Synthesis and release of chondroitin sulphate and heparan sulphate proteoglycans from mouse macrophagesin vitro

Macrophages were obtained from the mouse peritoneal cavity and culturedin vitro. The cells were exposed to³⁵S-sulphate for 20 h, and labelled proteoglycans were recovered from both medium and cell fractions by sodium dodecylsulphate solubilization. The cell fraction contained both proteoglycans and glycosaminoglycans, whereas only intact proteoglycans could be recovered from the medium fraction. ³⁵S-Glycosaminoglycans isolated from cell and medium fractions by papain digestion were shown to contain approximately 25% heparan sulphate and 75% galactosaminoglycans comprising 55% chondroitin sulphate and 20% dermatan sulphate. The galactosaminoglycans were shown by paper chromatography to contain more than 95% 4-sulphated units. Pulse-chase experiments showed that approximately 80% of the cell-associated material was released within 6 h of incubation.³⁵S-Proteoglycans released did not bind to the macrophages, but were recovered in a soluble form from the culture medium.Abbreviations CSPG chondroitin sulphate proteoglycan - HSPG heparan sulphate proteoglycan - SDS sodium dodecylsulphate - DME Dulbecco's Minimum Essential Medium - GAG glycosaminoglycan     

Differences in the distribution of O-sulphate groups of cell-surface and secreted heparan sulphate produced by human neuroblastoma cells in culture

Confluent cultures of a human neuroblastoma cell line (CHP100) were incubated for 48 h with d-[1-³H]glucosamine and sodium [³⁵S]sulphate. Radioactive glycosaminoglycans were analysed in the growth medium, rapid trypsin digest of the cell monolayer and a 1% (w/v) Triton/0.5 M NaOH extract of the final cell pellet. Sulphated glycosaminoglycans co-chromatographed when eluted by NaCL gradient from DEAE-cellulose. The medium contained mainly chondroitin sulphates, whereas the cell surface was enriched in heparan sulphate. Heparan sulphate was isolated as chondroitinase ABC-resistant material and treated with nitrous acid. Analysis of the scission products on Bio-Gel P-10 yielded fragments varying in size from single disaccharides to glycans consisting of nine disaccharide units. Cell-surface and medium heparan sulphate had respectively 52% and 54% N-sulphated glucosamine residues distributed in similar patterns along the polymer chain. The N:O-sulphate ratio of neuroblastoma heparan sulphate was 1.1:1. Analysis by high-voltage electrophoresis of di- and tetrasaccharide products produced by nitrous acid treatment showed that the distribution of $\text{‘O’-}\text{sulphate}$ groups differed strikingly between heparan sulphates from the medium and cell-surface compartments. A di-O-sulphated tetrasaccharide was identified in both heparan sulphate species. The absence of detectable amounts of ³⁵[S]sulphate associated with fragments larger than tetrasaccharide supports the close topographical association of N-sulphate and O-sulphate groups.     

Heterogeneity of cell-associated and secretory heparan sulphate proteoglycans produced by cultured human neuroblastoma cells

Biosynthetically radiolabelled heparan sulphate proteoglycans have been isolated from the growth medium and the cell lysate of a human neuroblastoma cell line (CHP100). Chromatography on Sepharose CL-4B identified two heparan sulphate proteoglycans in the medium (K_av 0.220 and 0.3890, whereas in the cell lysate the major proteoglycan species were more heterogenous and of a smaller overall molecular size (K_av 0.407) than the medium-derived counterparts. Chromatography on Sepharose CL-6B of free heparan sulphate glycosaminoglycan chains showed that the majority of cell-layer-derived material heparan sulphate 2, K_av=0.509) was smaller than medium heparan sulphates (heparan sulphate 1 and heparan sulphate 2, K_av 0.230 and 0.317). Analysis of the patterns of polymer sulphation by nitrous acid treatment, gel chromatography and high-voltage electrophoresis established that in each heparan sulphate fraction there was on average 1.1 sulphate residues per disaccharide with an N:O sulphate ratio of 1.1 Heparan sulphate in the medium had a high proportion of di-O-sulphated disaccharides in regions of the chain with repeat disaccharide sequences of structure GlcA-GlcNSO₃, whereas cell-associated material was enriched in di-O-sulphated tetrasaccharides of alternating sequences GlcA-GlcNAc-GlcA-GlcNSO₃. The identification of several populations of heparan sulphate proteoglycans differing in molecular size and glycosaminoglycan fine structure may reflect the functional diversity of this family of macromolecules in the nervous system.     

人主动脉硫酸乙酰肝素蛋白聚糖对培养人的脐静脉内皮细胞生长的影响

为探讨硫酸乙酰肝素蛋白聚糖（ＨＳＰＧ）对内皮细胞生长的作用,用解聚提取及离子交换柱层析法分离出人主动脉ＨＳＰＧ,用倒置显微镜、细胞计数、及￣３Ｎ－ＴｄＲ参入观察其对培养的第一代人脐静脉内皮细胞（ｈＵＶＦＣ）生长的影响。结果发现：（１）倒置显微镜下观察,加入ＨＳＰＧ（１．７０μｇ已糖醛酸／ｍｌ）的ｈＵＶＥＣ生长密度高于对照组（未加ＨＳＰＧ）．（２）随着培养时间增加（２４,４８及７２ｈ）．根据细胞计数计算出同一剂量的ＨＳＰＧ（１７．０μｇ已糖醛酸／ｍｌ）对ｈＵＶＥＣ的促增殖％增高（分别为１４％,３０％及３７％）。（３）随着加入ＨＳＰＧ浓度的升高（４．３,８．５及１７．０μｇ已糖醛酸／ｍｌ．培养７２ｈ）．根据￣３Ｈ－ＴｄＲ参入计算出ＨＳＰＧ对ｈＵＶＥＣ的促增殖％亦增高（分别为４９％,７１％及９８％）。故人主动脉ＨＳＰＧ对培养的人脐静脉内皮细胞有促增殖作用。     

Isolation and characterization of membrane-associated proteoglycans from normal and malignant human mammary epithelial cells

The plasma membrane-associated proteoglycans of a malignant human breast cell line (MDA-MB-231) were compared with the corresponding proteoglycans from a normal cell line (HBL-100). The labeled proteoglycans were isolated from the plasma membranes of cells grown in the presence of [³H]glucosamine and [³⁵S]Na₂SO₄ by extraction with guanidine hydrochloride and subsequently purified by DEAE-ion exchange chromatography. Their structural properties were established by treatment with nitrous acid, heparitinase and chondroitinase ABC, and by gel filtration before and after alkaline -elimination. About 18% of the proteoglycans synthesized by these cell lines were associated with the plasma membranes. The HBL plasma membranes contained 80% heparan sulfate and 20% chondroitin sulfate proteoglycans whereas MDA plasma membranes had 50% heparan sulfate and 50% chondroitin sulfate proteoglycans. The MDA plasma membrane contained two heparan sulfate proteoglycans, both having nearly the same molecular size as the two species secreted into the medium by these cells. The HBL plasma membrane also contained two hydrodynamic size heparan sulfate proteoglycans. The larger hydrodynamic size species has a slightly lower molecular size than that secreted into the medium, and the smaller hydrodynamic size species was not detectable in the medium. Even though the major chondroitin sulfate proteoglycans from MDA plasma membranes were smaller in size than those from HBL plasma membrane, a larger proportion of the glycosaminoglycan chains of the former were bigger than those from the latter.Abbreviations CHAPS 3-[(3-cholamidopropyl)dimethylammonio]propane-1-sulfonate - Di-OS 2-acetamido-2-deoxy-3-O-(-d-gluco-4-ene-pyranosyluronic acid)-d-galactose - Di-4S 2-acetamido-2-deoxy-3-O-(-d-gluco-4-ene-pyranosyluronic acid)-4-O-sulfo-d-galactose - Di-6S 2-acetamido-2-deoxy-3-O-(-d-gluco-4-ene-pyranosyluronic acid)-6-O-sulfo-d-galactose - Gdn-HCl guanidine hydrochloride - WGA wheat germ agglutinin     

Bovine aorta contains at least two related forms of heparan sulphate proteoglycan

• 1.1. The proteoglycan peak from anion exchange chromatography of an extract of bovine aorta was digested with chondroitinase ABC. The residual heparan sulphate proteoglycans were further purified by chromatography on Sepharose CL4B and DEAE-Sephacel to yield two species, of high and low charge density.
• 2.2. Higher molecular weight material had a higher proportion of high charge density proteoglycan, while the lower molecular weight species had a higher proportion of low charge density heparan sulphate proteoglycan.
• 3.3. The two species shared epitopes as they both reacted with an antibody to heparan sulphate proteoglycan from bovine glomerular basement membrane.
• 4.4. On electron microscopy, both high and low charge density proteoglycans were visualized as ‘tadpole-like’ molecules, which showed a tendency to aggregate via their globular heads.
• 5.5. Bovine aortic smooth muscle cells were cultured in the presence of [³⁵S]sulphate and [³H]glucosamine. Proteoglycans were isolated from medium and cell layer extract by the methods outlined above.
• 6.6. The major HSPG species isolated from medium were significantly larger than those from cell layer and displayed substantial heterogeneity in both size of HS chain after papain digestion and size of protein core after heparitinase digestion. 7. The major cell layer species yielded two HS species of widely differing mol. wt after papain digestion, and a very small protein core after heparitinase digestion. Therefore cell layer-associated HSPGs show a good deal more homogeneity than those found in the medium.
• 7.8. Further ion-exchange chromatography after digestion with chondroitinase ABC revealed HSPG species of lower charge density, possibly derived from a hybrid chondroitin sulphate-dermatan sulphate proteoglycan (CS/DSPG) after removal of the CS/DS chains.

     

Effect of low-density lipoproteins on the synthesis and secretion of proteoglycans by human endothelial cells in culture.

We studied the effect of low-density lipoproteins (LDL) on the synthesis and secretion of proteoglycans by cultured human umbilical-vein endothelial cells. Confluent cultures were incubated with [35S]sulphate or [3H]glucosamine in lipoprotein-deficient serum in the presence and in the absence (control) of LDL (100-400 micrograms/ml), and metabolically labelled proteoglycans in culture medium and cell layer were analysed. LDL increased accumulation of labelled proteoglycans in medium and cell fractions up to a concentration of 200 micrograms/ml. At this concentration of LDL the accumulations of proteoglycans in medium and cell layer were 65% and 32% respectively above control for 35S-labelled proteoglycans, and 55% and 28% respectively above control for 3H-labelled proteoglycans. At concentrations above this LDL was found to depress the accumulation of proteoglycans in medium and cell layer. Gel filtration on Sepharose CL-4B showed that in both control and LDL-treated cultures the cell layer contained a large (Kav. = 0) and a small (Kav. = 0.35) heparan sulphate proteoglycan, whereas the culture medium contained a large heparan sulphate proteoglycan (Kav. = 0) and a smaller isomeric chondroitin sulphate proteoglycan (control, Kav. = 0.35; LDL-treated, Kav. = 0.17). The relative increase in hydrodynamic size of the isomeric chondroitin sulphate proteoglycan (Mr 150,000 compared with 90,000) in the medium of cultures exposed to LDL was partly attributable to the larger size of the glycosaminoglycan side chains (Mr 39,000 compared with 21,000). The isomeric chondroitin sulphate proteoglycan in LDL-treated culture was relatively enriched in chondroitin 6-sulphate compared with that in control cultures (39% compared with 29%). Pulse-chase studies showed that LDL treatment did not alter the turnover rate of proteoglycans as compared with controls, implying that the elevation in proteoglycan accumulation in LDL-treated cultures was due to enhanced synthesis. These results demonstrate that LDL can modulate proteoglycan synthesis by cultured vascular endothelial cells, resulting in the secretion of a larger isomeric chondroitin sulphate proteoglycan enriched in chondroitin 6-sulphate.     

Structural requirements in heparin for binding and activation of FGF-1 and FGF-4 are different from that for FGF-2

Size- and structure-defined oligosaccharides from heparin, 2-O-desulphated(2-O-DS-) heparin, 6-O-desulphated (6-O-DS-) heparin, carboxy-reduced(CR-) heparin, and carboxyamidomethylsulphonated (AMS-) heparinwere utilized in characterizing the structural properties ofheparin to specifically bind to basic fibroblast growth factor(FGF-2) and to modulate the mitogenic activity of FGF-2 (Ishihara,M.et al., Glycobiology, 4, 451–458, 1994). The previousresults showed that both 2-O-sulphate groups and the negativecharge of the carboxy group in iduronate residues are requiredfor specific interaction with FGF-2, but the 6-O-sulphate groupsin N-sulphated glucosamine (GlcNS) residues do not influencethe interaction with FGF-2. In the present study, the same oligosaccharideswere fractionated on a FGF-1- or FGF-4-affinity column, andwere assessed as promotors of FGF-1- or FGF-4-induced proliferationof adrenocortical endothelial (ACE) cells and chlorate-treatedACE cells. The present results suggest that the smallest heparin-derivedoligosaccharide binding to these growth factors with the highestaffinity and promoting their mitogenic activities is a fullyN-sulphated decasaccharide enriched in 2-O- and 6-O- sulphateddisaccharide units. In contrast to our results with FGF-2, ahigh content of 6-O-sulphate groups in GlcNS residues is requiredfor specific interaction with FGF-1 and FGF-4. FGF-1 FGF-4 heparin heparan sulphate oligosaccharides     

Co-polymeric glycosaminoglycans in transformed cells. Transformation-dependent changes in the co-polymeric structure of heparan sulphate

1. Heparan sulphates from normal 3T3 fibroblasts are association-prone as indicated by their affinity for agarose gels substituted with cognate heparan sulphate species. Heparan sulphates from SV40-transformed or polyoma-virus-transformed cells have no affinity for the same gels. 2. Heparan sulphates from the medium, the pericellular and intracellular pools of normal, SV40-transformed and polyoma-transformed 3T3 cells were separated into four subfractions (HS1–HS4) by ion-exchange chromatography. In general, HS1–HS3 were found in cell-derived heparan sulphates, whereas HS3–HS4 were present in the medium. The heparan sulphates from transformed cells were more heterogeneous and of lower charge density than those from the normal counterpart. 3. Degradations via periodate oxidation/alkaline elimination yielded the oligomers glucosamine-(hexuronate–glucosamine)_n-R with n=1–5 and a large proportion of N-sulphate groups. There was a large contribution of fragments n=4–5 from heparan sulphates of normal cells. These fragments were less common in low-sulphated heparan sulphates of transformed cells. In the case of medium-drived heparan sulphates all species had a low content of fragments n=4–5. 4. The size distribution of (glucuronate–N-acetylglucosamine)_n regions was assessed after deaminative cleavage. It was broad and ranged from n=1–10 for all heparan sulphate species. In the case of medium-derived heparan sulphates there were distinct differences between normal and transformed cells. In the latter chains the N-acetyl-rich segments were both shorter and longer than in the normal case. The shape of the disaccharide peak was consistent with a lower content of O-sulphate in the heparan sulphates from transformed cells. 5. It was concluded that heparan sulphates from medium or transformed cells exhibit the greatest structural deviation from the normal case. The finding of lower proportions of extended, iduronate/glucuronate-bearing, N-sulphate-rich segments in heparan sulphates of transformed cells was particularly interesting in view of the fact that these elements have been associated with ability to self-interact.     

Isolation of heparan sulfate proteoglycan from beneath the monolayers of rat hepatocytes and its binding to type IV collagen

Primary cultures of rat hepatocytes maintained as monolayer in a serum-free medium synthesise and secrete sulphated proteoglycans. Nearly 5% of the total 35(S)-sulphated material was obtained in a soluble form from beneath the cell layer. A shift in gel filtration pattern on beta-elimination with alkali suggested that it is a sulphated proteoglycan. On ion exchange chromatography over Dowex AG 1 x 2, the major fraction was eluted with 1.25 M NaCl. Further, nearly 80% of the 35(S)-labeled material was susceptible to nitrous acid degradation and more than 90% of the material was resistant to chondroitinase ABC digestion suggesting that it is predominantly a heparan sulphate proteoglycan (HSPG). Since HSPG is a major component of basement membrane, its binding with collagen was studied by a solid phase binding assay. About 75% of the 35(S) HSPG bound to wells coated with type IV collagen whereas only about 20% bound to type I collagen at physiological pH. Binding to collagen IV was reduced by about 50% when free GAG chains were used indicating that the protein core is also involved in interaction with the collagen. These results indicate the possible role of this basal extracellular heparan sulphate proteoglycan in the basal lamina formation.     

Isolation and characterization of free glycans of the oligomannoside type from the extracellular medium of a plant cell suspension

The oligosaccharides Man₅GlcNAc and Man₃(Xyl)GlcNAc(Fuc)GlcNAc presumed to originate fromN-glycosyl proteins have been purified from an extracellular medium (concentration: 2–5 mg/l of 14 day cultures) of white campion (Silene alba) suspension culture. Their primary structures have been determined by¹H-400-MHz NMR spectroscopy and FAB-MS spectrometry. They are probably the result of an autophagic process including protein catabolism due to sucrose starvation. Additional identification of digalactosylglycerol (galactolipid breakdown) argues for this hypothesis.Abbreviations Fuc l-fucose - Man d-mannose - Xyl d-xylose - GlcNAc N-acetyl-d-glucosamine - Gal d-galactose - Glc d-glucose - FAB-MS fast atom bombardment mass spectrometry - NMR nuclear magnetic resonance     

Glycosaminoglycan—lipoprotein interactions: 2. Structure of heparin-releted variants with high affinity for low density lipoprotein

A particular heparan sulphate fraction which possessed the largest proportion of high affinity variants for human low density lipoprotein contained almost equal proportions of the repeating units l-iduronosyl(O-sulphate)N-sulphamidoglucosamine and d-glucoronosyl-N-acetylglucosamine. The heparan sulphate was fractionated on lipoprotein-agarose into three populations. Results of periodate oxidation—alkaline elimination indicated that the size of the completely N-sulphated block regions increased with increasing affinity. In contrast, the number of consecutive l-iduronosyl(O-sulphate)-containing repeats decreased with increasing affinity towards lipoprotein. After selective periodate oxidation—alkaline scission of d-glucoronic acid residues only a portion of the heparan sulphate fragments retained high affinity for lipoprotein. This portion consisted of fragments larger than dodecasaccharide which contained both l-iduronic acid-O-sulphate and non-sulphated uronic acid residues (−) 2:1). No affinity or little affinity was displayed by fragments (of comparable size) that contained only sulphated l-iduronic acid residues.     

N-acetylglucosaminyltransferase substrates prepared from glycoproteins by hydrazinolysis of the asparagine-N-acetylglucosamine linkage. Purification and structural determination of oligosaccharides with mannose andN-acetylglucosamine at the non-reducing termini

Sixteen asparagine-linked oligosaccharides ranging in size from (Man)₂(GlcNAc)₂ (Fuc)₁ to (GlcNAc)₆(Man)₃(GlcNAc)₂ were obtained from human ₁-acid glycoprotein and fibrinogen, hen ovomucoid and ovalbumin, and bovine fetuin, fibrin and thyroglobulin by hydrazinolysis, mild acid hydrolysis and glycosidase treatment. The oligosaccharides hadN-acetylglucosamine at the reducing termini and mannose andN-acetylglucosamine residues at the non-reducing termini and were prepared for use asN-acetylglucosaminyltransferase substrates. Purification of the oligosaccharides involved gel filtration and high performance liquid chromatography on reverse phase and amine-bonded silica columns. Structures were determined by 360 MHz and 500 MHz proton nuclear magnetic resonance spectroscopy, fast atom bombardment-mass spectrometry and methylation analysis. Several of these oligosaccharides have not previously been well characterized.Abbreviations bis bisecting GlcNAc - DMSO dimethylsulfoxide - FAB fast atom bombardment - Fuc l-fucose - Gal d-galactose - GLC gas-liquid chromatography - GlcNAc or Gn N-acetyl-d-glucosamine - HPLC high performance liquid chromatography - Man or M d-mannose - MES 2-(N-morpholino)ethanesulfonate - MS mass spectrometry - NMR nuclear magnetic resonance - PIPES piperazine-N,N-bis(2-ethane sulfonic acid) the nomenclature of the oligosaccharides is shown in Table 1.     

The transfer of platelet factor 4 from its proteoglycan carrier to natural and synthetic polymers

1. Transfer of dansyl-platelet factor 4 complexed with a series of glycosaminoglycans to heparin has been detected and studied by measuring changes in the anisotropy of the dansyl fluorescence. The protein was most easily transferred from chondroitin sulphate and least easily from heparan sulphaet. 2. The transfer of the dye-labelled protein from its biological chondroitin 4-sulphate proteoglycan carrier to natural and synthetic anionic polymers was similarly followed. The transfer to heparin and dermatan sulphate was shown to be the same whether 3 mM Ca²⁺ or 8 mM EGTA was present in the solution. 3. The shapes of the binding curves of the dansyl-factor to the polymers have been compared at I = 0.4 M. 4. The observed changes in anisotropy of dye fluorescence have been correlated with the charge density and the stereochemistry of the charged groups of the natural polymers. Large complexes are observed with polymers of high negative charge/weight ratios. Less charged polymers containing disaccharide units of iduronic acid and glucosamine N-sulphate will also form large complexes at I = 0.15 M. 5. It is demonstrated that the release of a platelet factor 4 proteoglycan complex in vivo would result in the transfer of the protein to heparin, moderate quantities of either dermatan or heparan sulphates would not prevent this transfer.     

Structural features in heparin which modulate specific biological activities mediated by basic fibroblast growth factor

The biological activity of basic fibroblast growth factor (bFGF)is influenced greatly by direct binding to heparin and heparansulphate (HS). Heparin-derived oligosaccharides have been utilizedto determine the structural requirements present in the polymerthat account for bind ing to bFGF. We had previously demonstratedthat fragments >6 mer can inhibit the interaction betweencell surface heparan sulphate proteoglycan (HSPG) and bFGF,and bFGF-induced proliferation of adrenocortical endothelial(ACE) cells. In contrast, oligosaccharides > 10 mer can enhancethe binding of bFGF to its high-affinity receptor or supportbFGF-induced mitogenesis in ACE cells (Ishihara et al., J. Biol.Chem., 268, 4675–4683, 1993). We have extended these studiesto size- and structure-defined oligosaccharides from heparin,2-O-desulphated (2-O-DS-) heparin, 6-O-desulphated (6-O-DS-)heparin, carboxyreduced (CR-) heparin and carboxy-amidomethylsulphonated(AMS-) heparin. Oligosaccharides from these polymers were fractionatedon a bFGF-affinity column and were assessed as inhibitors orenhancers of specific bFGF-derived biological activities. Theresults of these studies indicate that both 2-O-sulphate andthe negative charge of the carboxy group [L-iduronic acid (IdoA)residues] are required for specific interactions of heparin-derivedoligosaccharides with bFGF and for modulation of bFGF mitogenicactivity. In addition, the charge of the carboxy groups in uronicacids can be replaced by other functional groups with a negativecharge, such as the amidomethyl sulphonate moiety describedhere. basic fibroblast growth factor heparan sulphate heparin oligosaccharides     

N-Acetyl-4-deoxy-d-neuraminic acid is activated and transferred on to asialoglycoprotein

The sialic acid analogue,N-acetyl-4-deoxy-neuraminic acid, is readily activated by CMP-sialic acid synthase from bovine brain. We also show that sialyl-transfer from CMP-N-acetyl-4-deoxy-neuraminic acid to asialo- ₁-acid glycoprotein is achieved at a high rate using Gal1-4GlcNAc (2.6)-sialyltransferase from rat liver.In contrast toVibrio cholerae sialidase, fowl plague virus sialidase liberates boundN-acetyl-4-deoxy-neuraminic acid from the glycoprotein. Thus, as opposed to the general view, the action of neither synthase nor transferase depends on the presence of the hydroxy group at C-4 ofN-acetylneuraminic acid.Abbrevations BSA bovine serum albumin - DTE dithioerythritol - HPLC high performance liquid chromatography - NeuAc N-acetyl-d-neuraminic acid - 4-deoxy-NeuAc N-acetyl-4-deoxy-d-neuraminic acid - 4-epi-NeuAc 4-acetamido-3,5-dideoxy-d-glycero-d-talononulosonic acid - CMP-NeuAc Cytidine-5-monophospho-N-acetylneuraminic acid - CMP-4-deoxy-NeuAc Cytidine-5-monophospho-N-acetyl-4-deoxy-neuraminic acid - FPV-sialidase Fowl plague virus sialidase - VCN Vibrio cholerae neuraminidase     

Purification and characterization of sialidase fromClostridium sordellii G12

Sialidase secreted by the urease-positiveClostridium sordellii strain G12 was isolated from culture medium and purified to apparent homogeneity as estimated by Fast Protein Liquid Chromatography (FPLC) and sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE). For this purpose, ion-exchange chromatography, gel filtration, isoelectric focusing, and FPLC on ion-exchange resin and gel filtration materials were used. The sialidase was purified 159 300-fold from 5 l of culture medium, yielding 9 g of enzyme protein with a specific activity of 480 U/mg. For the denatured (SDS-PAGE) and native (FPLC) sialidase relative molecular masses of 40 000 and 38 500 Da, respectively, were estimated. The substrate specificity, kinetic data, and pH-optimum of the enzyme are similar to those of other bacterial sialidases. The influences of salt or serum proteins on enzyme activity are of interest.Abbreviations MU-Neu5Ac 4-methylumbelliferyl -d-N-acetylneuraminic acid - Ganglioside GD1a IV³NeuAc, ll³NeuAc-GgOse₄Cer - Neu5Ac2en 2-deoxy-2,3-didehydro-N-acetylneuraminic acid     

Biosynthesis of heparan sulfate

Microsomal preparations from Englebreth-Holm-Swarm mouse sarcoma were incubated with UDP-N-acetyl[³H] glucosamine and UDP-[¹⁴C]glucuronic acid to form proteoglycan containing [³H,¹⁴C]glycosaminoglycan with equimolar amounts of [³H]glucosamine and [¹⁴C]glucuronic acid. The labelled glycosaminoglycan was totally resistant to degradation by testicular hyaluronidase, but could be degraded readily by a crudeFlavobacter heparinum enzyme preparation which is capable of degrading heparin and heparan sulfate. Chromatography of the [³H,¹⁴C]glycosaminoglycan on DEAE-cellulose provided a pattern with three peaks: the first appearing before hyaluronic acid, the second and largest appearing at the site of hyaluronic acid, and a third appearing slightly beyond hyaluronic acid but before a standard of chondroitin sulfate. When 3-phosphoadenosine 5-phosphosulfate was also included in the reaction mixture, a change appeared in the [³H,¹⁴C]glycosaminoglycan so that chromatography on DEAE-cellulose presented a pattern with a significant amount of material which cochromatographed in the area where heparan sulfate would be found. There was no material that co-chromatographed with the more highly sulfated substance, heparin. This indicates that the microsomal preparation from the Englebreth-Holm-Swarm sarcoma is capable of producing a heparan sulfate-like molecule and is controlled in its sulfation of precursors so that heparin is not formed.     

Interaction between heparan sulphate chains II. Structural characterization of iduronate- and glucuronate-containing sequences in aggregating chains

A heparan sulphate fraction (uronic acid composition: 20% sulphated iduronate, 15% iduronate and 65% glucuronate of total uronate) was separated into aggregating and non-aggregating chains by gel chromatography. ¹³C-NMR analyses revealed that non-aggregating chains had a higher degree of sulphation than did aggregating chains. In aggregating chains, there was more N-acetyl-glucosamine than N-sulphamidoglucosamine; the extent of C-6 sulphation of the latter moiety was low and most of the iduronate residues were non-sulphated. In non-aggregating chains, the N-acetyl-to-N-sulphate ratio was approx. 2 : 1, the N-sulphated glucosamines were also largely C-6 sulphated and the sulphated iduronates were concentrated to these species.Both preparations were subjected to deaminative cleavage which produces fragments like uronate-(N-acetylglucosamine-uronate)_n-anhydromannose. Tetrasaccharides (n = 1) were further fractionated into non-, mono-, di- and trisulphated species by ion-exchange chromatography. The tetrasaccharides have the general carbohydrate structure uronate-N-acetylglucosamine-glucuronate-anhydromannose. Non-reducing terminal glucuronate was removed by β-glucuronidase. The results showed that saccharides containing glucuronate in both positions were more prevalent in the products of aggregating chains. The β-glucuronidase-resistant saccharides (carrying either sulphated or non-sulphated iduronate in non-reducing terminal position) were oxidised with periodate under conditions where non-sulphated residues are degraded, whereas sulphated residues are resistant. Mono-sulphated and di-sulphated tetrasaccharides from aggregating chains were extensively degraded indicating that iduronate-N-acetylglucosamine-glucuronate-anhydromannose was the major sequence.In saccharides from non-aggregating chains iduronate was frequently sulphated. The results of this and previous investigations (Fransson, L.-Å., Nieduszynski, I.A. and Sheehan, J.K. (1980) Biochim. Biophys. Acta 630, 287–300) indicate that an alternating arrangement of iduronate and glucuronate in aggregating chains is present both in N-sulphated block regions and in regionsthat carry alternating N-acetyl- and N-sulphated glucosamine.     

Comparative study on glycosaminoglycans synthesized in peripheral and peritoneal polymorphonuclear leucocytes from guinea pigs.

Glycosaminoglycans synthesized in polymorphonuclear (PMN) leucocytes isolated from blood (peripheral PMN leucocytes) and in those induced intraperitoneally by the injection of caseinate (peritoneal PMN leucocytes) were compared. Both peripheral and peritoneal PMN leucocytes were incubated in medium containing [35S]sulphate and [3H]glucosamine. Each sample obtained after incubation was separated into cell, cell-surface and medium fractions by trypsin digestion and centrifugation. The glycosaminoglycans secreted from peripheral and peritoneal PMN leucocytes were decreased in size by alkali treatment, indicating that they existed in the form of proteoglycans. Descending paper chromatography of the unsaturated disaccharides obtained by the digestion of glycosaminoglycans with chondroitinase AC and chondroitinase ABC identified the labelled glycosaminoglycans of both the cell and the medium fractions in peripheral PMN leucocytes as 55-58% chondroitin 4-sulphate, 16-19% chondroitin 6-sulphate, 16-19% dermatan sulphate and 6-8% heparan sulphate. Oversulphated chondroitin sulphate and oversulphated dermatan sulphate were found only in the medium fraction. In peritoneal PMN leucocytes there is a difference in the composition of glycosaminoglycans between the cell and the medium fractions; the cell fraction was composed of 60% chondroitin 4-sulphate, 5.5% chondroitin 6-sulphate, 16.8% dermatan sulphate and 13.9% heparan sulphate, whereas the medium fraction consisted of 24.5% chondroitin 4-sulphate, 28.2% chondroitin 6-sulphate, 33.7% dermatan sulphate and 10% heparan sulphate. Oversulphated chondroitin sulphate and oversulphated dermatan sulphate were found in the cell, cell-surface and medium fractions. On the basis of enzymic assays with chondro-4-sulphatase and chondro-6-sulphatase, the positions of sulphation in the disulphated disaccharides were identified as 4- and 6-positions of N-acetylgalactosamine. Most of the 35S-labelled glycosaminoglycans synthesized in peripheral PMN leucocytes were retained within cells, whereas those in peritoneal PMN leucocytes were secreted into the culture medium. Moreover, the amount of glycosaminoglycans in peritoneal PMN leucocytes was significantly less than that in peripheral PMN leucocytes. Assay of lysosomal enzymes showed that these activities in peritoneal PMN leucocytes were 2-fold higher than those in peripheral PMN leucocytes.