Lowered expression of heparan sulfate/heparin biosynthesis enzyme N-deacetylase/n-sulfotransferase 1 results in increased sulfation of mast cell heparin

Deficiency of the heparan sulfate biosynthesis enzyme N-deacetylase/N-sulfotransferase 1 (NDST1) in mice causes severely disturbed heparan sulfate biosynthesis in all organs, whereas lack of NDST2 only affects heparin biosynthesis in mast cells (MCs). To investigate the individual and combined roles of NDST1 and NDST2 during MC development, in vitro differentiated MCs derived from mouse embryos and embryonic stem cells, respectively, have been studied. Whereas MC development will not occur in the absence of both NDST1 and NDST2, lack of NDST2 alone results in the generation of defective MCs. Surprisingly, the relative amount of heparin produced in NDST1(+/-) and NDST1(-/-) MCs is higher (≈30%) than in control MCs where ≈95% of the (35)S-labeled glycosaminoglycans produced is chondroitin sulfate. Lowered expression of NDST1 also results in a higher sulfate content of the heparin synthesized and is accompanied by increased levels of stored MC proteases. A model of the GAGosome, a hypothetical Golgi enzyme complex, is used to explain the results.     

Altered expression of glycosaminoglycans in metastatic 13762NF rat mamma adenocarcinoma cells

A difference in the expression and metabolism of sulfated glycosaminoglycans between rat mammary tumor cells derived from a primary tumor and those from its metastatic lesions has been observed. Cells from the primary tumor possessed about equal quantities of chondroitin sulfate and heparan sulfate on their cell surfaces but released fourfold more chondroitin sulfate than heparan sulfate into their medium. In contrast, cells from distal metastatic lesions expressed approximately 5 times more heparan sulfate than chondroitin sulfate in both medium and cell surface fractions. This was observed to be the result of differential synthesis of the glycosaminoglycans and not of major structural alterations of the individual glycosaminoglycans. The degree of sulfation and size of heparan sulfate were similar for all cells examined. However, chondroitin sulfate, observed to be only chondroitin 4-sulfate, from the metastases-derived cells had a smaller average molecular weight on gel filtration chromatography and showed a decreased quantity of sulfated disaccharides upon degradation with chondroitin ABC lyase compared to the primary tumor derived cells. Major qualitative or quantitative alterations were not observed for hyaluronic acid among the various 13762NF cells. The metabolism of newly synthesized sulfated glycosaminoglycans was also different between cells from primary tumor and metastases. Cells from the primary tumor continued to accumulate glycosaminoglycans in their medium over a 72-h period, while the accumulation of sulfated glycosaminoglycans in the medium of metastases-derived cells showed a plateau after 18-24 h. A pulse-chase kinetics study demonstrated that both heparan sulfate and chondroitin sulfate were degraded by the metastases-derived cells, whereas the primary tumor derived cells degraded only heparan sulfate and degraded it at a slower rate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sulfated glycosaminoglycans synthesized by human smooth muscle cells isolated from different organs

The sulfated glycosaminoglycans synthesized by human smooth muscle cells isolated from different organs were identified on the basis of electrophoretic mobility, enzymatic degradation with specific mucopolysaccharidases and by the type of degradation products formed. The results obtained indicated that chondroitin sulfate and heparan sulfate were the main glycosaminoglycans found, that most of the labeled glycosaminoglycans were found in the pericellular pool, and that no marked differences were observed in the sulfated glycosaminoglycan composition of the smooth muscle cells obtained from different organs. 'Liver connective tissue cells', isolated from pathological livers (which had been shown to possess biochemical and physiological features typical of smooth muscle cells) showed a pattern of glycosaminoglycan synthesis similar to that of the smooth muscle cells.     

Characterization of extracellular matrix-associated glycosaminoglycans produced by untransformed and transformed bovine corneal endothelial cells in culture

A cloned bovine corneal endothelial cell line was transformed in vitro by simian virus 40, and the subendothelial extracellular matrix-associated sulfated glycosaminoglycans synthesized by the cells were isolated and compared with their untransformed counterpart. The transformed endothelial cells grew at faster rates to higher stationary cell densities in the absence of fibroblast growth factor than did the untransformed cells. On a per-cell basis, the transformed cells produced slightly lower amounts of sulfated glycosaminoglycans. The rate of production of sulfated glycosaminoglycans in extracellular matrix increased during seven days of culture. At confluency the extracellular matrix-associated sulfated glycosaminoglycans synthesized by the untransformed endothelial cells consisted of about 80% heparan sulfate and about 20% chondroitin sulfate. Extracellular matrix-associated sulfated glycosaminoglycans of transformed endothelial cells were composed of about 70% heparan sulfate and about 30% chondroitin sulfate plus dermatan sulfate. High-speed gel permeation chromatography profiles on Fractogel TSK HW-55(S) of matrix-associated heparan sulfate from untransformed and transformed endothelial cells were very similar, and gave single peaks (Kav = 0.19). Apparent Mr estimated from the eluting position of the peaks were approximately 47000. Heparan sulfate from both untransformed and transformed endothelial cells was degraded by incubation with a metastatic B16 melanoma cell lysate containing heparanase (heparan-sulfate-specific endo-beta-glucuronidase). The eluting position of the heparan sulfate degradation products on gel permeation column were similar (Kav = 0.43). Size analysis and anion-exchange chromatography of the degradation products after nitrous acid deamination at low pH indicated that the degree of N-sulfation of heparan sulfate was similar in untransformed and transformed endothelial cells. The results indicated that transformation of endothelial cells only slightly changes the molecular nature of subendothelial matrix-associated sulfated glycosaminoglycans.     

Disaccharide Composition of Heparan Sulfates: Brain, Nervous Tissue Storage Organelles, Kidney, and Lung

Abstract: We have characterized the structural properties of heparan sulfates from brain and other tissues after de-polymerization with a mixture of three heparin and heparan sulfate lyases from Flavobacterium heparinum. The resulting disaccharides were separated by HPLC and identified by comparison with authentic standards. In rat, rabbit, and bovine brain, 46–69% of the heparan sulfate disaccharides are N-acetylated and unsulfated, and 17–21% contain a single sulfate residue in the form of a sulfoamino group. In rabbit, bovine, and 1-day postnatal rat brain, disaccharides containing both a sulfated uronic acid and N-sulfate account for an additional 10–14%, together with smaller and approximately equall proportions (5–9%) of mono-, di-, and trisulfated disaccharides having sulfate at the 6-position of the glucosamine residue. Kidney and lung heparan sulfates are distinguished by high concentrations of disaccharides containing 6-sulfated N-acetylglucosamine residues. In chromaffin granules, the catecholamine-and peptide-storing organelles of adrenal medulla, where heparan sulfate accounts for a minor portion (5–10%) of the glycosaminoglycans, we have determined that bovine chromaffin granule membranes contain heparan sulfate in which almost all of the disaccharides are either unsulfated (71 %) or monosulfated (18%). In sympathetic nerves, norepinephrine is stored in large densecored vesicles that in biochemical composition and properties closely resemble adrenal chromaffin granules. However, in contrast to chromaffin granules, heparan sulfate accounts for ～ 75% of the total glycosaminoglycans in large dense-cored vesicles and more closely resembles heparin, insofar as it contains only 21 % unsulfated disaccharides, 10% mono-and disulfated disaccharides, and 69% trisulfated disaccharides. Our results therefore reveal significant differences among heparan sulfates from different sources, supporting other evidence that structural variations in heparan sulfate may be related to specific biological functions, such as the switching in the neural response from fibroblast growth factor-2 to fibro-blast growth factor-1 resulting from developmental changes in the glycosaminoglycan chains of a heparan sulfate proteoglycan.     

Metabolism of sulfated glycosaminoglycans in rat hepatocytes. Synthesis of heparan sulfate and distribution into cellular and extracellular pools

Primary cultures of rat hepatocytes grown in a serum-free medium supplemented with [35S]sulfate synthesize 35S-labelled glycosaminoglycans at an almost constant rate for 58 h. Approx. 57% of the newly synthesized 35S-labelled glycosaminoglycans remain within the hepatocytes, approx. 30% become associated with the cell surface and only 13% are secreted into the medium. The amount of cell-surface-associated 35S-labelled glycosaminoglycans became constant within 36 h, whereas no equilibrium was reached in the intra- and extracellular pool. During a 24 h chase more than 50% of the intracellular and cell-surface-associated 35S-labelled glycosaminoglycans disappears, more than 80% of this material is degraded and radioactivity is recovered as inorganic sulfate. A minor part is released into the medium in a macromolecular form. Heparan sulfate accounts for more than 95% of the 35S-labelled glycosaminoglycans in each of the three pools. It is distinguished from heparan sulfates from other sources by the presence of unsubstituted glucosamine residues. In all three pools, heparan sulfate chains of mean molecular weights between 24 000 and 30 000 are part of an alkali labile proteoglycan. Intra- and extracellularly, however, part of the heparan sulfate appears to have little, if any, protein attached. Hepatocytes contain heparan sulfate-degrading endoglycosidase activity, which may contribute to the variation of molecular weights observed for the heparan sulfate.     

Heparan sulfate and control of endothelial cell proliferation: increased synthesis during the S phase of the cell cycle and inhibition of thymidine incorporation induced by ortho-nitrophenyl-beta-D-xylose

The effect of xylosides on the synthesis of [35S]-sulfated glycosaminoglycans by endothelial cells in culture was investigated. Ortho-nitrophenyl-beta-D-xylose (10(-3)M) produces a dramatic enhancement on the synthesis of heparan sulfate and chondroitin sulfate secreted to the medium (20- and 100-fold, respectively). Para-nitrophenylxyloside, at the same concentration, produces an enhancement of only 37- and 3-fold of chondroitin sulfate and heparan sulfate, respectively. These differences of action seem to be related with the higher lipophilic character of ortho-nitrophenyl-xyloside. A lower enhancement of the synthesis of the two glycosaminoglycans is also observed with 2-naphtol beta-D-xylose and cis/trans-decahydro-2-naphtol beta-D-xylose. Besides stimulating the synthesis, O-nitrophenyl-beta-D-xylose as PMA [J. Cell. Biochem. 70 (1998) 563] also inhibits [3H]-thymidine incorporation by quiescent endothelial cells stimulated for growth by fetal calf serum (FCS). The combination of xylosides with PMA produced some cumulative effect. PMA stimulates the synthesis of heparan sulfate mainly at G1 phase whereas the highest enhancement of synthesis produced by the xylosides is in the S phase of the endothelial cell cycle.     

The effect of sexual hormones on the sulfated glycosaminoglycan pattern of male genital accessory organs

The absolute and relative amounts of glycosaminoglycans and [35S]sulfate uptake were investigated in several tissues of male guinea pigs and rats under different sexual hormonal conditions (castration, estrogen treatment, or both). The hormonal effects, regarding the pattern of sulfated glycosaminoglycans, were specifically observed in the target organs (vas deferens and seminal vesicles) of both animals. Castration, in both species, decreases the amount of heparan sulfate and chondroitin sulfate, while diethylstilbestrol (DES) treatment causes different effects on rat and guinea pig target organs. In rats the effect of estrogen administration and surgical castration was essentially the same, and in guinea pigs DES increased the content of dermatan sulfate and chondroitin sulfate. The modifications in the specific patterns of the sulfated glycosaminoglycans suggest that these compounds are under sexual hormonal control only in the target organs, and show a specific pattern of distribution according to the tissue layer.     

Ninhydrin Positive Compounds Produced by Heating of Poly-l-amino Acids,Tripeptides and an Amino Acid Mixture

Glycosaminoglycans were isolated from bovine and human milk fat globule membrane (MFGM) preparations and characterized. The amount of glycosaminoglycans in human MFGM protein fraction was 5 ~ 10 times higher than that in bovine MFGM protein fraction. On Dowex-1 column chromatography most of the glycosaminoglycans were eluted with 1 M and 3 M NaCl. Cellulose acetate electrophoresis, chondroitinase digestion and nitrous acid degradation indicated that the major glycosaminoglycan in both bovine and human MFGM was heparan sulfate. Bovine MFGM was shown to contain chondroitin sulfate A/C in addition to heparan sulfate. The glycosaminoglycan composition of bovine colostral MFGM was similar to that of bovine normal MFGM. The glycosaminoglycan fraction prepared from MFGM of human colostrum, however, did not contain heparan sulfate, suggesting some compositional changes in glycosaminoglycans during the lactation period.     

Metabolism of sulfated glycosaminoglycans in cultivated bovine arterial cells. I. Characterization of different pools of sulfated glycosaminoglycans.

"Fibroblast-like" cells from the intimal layer of bovine aorta were grown in culture. The formation, composition, molecular weight and turnover rate of different pools of glycosaminoglycans were investigated in cultures incubated in the presence [35S]sulfate or [14C]glucosamine. The newly synthesized glycosaminoglycans are distributed into an extracellular pool (37 - 58%), a cell-membrane associated or pericellular pool (23 - 33%), and an intracellular pool (19 - 30%), each pool exhibiting a characteristic distribution pattern of chondroitin sulfate, dermatan sulfate, heparan sulfate and hyaluronate. The distribution pattern of the extracellular glycosaminoglycans resembles closely that found in bovine aorta. A small subfraction of the pericellular pool - tentatively named "undercellular" pool--has been characterized by its high heparan sulfate content. The intracellular and pericellular [35S]glycosaminoglycan pools reach a constant radioactivity after 8-12 h and 24 h, respectively, whereas the extracellular [35S]glycosaminoglycans are secreted into the medium at a linear rate over a period of at least 6 days. The intracellular glycosaminoglycans are mainly in the process of degradation, as indicated by their low molecular weight and by their half-life of 7 h, but intracellular dermatan sulfate is degraded more rapidly (half-life 4-5 h) than intracellular chondroitin sulfate and heparan sulfate (half-life 7-8 h). Glycosaminoglycans leave the pericellular pool with a half-life of 12-14 h by 2 different routes: about 60% disappear as macromolecules into the culture medium, and the remainder is pinocytosed and degraded to a large extent. Extracellular and at least a part of the pericellular glycosaminoglycans are proteoglycans. Even under dissociative conditions (4M guanidinium chloride) their hydrodynamic volume is sufficient for partial exclusion from Sepharose 4B gel. The existence of topographically distinct glycosaminoglycan pools with varying metabolic characteristics and differing accessibility for degradation requiresa reconsideration and a more reserved interpretation of results concerning the turnover rates of glycosaminoglycans as determined in arterial tissue.     

Dermatan sulfate is the predominant antithrombotic glycosaminoglycan in vessel walls: implications for a possible physiological function of heparin cofactor II

The role of different glycosaminoglycan species from the vessel walls as physiological antithrombotic agents remains controversial. To further investigate this aspect we extracted glycosaminoglycans from human thoracic aorta and saphenous vein. The different species were highly purified and their anticoagulant and antithrombotic activities tested by in vitro and in vivo assays. We observed that dermatan sulfate is the major anticoagulant and antithrombotic among the vessel wall glycosaminoglycans while the bulk of heparan sulfate is a poorly sulfated glycosaminoglycan, devoid of anticoagulant and antithrombotic activities. Minor amounts of particular a heparan sulfate (< 5% of the total arterial glycosaminoglycans) with high anticoagulant activity were also observed, as assessed by its retention on an antithrombin-affinity column. Possibly, this anticoagulant heparan sulfate originates from the endothelial cells and may exert a significant physiological role due to its location in the interface between the vessel wall and the blood. In view of these results we discuss a possible balance between the two glycosaminoglycan-dependent anticoagulant pathways present in the vascular wall. One is based on antithrombin activation by the heparan sulfate expressed by the endothelial cells. The other, which may assume special relevance after vascular endothelial injury, is based on heparin cofactor II activation by the dermatan sulfate proteoglycans synthesized by cells from the subendothelial layer.     

Proteoglycan and glycosaminoglycan synthesis by cultured rat mesangial cells

The synthesis of metabolically labeled proteoglycans and glycosaminoglycans from medium, cell layer and substrate attached material by rat glomerular mesangial cells in culture was characterized. The cellular localization of the labeled proteoglycans and glycosaminoglycans was determined by treating the cells with Flavobacterial heparinase. Of the total sulfated glycosaminoglycans, 33% were heparan sulfate; 55% of the cell layer material was heparan sulfate; 80% of sulfated proteins in the medium were chondroitin sulfate/dermatan sulfate. Putative glycosaminoglycan free chains of heparan sulfate and chondroitin sulfate were found in both the medium and cell layer; 95% of total proteoglycans and most (90%) of the putative heparan sulfate free chains were removed from the cell layer by the heparinase, whereas only 50% of the chondroitin sulfate and 25% of dermatan sulfate were removed. Large amounts of hyaluronic acid labeled with 3H glucosamine were found in the cell layer. In summary, approximately 60% of total sulfated glycoproteins was in the form of putative glycosaminoglycan free chains. Thus rat mesangial cells may synthesize large amounts of putative glycosaminoglycan free chains, which may have biological functions in the glomerulus independent of proteoglycans.     

Heparin induces changes in the synthesis of porcine aortic endothelial cell heparan sulfate proteoglycans

Heparin is known to bind to cultured endothelial cells. This report documents that addition of heparin to endothelial cells results in an alteration of the heparan sulfate proteoglycan synthetic pattern. Specifically, the addition of saturating amounts of heparin to confluent cultures of porcine aortic endothelial cells results in an increase in the amount of radiolabeled heparan sulfate proteoglycan secreted into the growth medium. The increase is apparent as early as 8 h after heparin administration. Although there is often a decrease in the amount of cell surface heparan sulfate proteoglycan produced, it is not sufficient to account for the increase in the secreted form. Of the other glycosaminoglycans tested, only dextran sulfate and commercial heparan sulfate induce changes in heparan sulfate proteoglycan synthesis and secretion. Chondroitin sulfate glycosaminoglycans do not elicit this synthetic change. These data indicate that endothelial cells can alter the synthesis of heparan sulfate proteoglycans in response to extracellular signals including heparin and related glycosaminoglycans.     

The cell surface proteoglycan from mouse mammary epithelial cells bears chondroitin sulfate and heparan sulfate glycosaminoglycans

The cell surface proteoglycan fraction isolated by mild trypsin treatment of NMuMG mouse mammary epithelial cells contains largely heparan sulfate, but also 15-24% chondroitin sulfate glycosaminoglycans. We conclude that this fraction contains a unique hybrid proteoglycan bearing both heparan sulfate and chondroitin sulfate glycosaminoglycans because (i) the proteoglycan behaves as a single species by sizing, ion exchange and collagen affinity chromatography, and by isopycnic centrifugation, even in the presence of 8 M urea or 4 M guanidine hydrochloride, (ii) the behavior of the chondroitin sulfate in these separation techniques is affected by heparan sulfate-specific probes and vice versa, and (iii) proteoglycan core protein bearing both heparan sulfate and chondroitin sulfate is recognized by a single monoclonal antibody. Removal of both types of glycosaminoglycan reduces the proteoglycan to a core protein of approximately 53 kDa. The proteoglycan fraction is heterogeneous in size, largely due to a variable number and/or length of the glycosaminoglycan chains. We estimate that one or two chondroitin sulfate chains (modal Mr of 17,000) exist on the proteoglycan for every four heparan sulfate chains (modal Mr of 36,000). Synthesis of these chains is reportedly initiated on an identical trisaccharide that links the chains to the same amino acid residues on the core protein. Therefore, some regulatory information, perhaps residing in the amino acid sequence of the core protein, must determine the type of chain synthesized at any given linkage site. Post-translational addition of these glycosaminoglycans to the protein may provide information affecting its ultimate localization. It is likely that the protein is directed to specific sites on the cell surface because of the ability of the glycosaminoglycans to recognize and bind extracellular components.     

Mosquito heparan sulfate and its potential role in malaria infection and transmission

Heparan sulfate has been isolated for the first time from the mosquito Anopheles stephensi, a known vector for Plasmodium parasites, the causative agents of malaria. Chondroitin sulfate, but not dermatan sulfate or hyaluronan, was also present in the mosquito. The glycosaminoglycans were isolated, from salivary glands and midguts of the mosquito in quantities sufficient for disaccharide microanalysis. Both of these organs are invaded at different stages of the Plasmodium life cycle. Mosquito heparan sulfate was found to contain the critical trisulfated disaccharide sequence, -->4)beta-D-GlcNS6S(1-->4)-alpha-L-IdoA2S(1-->, that is commonly found in human liver heparan sulfate, which serves as the receptor for apolipoprotein E and is also believed to be responsible for binding to the circumsporozoite protein found on the surface of the Plasmodium sporozoite. The heparan sulfate isolated from the whole mosquito binds to circumsporozoite protein, suggesting a role within the mosquito for infection and transmission of the Plasmodium parasite.     

Structural analysis of glycosaminoglycans in Drosophila and Caenorhabditis elegans and demonstration that tout-velu, a Drosophila gene related to EXT tumor suppressors, affects heparan sulfate in vivo

We have devised a sensitive method for the isolation and structural analysis of glycosaminoglycans from two genetically tractable model organisms, the fruit fly, Drosophila melanogaster, and the nematode, Caenorhabditis elegans. We detected chondroitin/chondroitin sulfate- and heparan sulfate-derived disaccharides in both organisms. Chondroitinase digestion of glycosaminoglycans from adult Drosophila produced both nonsulfated and 4-O-sulfated unsaturated disaccharides, whereas only unsulfated forms were detected in C. elegans. Heparin lyases released disaccharides bearing N-, 2-O-, and 6-O-sulfated species, including mono-, di-, and trisulfated forms. We observed tissue- and stage-specific differences in both chondroitin sulfate and heparan sulfate composition in Drosophila. We have also applied these methods toward the analysis of tout-velu, an EXT-related gene in Drosophila that controls the tissue distribution of the growth factor Hedgehog. The proteins encoded by the vertebrate tumor suppressor genes EXT1 and 2, show heparan sulfate co-polymerase activity, and it has been proposed that tout-velu affects Hedgehog activity via its role in heparan sulfate biosynthesis. Analysis of total glycosaminoglycans from tout-velu mutant larvae show marked reductions in heparan sulfate but not chondroitin sulfate, consistent with its proposed function as a heparan sulfate co-polymerase.     

Heparan sulfate proteoglycans are essential for FGF receptor signaling during Drosophila embryonic development.

The Drosophila sugarless and sulfateless genes encode enzymes required for the biosynthesis of heparan sulfate glycosaminoglycans. Biochemical studies have shown that heparan sulfate glycosaminoglycans are involved in signaling by fibroblast growth factor receptors, but evidence for such a requirement in an intact organism has not been available. We now demonstrate that sugarless and sulfateless mutant embryos have phenotypes similar to those lacking the functions of two Drosophila fibroblast growth factor receptors, Heartless and Breathless. Moreover, both Heartless- and Breathless-dependent MAPK activation is significantly reduced in embryos which fail to synthesize heparan sulfate glycosaminoglycans. Consistent with an involvement of Sulfateless and Sugarless in fibroblast growth factor receptor signaling, a constitutively activated form of Heartless partially rescues sugarless and sulfateless mutants, and dosage-sensitive interactions occur between heartless and the heparan sulfate glycosaminoglycan biosynthetic enzyme genes. We also find that overexpression of Branchless, the Breathless ligand, can partially overcome the requirement of Sugarless and Sulfateless for Breathless activity. These results provide the first genetic evidence that heparan sulfate glycosaminoglycans are essential for fibroblast growth factor receptor signaling in a well defined developmental context, and support a model in which heparan sulfate glycosaminoglycans facilitate fibroblast growth factor ligand and/or ligand-receptor oligomerization.     

Disaccharide analysis and molecular mass determination to microgram level of single sulfated glycosaminoglycan species in mixtures following agarose-gel electrophoresis.

The separation of sulfated glycosaminoglycans in mixtures by agarose-gel electrophoresis and the recovery of single polysaccharide bands has been applied to the characterization of polysaccharides extracted from tissues without previous purification of single species. Sulfated glycosaminoglycans, heparin with its two components, slow-moving and fast-moving, heparan sulfate, dermatan sulfate, and chondroitin sulfate, were separated to microgram level by conventional agarose-gel electrophoresis. After their separation, they were fixed in the agarose-gel matrix by precipitation in a cetyltrimethylammonium bromide solution, making them visible on a dark background. After recovery of gel containing the fixed bands, high temperatures (90 degrees C for 15 min) were necessary to dissolve the gel matrix, and a solution of NaCl (3 M) was used to release sulfated polysaccharides from the complex with cetyltrimethylammonium. After precipitation of glycosaminoglycans in the presence of ethanol, the recovery of slow-moving heparin, fast-moving heparin, heparan sulfate, dermatan sulfate, and chondroitin sulfate was from 1 to 10 microg, with a percentage greater than 45% and a purity above 90%. Sulfated glycosaminoglycans in mixtures recovered from gel matrix as single species were evaluated for purity and characterized for unsaturated disaccharides after treatment with bacterial lyases (heparinases for heparin and heparan sulfate samples, and chondroitinases for dermatan sulfate and chondroitin sulfate) and molecular mass. Bovine lung and heart Glycosaminoglycans were extracted and separated into single species by agarose-gel electrophoresis and recovered from gel matrix after treatment in cetyltrimethylammonium solution. Unsaturated disaccharides pattern, the sulfate to carboxyl ratio, and the molecular mass of each single polysaccharide species were determined.     

Removal of Heparan Sulfate Chains Halted Epithelial Branching Morphogenesis of the Developing Mouse Submandibular Gland in vitro

The physiological function of heparan sulfate chains in the mouse embryonic submandibular gland was studied by the use of heparitinases purified from Flavobacteriu heparinum . Heparitinase I, which catalyzes the cleavage of specific glycosaminidic linkages adjacent to non-or monosulfated disaccharides of heparan sulfate chains, in the culture medium of the mid and late 12-day gland inhibited the branch-initiation and changed their round epithelial shape to elongated one, together with a concommitant reduction in lobular growth. [³H]Thymidine incorporation experiments indicated that heparitinase I treatment blocked 24% of the DNA synthesis compared with controls. Analysis of ³⁵S-inorganic sulfate labeled glycosaminoglycans extracted from cultured rudiments revealed that the glands with heparitinase I contained no heparan sulfate, while in the glands without the enzyme more than 20% of total glycosaminoglycans was heparan sulfate.
The heparitinase effect on morphogenesis was mimicked by the addition of heparan sulfate (1 mg ml⁻¹) or heparin (75 μg ml⁻¹), but not by chondroitin sulfate (1 mg ml⁻¹) in the culture medium. Transmission electron microscopic study indicated that at the epithelial-mesenchymal interface close contacts between the fibroblast and epithelial cells were much fewer in heparitinase-treated glands than in controls. Immunohistochemical analysis demonstrated that the core protein of basement membrane heparan sulfate proteoglycan and type IV collagen accumulated abnormally inside the epithelial lobules of glands cultured with heparitinase I. These results strongly suggested that glycosaminoglycan chains of heparan sulfate or heparin is involved in the epithelial morphogenesis of the mouse embryonic submandibular gland.     

The Circulating Glycosaminoglycan Signature of Respiratory Failure in Critically Ill Adults

Systemic inflammatory illnesses (such as sepsis) are marked by degradation of the endothelial glycocalyx, a layer of glycosaminoglycans (including heparan sulfate, chondroitin sulfate, and hyaluronic acid) lining the vascular lumen. We hypothesized that different pathophysiologic insults would produce characteristic patterns of released glycocalyx fragments. We collected plasma from healthy donors as well as from subjects with respiratory failure due to altered mental status (intoxication, ischemic brain injury), indirect lung injury (non-pulmonary sepsis, pancreatitis), or direct lung injury (aspiration, pneumonia). Mass spectrometry was employed to determine the quantity and sulfation patterns of circulating glycosaminoglycans. We found that circulating heparan sulfate fragments were significantly (23-fold) elevated in patients with indirect lung injury, while circulating hyaluronic acid concentrations were elevated (32-fold) in patients with direct lung injury. N-Sulfation and tri-sulfation of heparan disaccharides were significantly increased in patients with indirect lung injury. Chondroitin disaccharide sulfation was suppressed in all groups with respiratory failure. Plasma heparan sulfate concentrations directly correlated with intensive care unit length of stay. Serial plasma measurements performed in select patients revealed that circulating highly sulfated heparan fragments persisted for greater than 3 days after the onset of respiratory failure. Our findings demonstrate that circulating glycosaminoglycans are elevated in patterns characteristic of the etiology of respiratory failure and may serve as diagnostic and/or prognostic biomarkers of critical illness.