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The glycosaminoglycans (GAGs) are long, linear, sulfated and highly charged heterogeneous polysaccharides that are expressed throughout the body in different forms, depending on the development or pathological state of the organ/organism. The GAGs and their interaction with proteins are discussed with a special emphasis on the chemokine system.

Osmotically inactive skin [Na.sup.+] storage is characterized by [Na.sup.+] accumulation without water accumulation in the skin. Negatively charged glycosaminoglycans (GAGs) may be important in skin [Na.sup.+] storage. We investigated changes in skin GAG content and key enzymes of GAG chain polymerization during osmotically inactive skin [Na.sup.+] storage. Female Sprague-Dawley rats were fed a 0.1% or 8% NaCl diet for 8 wk. Skin GAG content was measured by Western blot analysis, mRNA content of key dermatan sulfate polymerization enzymes was measured by real-time PCR. The [Na.sup.+] concentration in skin was determined by dry ashing. Skin [Na.sup.+] concentration during osmotically inactive [Na.sup.+] storage was 180-190 mmol/l. Increasing skin [Na.sup.+] coincided with increasing GAG content in cartilage and skin. Dietary NaCl loading coincided with increased chondroitin synthase mRNA content in the skin, whereas xylosyl transferase, biglycan, and decorin content were unchanged. We conclude that osmotically inactive skin [Na.sup.+] storage is an active process characterized by an increased GAG content in the reservoir tissue. Inhibition or disinhibition of GAG chain polymerization may regulate osmotically inactive [Na.sup.+] storage. hypertension; extracellular matrix; chondroitin synthase; elongation enzymes

A study used ultraviolet (UV) visible spectroscopy to show that the chloramides derived from N-acetyl function of the glycosamine rings were formed by the reaction of HOCl with glycosaminoglycans and model compounds. Results indicate that selective fragmentation takes place at disaccharides intervals due to the analogous reactions with glycosaminoglycans and HOCL- mediated glycosaminoglycan fragmentation can play an important role in disease progression and resolution.

Host cell binding is an essential step in colonization by many bacterial pathogens, and the Lyme disease agent, Borrelia burgdorferi, which colonizes multiple tissues, is capable of attachment to diverse cell types. Glycosaminoglycans (GAGs) are ubiquitously expressed on mammalian cells and are recognized by multiple B. burgdorferi surface proteins. We previously showed that B. burgdorferi strains differ in the particular spectrum of GAGs that they recognize, leading to differences in the cultured mammalian cell types that they efficiently bind. The molecular basis of these binding specificities remains undefined, due to the difficulty of analyzing multiple, potentially redundant cell attachment pathways and to the paucity of genetic tools for this pathogen. In the current study, we show that the expression of decorin-binding protein (Dbp) A and/or DbpB, two B. burgdorferi surface proteins that bind GAGs, is sufficient to convert a high-passage nonadherent B. burgdorferi strain into one that efficiently binds 293 epithelial cells. Epithelial cell attachment was mediated by dermatan sulfate, and, consistent with this GAG-binding specificity, these recombinant strains did not bind EA-Hy926 endothelial cells. The GAG-binding properties of bacteria expressing DbpB or DbpA were distinguishable, and DbpB but not DbpA promoted spirochetal attachment to C6 glial cells. Thus, DbpA and DbpB may each play central but distinct roles in cell type-specific binding by Lyme disease spirochetes. This study illustrates that transformation of high-passage B. burgdorferi strains may provide a relatively simple genetic approach to analyze virulence-associated phenotypes conferred by multiple bacterial factors. cell adhesion | proteoglycans | glycosaminoglycans

Research has been conducted on carbohydrate sulfation. The study of the sulfotransferase subtype acting on GalNAc, Gal and GlcNAc residue 6-position has been carried out and the role of carbohydrate sulfotransferases in glycosaminoglycans' inflammatory response and biosynthesis has been investigated and discussed.

Results reveal that among the glycosaminoglycans heparin binds with high affinity to the surface antigen circumsporozoite protein of malaria sporozoites. Data indicate that a decasaccharide represents the minimum sized sequence that can bind to circumsporozoite.

Fibroblast growth factor (FGF) family plays key roles in development, wound healing, and angiogenesis. Understanding of the molecular nature of interactions of FGFs with their receptors (FGFRs) has been seriously limited by the absence of structural information on FGFR or FGF-FGFR complex. in this study, based on an exhaustive analysis of the primary sequences of the FGF family, we determined that the residues that constitute the primary receptor-binding site of FGF-2 are conserved throughout the FGF family, whereas those of the secondary receptor binding site of FGF-2 are not. We propose that the FGF-FGFR interaction mediated by the 'conserved' primary site interactions is likely to be similar if not identical for the entire FGF family, whereas the 'variable' secondary sites, on both FGF as well as FGFR mediates specificity of a given FGF to a given FGFR isoform. Furthermore, as the pro-inflammatory cytokine interleukin 1 (IL-1) and FGF-2 share the same structural scaffold, we find that the spatial orientation of the primary receptor-binding site of FGF-2 coincides structurally with the IL-1[Beta] receptor-binding site when the two molecules are superimposed. The structural similarities between the IL-1 and the FGF system provided a framework to elucidate molecular principles of FGF-FGFR interactions. In the FGF-FGFR model proposed here, the two domains of a single FGFR wrap around a single FGF-2 molecule such that one domain of FGFR binds to the primary receptor-binding site of the FGF molecule, while the second domain of the same FGFR binds to the secondary receptor-binding site of the same FGF molecule. Finally, the proposed model is able to accommodate not only heparin-like glycosaminoglycan (HLGAG) interactions with FGF and FGFR but also FGF dimerization or oligomerization mediated by HLGAG.

Sulfated glycosaminoglycans (GAGs) are linear polysaccharides of repeating disaccharide sequences on which are superimposed highly complex and variable patterns of sulfation, especially in heparan sulfate (HS). HS and the structurally related heparin exert important biological functions, primarily by interacting with proteins and regulating their activities. Evidence is accumulating that these interactions depend on specific saccharide sequences, but the lack of simple, direct techniques for sequencing GAG saccharides has been a major obstacle to progress. We describe how HS and heparin saccharides can be sequenced rapidly by using an integrated strategy with chemical and enzymic steps. Attachment of a reducing-end fluorescent tag establishes a reading frame. Partial selective chemical cleavage at internal N-sulfoglucosamine residues with nitrous acid then creates a set of fragments of defined sizes. Subsequent digestion of these fragments with combinations of exosulfatases and exoglycosidases permits the selective removal of specific sulfates and monosaccharides from their nonreducing ends. PAGE of the products yields a pattern of fluorescent bands from which the saccharide sequence can be read directly. Data are presented on sequencing of heparin tetrasaccharides and hexasaccharides of known structure; these data show the accuracy and versatility of this sequencing strategy. Data also are presented on the application of the strategy to the sequencing of an HS decasaccharide of unknown structure. Application and further development of this sequencing strategy, called integral glycan sequencing, will accelerate progress in defining the structure-activity relationships of these complex GAGs and lead to important insights into their biological functions.

The regio- and stereoselective synthesis of beta-D-gluco-, alpha-L-ido- and alpha-L-altropyranosiduronic acids from delta(super 4)-uronate monosaccharides was described. The trans-diaxial bromohydrin analogues were produced by bromination of the C-4,5 double bond, and these compounds were converted to the corresponding epoxides in high yields. Direct reduction of the epoxides using borane-tetrahydrofuran system resulted in the corresponding glucuronic acids in low to moderate yields.

Heparin-like glycosaminoglycans, acidic complex polysaccharides present on cell surfaces and in the extracellular matrix, regulate important physiological processes such as anticoagulation and anglogenesis. Heparin-like glycosaminoglycan degrading enzymes or heparinases are powerful tools that have enabled the elucidation of important biological properties of heparin-like glycosaminoglycans in vitro and in vivo. With an overall goal of developing an approach to sequence heparin-like glycosaminoglycans using the heparinases, we recently have elaborated a mass spectrometry methodology to elucidate the mechanism of depolymerization of heparin-like glycosaminoglycans by heparinase I. In this study, we investigate the mechanism of depolymerization of heparin-like glycosaminoglycans by heparinase II, which possesses the broadest known substrate specificity of the heparinases. We show here that heparinase II cleaves heparin-like glycosaminoglycans endolytically in a nonrandom manner. In addition, we show that heparinase II has two distinct active sites and provide evidence that one of the active sites is heparinase I-like, cleaving at hexosamine-sulfated iduronate linkages, whereas the other is presumably heparinase III-like, cleaving at hexosamine-glucuronate linkages. Elucidation of the mechanism of depolymerization of heparin-like glycosaminoglycans by the heparinases and mutant heparinases could pave the way to the development of much needed methods to sequence heparin-like glycosaminoglycans.

The binding site on the chemokine interleukin-8 (IL-8) for the glycosaminoglycan heparin was characterized through a systematic series of site-directed mutants of IL-8 in which the basic residues were replaced by alanine. Findings revealed a heparin-binding surface on IL-8 that includes the C-terminal alpha-helix and the proximal loop around residues 18-23. The heparin-binding site is spatially differentiated from the residues having to do with receptor binding.

A study was conducted to test whether pigment epithelium-derived factor (PEDF) associates with the bovine interphotoreceptor matrix by ionic interactions. Chemical modification, limited proteolysis and a predictive spatial framework were utilized to determine a glycosaminoglycan-binding region on the amino acid sequence of the PEDF. Results indicated that PEDF does not support a homology with antithrombin III in basic residues at helix D.

Research was conducted to examine the intracellular signaling pathway by which platelet-derived growth factor (PDGF) homodimer PDGF-BB-induced GAG synthesis is relayed in primary cultures of fetal lung fibroblasts. Their activities after PDGF-BB stimulation were measured. Results indicate that P13K acts as a downstream mediator of PDGF-BB-induced GAG synthesis in fetal rat lung fibroblasts.

Heparinase I from Flavobacterium heparinum has important uses for elucidating the complex sequence heterogeneity of heparin-like glycosaminoglycans (HLGAGs). Understanding the biological function of HLGAGs has been impaired by the limited methods for analysis of pure or mixed oligosaccharide fragments. Here, we use methodologies involving MS and capillary electrophoresis to investigate the sequence of events during heparinase I depolymerization of HLGAGs. In an initial step, heparinase I preferentially cleaves exolytically at the nonreducing terminal linkage of the HLGAG chain, although it also cleaves internal linkages at a detectable rate. In a second step, heparinase I has a strong preference for cleaving the same substrate molecule processively, i.e., to cleave the next site toward the reducing end of the HLGAG chain. Computer simulation showed that the experimental results presented here from analysis of oligosaccharide degradation were consistent with literature data for degradation of polymeric HLGAG by heparinase I. This study presents direct evidence for a predominantly exolytic and processive mechanism of depolymerization of HLGAG by heparinase I.

Difficulties in determining composition and sequence of glycosaminoglycans, such as those related to heparin, have limited the investigation of these biologically important molecules. Here, we report methodology, based on matrix-assisted laser desorption ionization MS and capillary electrophoresis, to follow the time course of the enzymatic degradation of heparin-like glycosaminoglycans through the intermediate stages to the end products. MS allows the determination of the molecular weights of the sulfated carbohydrate intermediates and their approximate relative abundances at different time points of the experiment. Capillary electrophoresis subsequently is used to follow more accurately the abundance of the components and also to measure sulfated disaccharides for which MS is not well applicable. For those substrates that produce identical or isomeric intermediates, the reducing end of the carbohydrate chain was converted to the semicarbazone. This conversion increases the molecular weight of all products retaining the reducing terminus by the 'mass tag' (in this case 56 Da) and thus distinguishes them from other products. A few picomoles of heparin-derived, sulfated hexa- to decasaccharides of known structure were subjected to heparinase I digestion and analyzed. The results indicate that the enzyme acts primarily exolytically and in a processive mode. The methodology described should be equally useful for other enzymes, including those modified by site-directed mutagenesis, and may lend to the development of an approach to the sequencing of complex glycosaminoglycans.

A study was conducted on the interaction between glycosaminoglycans and amyloid-associated immunoglobulin light chains using size-exclusion high-performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The findings show that heparin 16,000 and chondroitin sulfate B and C precipitated both human intact light chains and recombinant light chain variable domains. The strongest interactions were observed with proteins that had formed amyloid.

The role of the chondroitin-dermatan sulfate-containing proteoglycans in the contractile and elastic properties of small resistance arteries from rat mesentery was examined. Functional and mechanical tests on resistance-sized mesenteric arteries were carried out. Results of the experiments revealed that significant alterations in myogenic behavior are associated with changes in passive mechanics. These alterations are caused by stress transfers between cells and collagen fibrils.

The adhesion of hydroxyapatite crystals to anionic sites on the surface of renal epithelial cells was investigated using a tissue culture model system to study the interaction between hydroxyapatite (HA) crystals and renal epithelial cells. Results of the study revealed that the quantity, structure and function of soluble anions in tubular fluid are significant determinants of HA crystal retention. Furthermore, anionic cell surface siali acid residues function as HA crystal receptor sites.

Effects of restricting uterine space on physical, biochemical, and histochemical characteristics of ovine placental tissues were studied. Ewes (n = 20) were unilaterally ovariectomized, assigned to either control (C; n = 10) or unilateral (UPx; n = 10) pregnancy groups, mated (d 0), and hysterectomized on either d 60, 90, or 120. Placental and fetal weights and placentome wet weights (PWT) in three placental areas (AI, AII, AIII) were recorded. Placentome tissue concentrations of RNA, DNA, hyaluronic acid (HA), and protein (TP) were determined. Overall, placentome numbers were reduced (P < .02) 23%, but individual PWT increased (P < .05) 27% in UPx ewes. In UPx ewes, neither total placental nor placentome weights, fetal weights, fetal crown-rump lengths, nor PWT:fetal weight ratios were affected by treatment (Trt). In the C and UPx groups, PWT increased (P < .01) from d 60 to 90. However, compensatory growth was confined to placental areas AII and Alii in UPx groups (treatment x area, P < .01). Treatment did not affect concentrations of RNA, DNA, TP, or HA. However, RNA, DNA, and TP increased from d 60 to 120 (P < .01), but HA decreased (P < .01). Histologically, placentome cellularity increased from d 60 to 120 as area occupied by individual fetal chorioallantoic villi (FV) decreased. The FV stained with Alcian Blue 8X. Alcianophilia was attenuated at low pH and eliminated by pretreatment with hyaluronidase, indicating the presence of HA. Thus, increased placentomal cellularity was accompanied by loss of HA from fetal allantoic mesenchyme. Mechanisms regulating loss of HA from FV may support placental maturation and fetal growth. Key Words: Sheep, Placenta, Growth, Glycosaminoglycans, Hyaluronic Acid

The binding of the long platelet-derived growth factor A (PDGF-A) isoform to a heparin, a glycosaminoglycan, was studied. To this end, the rA125 and the rA109 variants of PDGF-A were expressed in Escherichia coli and characterized using surface plasmon resonance. The results revealed the high affinity binding of the long homo dimer, rA125, to low molecular weight heparin. This was attributed to the presence of an amino acid carboxyl-terminal extension that is absent in the short homodimer.

A model based on an X-ray crystal structure of Congo Red described the sulfonated azo dye's active site binding with the HIV-1 protease. Model behavior indicated a higher affinity for the Evans Blue dye compared to Congo Red. Analysis of the interactions involving sulfonate and sulfate groups with functional groups in protein yielded six complexes that were formed by the activity between an amino acid and a sulfonated azo dye.

Methylated and acetylated derivates of Xyl-beta-1 to 6Gal-beta-O-2-naphthol act as primers of glycosaminoglycan chains and inhibit the synthesis of glycoconjugates. The acetylated product Gal-beta-1 to 4GlcNAc-beta-O-naphthalenemethanol also inhibits the expression of sialyl Lewis X in HeLa cells. The unmodified disaccharides are not taken up by the cells. The biology of glycoconjugates can be studied by the inhibition of glycosylation.

An investigation of human orbital fibroblast cultures reveals that synthesis of hyaluronan, a glycosaminoglycan, in these cultures depends on the 50 kDa glycoprotein lymphokine leukoregulin, the extent of synthesis increase by leukoregulin depending on concentration and time. The irregular glycosaminoglycan deposits in orbital connective tissues during Grave's ophthalmopathy can be studied using leukoregulin as a marker signal.

Administration of chondroitin sulfate (CS) glycosaminoglycan into retinal ventricular surfaces in an organ culture results in the repolarization of retinal ganglion cell bodies and axonal fibers in the neuroepithelium. Extracellular CS milieu level regulates the radial formation of the axon. The basal lamina of the retina remain intact and axon development is not confined to the neuronal vitreal endfoot site of the retina.

The synthesis of glycosaminoglycans (GAG) in a freshwater mussel was studied in organ culture using labeled precursors. The major GAGs synthesized were determined and characterized by chemical and enzymatic methods. They were shown to be heparin and an unusual type of heparan sulfate. Gills produced about 50% of each polymer; mantles synthesized little heparin and mostly the heparan-sulfate-like compound, which is similar to a GAG isolated previously from lobsters. No significant amounts of chondroitin sulfates were present. Histological data showed that the sulfate-labeled GAGs were present mainly in exterior pericellular and basement membrane locations of gills and mantle. That is, they would be in contact with the external aqueous environment, suggesting a potential role in calcium transport and storage.

1H and 13C NMR spectroscopy are used to investigate the interaction of histamine with beef lung heparin. Results reveal that histamine is bound by heparin in an intensely pD-dependent manner. At low pD, some histamine binds territorially in the condensation volume around heparin. At increasing pD and as the carboxylic group is titrated, a binding region with a high affinity for the imidazolium ring is generated. NMR results also indicate the presence of a binding site with a high attraction for the imidazolium ring.