited the binding of lipoprotein lipase by endothelial cells, but the removal of chondroitin sulfate by chondroitin ABC lyase had no effect on this binding. These results provide direct evidence for lipoprotein lipase attachment to endothelial cells through heparan sulfate on the cell surface, and provide evidence for the release of lipoprotein lipase by heparin through a detachment from this binding site.

The extracellular matrix through which growth cones navigate contains molecules, such as chondroitin sulfate proteoglycan, that can inhibit growth cone advance and induce branching and turning. Growth cone turning is accompanied by rearrangement of the cytoskeleton. To identify changes in the organization of actin filaments and microtubules that occur as growth cones turn, we used time-lapse phase contrast videomicroscopy to observe embryonic chick dorsal root ganglion neuronal growth cones at a substratum border between fibronectin and chondroitin sulfate proteoglycan, in the presence and absence of cytochalasin B. Growth cones were fixed and immunocytochemically labeled to identify actin filaments and dynamic and stable microtubules. Our results suggest that microtubules are rearranged within growth cones to accomplish turning to avoid chondroitin sulfate proteoglycan. Compared to growth cones migrating on fibronectin, turning growth cones were more narrow, and they contained dynamic microtubules that were closer to the leading edge and were more bundled. Cytochalasin B-treated growth cones sidestepped laterally along the border instead of turning, and in sidestepping growth cones, microtubules were not bundled and aligned. We conclude that actin filament bundles are required for microtubule reorientation and growth cone turning to avoid chondroitin sulfate proteoglycan.

Fractionation of proteins from perinatal rat brain was monitored using a neurite outgrowth assay. Two neurite-promoting proteins, HB-GAM (heparin-binding growth-associated molecule; also known as pleiotrophin) and amphoterin, were isolated, cloned and produced by baculovirus expression for structural and functional studies. HB-GAM is highly expressed in embryonic and early post-natal fiber pathways of the nervous system, and it enhances axonal growth/guidance by binding to N-syndecan (syndecan-3) at the neuron surface. N-syndecan in turn communicates with the cytoskeleton through the cortactin/src-kinase pathway to enhance neurite extension. In addition to N-syndecan, the chondroitin sulfate proteoglycan RPTP beta/zeta (receptor-type tyrosine phosphatase beta/zeta) is implicated in the receptor mechanism of HB-GAM. HB-GAM is also prominently expressed in developing and regenerating bone as a matrix-bound cue for migration of osteoblasts/osteoblast precursors to the site of bone deposition. HB-GAM is suggested to regulate motility in osteoblasts through a similar mechanism as in neurons. Structural studies using heteronuclear NMR reveal two similar protein domains in HB-GAM, both consisting of three anti-parallel beta-strands. Search of sequence databases shows that the beta structures of HB-GAM and of the similar domains of MK (midkine) correspond to the thrombospondin type I (TSR) sequence motif. We suggest that the TSR sequence motif, found in several neurite outgrowth-promoting and other cell surface and matrix-binding proteins, defines a beta structure similar to those found in HB-GAM and MK. In general, amphoterin is highly expressed in immature and transformed cells. We suggest a model, according to which amphoterin is an autocrine/paracrine regulator of invasive migration. Amphoterin binds to RAGE (receptor of advanced glycation end products), an immunoglubulin superfamily member related to N-CAM (neural cell adhesion molecule), that communicates with the GTPases Cdc42 and Rac to regulate cell motility. In addition, ligation of RAGE by amphoterin activates NF-kappaB to regulate transcription.

Animal cells utilize beta-D-xylosides as primers for glycosaminoglycan synthesis. However, most xylosides preferentially stimulate chondroitin sulfate synthesis and only weakly prime heparan sulfate synthesis. To test if the structure of the aglycone determines the type of glycosaminoglycan made, the priming activity of methyl, n-octyl, p-nitrophenyl, 4-methylumbelliferyl, trans,trans-farnesyl, cholesteryl, and estradiol beta-D-xylosides was compared. Their potency was tested in pgsA-745 cells, a Chinese hamster ovary cell mutant unable to initiate glycosaminoglycan synthesis due to a defect in xylosyltransferase. All of the xylosides stimulated chondroitin sulfate synthesis in the mutant, but only estradiol beta-D-xyloside primed heparan sulfate synthesis efficiently. When incubated with 30 microM estradiol beta-D-xyloside, mutant cells made about 3-fold more glycosaminoglycan than untreated wild-type cells and as much as 50% was heparan sulfate. Estradiol beta-D-xyloside also induced heparan sulfate synthesis in cycloheximide-treated wild-type Chinese hamster ovary cells, bovine aortic endothelial cells, baby hamster kidney cells, and Balb/c 3T3 fibroblasts. In addition to stimulating heparan sulfate synthesis, low concentrations of estradiol beta-D-xyloside inhibited the formation of endogenous heparan sulfate proteoglycans.

Dentin sialoprotein (DSP) is a glycoprotein that is critical for proper tooth dentin formation, but little is known about the nature of its carbohydrate attachments and other post-translational modifications. We have isolated DSP from pig dentin and demonstrate that it is a proteoglycan. Polyclonal antibodies were raised in chicken against recombinant pig DSP, and used to identify native DSP in fractions of tooth dentin proteins extracted from developing pig molars. Amino acid analyses and characterization of lysylendopeptidase cleavage products confirmed that the purified protein was DSP, and that Arg391 is at the DSP C terminus. On SDS-PAGE and on urea gels, DSP appeared as a smear extending from 280 to 100 kDa, but in the presence of beta-mercaptoethanol the top of the DSP smear disappeared. The high molecular weight material was likely comprised of covalent DSP dimers connected by a disulfide bridge at Cys205. Oligosaccharides were released from DSP following N- and O-linked glycosidase digestions, but these digestions had little effect on the apparent molecular weight of DSP on SDS-PAGE, when compared with the significant reduction following chondroitinase ABC digestion. Glycosaminoglycanases with assorted glycosaminoglycan (GAG) cleavage specificities coupled with Western analyses of the cleaved GAG "stubs" demonstrated that the DSP GAG attachments contain chondroitin 6-sulfate, but not keratan sulfate, heparan sulfate, chondroitin, or chondroitin 4-sulfate. DSP binds biotin-labeled hyaluronic acid, and such binding is inhibited by the addition of unlabeled hyaluronic acid. We conclude that DSP is a proteoglycan and that GAG attachments are the predominant structural feature of porcine DSP.

Chondroitin sulfate glycosaminoglycans are sulfated polysaccharides involved in cell division, neuronal development, and spinal cord injury. Here, we report the synthesis and identification of a chondroitin sulfate tetrasaccharide that stimulates the growth and differentiation of neurons. These studies represent the first, direct investigations into the structure-activity relationships of chondroitin sulfate using homogeneous synthetic molecules and define a tetrasaccharide as a minimal motif required for activity.

Chondroitin sulfate (CS) plays critical roles in central nervous system development and regeneration, and individual modifications of CS form a "sulfation code" that regulates growth factor signaling or neuronal growth. Although we have shown that CS-E polysaccharide, but not CS-A or -C polysaccharide, has an inherent ability to promote neurite outgrowth toward primary neurons, its molecular mechanism remains elusive. Here, we show the involvement of a plasma membrane-tethered cell adhesion molecule, contactin-1 (CNTN-1), in CS-E-mediated neurite extension in a mouse neuroblastoma cell line and primary hippocampal neurons. CS-E, but not CS-A, -C, or heparan sulfate, engaged CNTN-1 with significant affinity and induced intracellular signaling downstream of CNTN-1, indicating that CS-E is a selective ligand for a potential CS receptor, CNTN-1, leading to neurite outgrowth. Our data provide the first evidence that biological functions of CS are exerted through the CS receptor-mediated signaling pathway(s).

The structure of a unique focose-branched chondroitin sulfate isolated from the body wall of a sea cucumber was examined in detail. This glycosaminoglycan contains side chain disaccharide units of sulfated fucopyranosyl units linked to approximately one-half of the glucuronic acid moieties through the O-3 position of the acid. The intact polysaccharide is totally resistant to chondroitinase degradation, whereas, after defucosylation, it is partially degraded by the enzyme. However, only after an additional step of desulfation, the chondroitin from sea cucumber is almost totally degraded by chondroitinase AC or ABC. This result, together with the methylation and NMR studies of the native and chemically modified polysaccharide, suggest that besides the fucose branches, the sea cucumber chondroitin sulfate contains sulfate esters at position O-3 of the beta-D-glucuronic acid units. Furthermore, the proteoglycan from the sea cucumber chondroitin sulfate is recognized by anti-Leu-7 monoclonal antibody, which specifically recognizes 3-sulfoglucuronic acid residues. In analogy with the fucose branched units, the 3-O-sulfo-beta-D-glucuronosyl residues are resistant to chondroitinase degradation. Regarding the position of the glycosidic linkage and site of sulfation in the fucose branches, our results suggest high heterogeneity. Tentatively, it is possible to suggest the preponderance of disaccharide units formed by 3,4-di-O-sulfo-alpha-L-fucopyranosyl units glycosidically linked through position 1----2 to 4-O-sulfo-alpha-L-fucopyranose. Finally, the presence of unusual 4/6-disulfated disaccharide units, together with the common 6-sulfated and non-sulfated units, was detected in the chondroitin sulfate core of this polysaccharide.

Footpad adhesion sites pinch off from the rest of the cell surface during EGTA-mediated detachment of normal or virus-transformed murine cells from their tissue culture substrates. In these studies, highly purified trypsin and testicullar hyaluronidase were used to investigate the selective destruction or solubilization of proteins and polysaccharides in this substrate-attached material (SAM). Trypsin-mediated detachment of cells or trypsinization of SAM after EGTA-mediated detachment of cells resulted in the following changes in SAM composition: (a) solubilization of 50-70% of the glycosaminoglycan polysaccharide with loss of only a small fraction of the protein, (b) selective loss of one species of glycosaminoglycan-associated protein in longterm radiolabeled preparations, (c) no selective loss of the LETS glycoprotein or cytoskeletal proteins in longterm radiolabeled preparations, and (d) selective loss of one species of glycosaminoglycan-associated protein, a protion of the LETS glycoprotein, and proteins Cd (mol wt 47,000 and Ce' (mol wt 39,000) in short term radiolabeled preparations. Digestion of SAM with testicular hyaluronidase resulted in: (a) almost complete solubilization of the hyaluronate and chondroitin sulfate moieties from long term radiolabeled SAM with minimal loss of heparan sulfate, (b) solubilization of a small portion of the LETS glycoprotein and the cytoskeletal proteins from longterm radiolabeled SAM, (c) resistance to solubilization of protein and polysaccharide in reattaching cell SAM which contains principally heparan sulfate, and (d) complete solubilization of the LETS glycoprotein in short term radiolabeled preparations with no loss of cytoskeletal proteins. Thus, there appear to be two distinct pools of LETS in SAM, one associated in some unknown fashion with hyaluronate-chondroitin sulfate complexes, and a second associated with some other component in SAM, perhaps heparan sulfate. These data, together with other results, suggest that the cell-substrate adhesion process may be mediated principally by a heparan sulfate--LETS complex and that hyaluronate-chondroitin sulfate complexes may be important in the detachability of cells from the serum-coated substrate by destabilizing LETS matrices at posterior footpad adhesion sites.

Corneal cross-linking using riboflavin and ultraviolet-A (RFUVA) is a clinical treatment targeting the stroma in progressive keratoconus. The stroma contains keratocan, lumican, mimecan, and decorin, core proteins of major proteoglycans (PGs) that bind collagen fibrils, playing important roles in stromal transparency. Here, a model reaction system using purified, non-glycosylated PG core proteins in solution in vitro has been compared with reactions inside an intact cornea, ex vivo, revealing effects of RFUVA on interactions between PGs and collagen cross-linking. Irradiation with UVA and riboflavin cross-links collagen α and β chains into larger polymers. In addition, RFUVA cross-links PG core proteins, forming higher molecular weight polymers. When collagen type I is mixed with individual purified, non-glycosylated PG core proteins in solution in vitro and subjected to RFUVA, both keratocan and lumican strongly inhibit collagen cross-linking. However, mimecan and decorin do not inhibit but instead form cross-links with collagen, forming new high molecular weight polymers. In contrast, corneal glycosaminoglycans, keratan sulfate and chondroitin sulfate, in isolation from their core proteins, are not cross-linked by RFUVA and do not form cross-links with collagen. Significantly, when RFUVA is conducted on intact corneas ex vivo, both keratocan and lumican, in their natively glycosylated form, do form cross-links with collagen. Thus, RFUVA causes cross-linking of collagen molecules among themselves and PG core proteins among themselves, together with limited linkages between collagen and keratocan, lumican, mimecan, and decorin. RFUVA as a diagnostic tool reveals that keratocan and lumican core proteins interact with collagen very differently than do mimecan and decorin.

Proteoglycans were extracted in good yield from the proximal, fibrous portion of adult bovine tendon with 4 m guanidine HCl. They comprise less than 1% of the dry weight of the tissue. Using CsCl density gradient centrifugation, gel chromatography, and ion exchange chromatography, two populations of proteoglycans were separated and purified from other tissue proteins. One was a large, chondroitin sulfate proteoglycan with high buoyant density in CsCl. This component appeared to be composed of two or three subpopulations as detected by agarose/polyacrylamide electrophoresis, although they could not be effectively separated from one another for individual characterization. As a group, the large proteoglycans eluted from Sepharose CL-2B with Kav from 0.1-0.5 and their core protein had Mr greater than 200,000 with high contents of glutamic acid, serine, and glycine. The glycosaminoglycan chains had a weight average Mr of 17,000 and more than 98% of the uronic acid was glucuronic acid. This group comprised only 12% of the total proteoglycan of the tissue. The other 88% of the proteoglycans appeared to represent one group of small molecules that eluted from Sepharose CL-2B at Kav = 0.70. They demonstrated buoyant densities in a CsCl gradient ranging from greater than or equal to 1.51 to 1.30 g/ml. Their core protein had an apparent Mr = 48,000 following removal of the glycosaminoglycan chains by digestion with chondroitinase ABC. This core protein had a particularly high content of aspartic acid/asparagine and leucine. The glycosaminoglycan chains had a weight average Mr of 37,000 and were dermatan sulfate containing 73% iduronic acid. Those molecules found at highest buoyant density appeared to have additional glycosaminoglycan chains that were shorter. Proteoglycans were also extracted from the pressure-bearing distal region of this tendon, where contents of proteoglycan per wet weight of tissue were 3-fold higher and as much as 50% of this was as large as the large proteoglycans from the proximal tissue. Preparations of large proteoglycans from both tendon regions contained molecules capable of interacting with hyaluronic acid.

To meet the growing need for tissue replacement materials for our aging population, the development of new adaptive biomaterials is essential. The tissues with the highest demand for implant materials are skin and bone. These tissues share various similarities, including signaling pathways and extracellular matrix composition. Glycosaminoglycans such as hyaluronan and chondroitin sulfate are the major organic extracellular matrix components. They modulate the attraction of skin and bone precursor cells and their subsequent differentiation and gene expression and regulate the action of proteins essential to bone and skin regeneration. The precise action of glycosaminoglycans varies according to their structural composition mainly in respect to the degree of sulfation and polymer length. Changes in the glycosaminoglycan composition are frequently seen in physiological and pathological remodeling processes, such as bone formation or scaring. Here, we review the current state of knowledge of how the most common glycosaminoglycan, chondroitin sulfate and hyaluronan, interact with bone and skin cells, and summarize their potential in tissue engineering for skeletal and skin diseases.

Heparin is an excellent inhibitor of P- and L-selectin binding to the carbohydrate determinant, sialyl Lewis(x). As a consequence of its anti-selectin activity, heparin attenuates metastasis and inflammation. Here we show that fucosylated chondroitin sulfate (FucCS), a polysaccharide isolated from sea cucumber composed of a chondroitin sulfate backbone substituted at the 3-position of the beta-D-glucuronic acid residues with 2,4-disulfated alpha-L-fucopyranosyl branches, is a potent inhibitor of P- and L-selectin binding to immobilized sialyl Lewis(x) and LS180 carcinoma cell attachment to immobilized P- and L-selectins. Inhibition occurs in a concentration-dependent manner. Furthermore, FucCS was 4-8-fold more potent than heparin in the inhibition of the P- and L-selectin-sialyl Lewis(x) interactions. No inhibition of E-selectin was observed. FucCS also inhibited lung colonization by adenocarcinoma MC-38 cells in an experimental metastasis model in mice, as well as neutrophil recruitment in two models of inflammation (thioglycollate-induced peritonitis and lipopolysaccharide-induced lung inflammation). Inhibition occurred at a dose that produces no significant change in plasma activated partial thromboplastin time. Removal of the sulfated fucose branches on the FucCS abolished the inhibitory effect in vitro and in vivo. Overall, the results suggest that invertebrate FucCS may be a potential alternative to heparin for blocking metastasis and inflammatory reactions without the undesirable side effects of anticoagulant heparin.

Glycocalyx on the surface of endothelium has been suggested to be involved in vascular permeability and anticoagulation. In the present study, we demonstrated that fluid laminar shear stress enhanced a glycosaminoglycan (GAG) synthesis in porcine aortic endothelial cells, in vitro. Shear stress (15, 40 dyn/cm2) for 24 hours significantly increased GAG synthesis, assayed by [35S]sulfate incorporation, in "medium" fraction and "trypsinated" fraction which includes GAGs derived from the cell surface and from the solubilized matrix. Increased GAGs in the trypsinated and medium fractions consisted of mainly heparan sulfate and chondroitin/dermatan sulfate, respectively. Both heparan and chondroitin/dermatan sulfate increases are required to expose the cells to shear stress for more than 24 hours. Shear-stress-induced increase in GAG synthesis was concomitant with a decrease in DNA synthesis and an increase in protein synthesis. These data indicate that relatively high shear stress may suppress atherogenesis by changing endothelial GAG synthesis.