Birgitta Havsmark

Prion or amyloid-b-derived Cu(II)- or free Zn(II)-ions support S-nitroso-dependent autocleavage of glypican-1 heparan sulfate.

Copper are generally bound to proteins, e.g. the prion and the amyloid β proteins. We have previously shown that copper ions are required to nitrosylate thiol groups in the core protein of glypican-1, a heparan sulfate-substituted proteoglycan. When S-nitrosylated glypican-1 is then exposed to an appropriate reducing agent, such as ascorbate, nitric oxide is released and autocatalyzes deaminative cleavage of the glypican-1 heparan sulfate side chains at sites where the glucosamines are N-unsubstituted. These processes take place in a stepwise manner, whereas glypican-1 recycles via a caveolin-1-associated pathway where copper ions could be provided by the prion protein. Here we show, by using both biochemical and microscopic techniques, that (a) the glypican-1 core protein binds copper(II) ions, reduces them to copper(I) when the thiols are nitrosylated and reoxidizes copper(I) to copper(II) when ascorbate releases nitric oxide; (b) maximally S-nitrosylated glypican-1 can cleave its own heparan sulfate chains at all available sites in a nitroxyl ion-dependent reaction; (c) free zinc(II) ions, which are redox inert, also support autocleavage of glypican-1 heparan sulfate, probably via transnitrosation, whereas they inhibit copper(II)-supported degradation; and (d) copper(II)-loaded but not zinc(II)-loaded prion protein or amyloid β peptide support heparan sulfate degradation. As glypican-1 in prion null cells is poorly S-nitrosylated and as ectopic expression of cellular prion protein restores S-nitrosylation of glypican-1 in these cells, we propose that one function of the cellular prion protein is to deliver copper(II) for the S-nitrosylation of recycling glypican-1.

Involvement of glycosylphosphatidylinositol-linked ceruloplasmin in the copper/zinc-nitric oxide-dependent degradation of glypican-1 heparan sulfate in rat C6 glioma cells

The core protein of glypican-1, a glycosylphosphatidylinositol-linked heparan sulfate proteoglycan, can bind Cu(II) or Zn(II) ions and undergo S-nitrosylation in the presence of nitric oxide. Cu(II)-to-Cu(I)-reduction supports extensive and permanent nitrosothiol formation, whereas Zn(II) ions appear to support a more limited, possibly transient one. Ascorbate induces release of nitric oxide, which catalyzes deaminative degradation of the heparan sulfate chains on the same core protein. Although free Zn(II) ions support a more limited degradation, Cu(II) ions support a more extensive self-pruning process. Here, we have investigated processing of glypican-1 in rat C6 glioma cells and the possible participation of the copper-containing glycosylphosphatidylinositol-linked splice variant of ceruloplasmin in nitrosothiol formation. Confocal microscopy demonstrated colocalization of glypican-1 and ceruloplasmin in endosomal compartments. Ascorbate induced extensive, Zn(II)-supported heparan sulfate degradation, which could be demonstrated using a specific zinc probe. RNA interference silencing of ceruloplasmin expression reduced the extent of Zn(II)-supported degradation. In cell-free experiments, the presence of free Zn(II) ions prevented free Cu(II) ion from binding to glypican-1 and precluded extensive heparan sulfate autodegradation. However, in the presence of Cu(II)-loaded ceruloplasmin, heparan sulfate in Zn(II)-loaded glypican-1 underwent extensive, ascorbate-induced degradation. We propose that the Cu(II)-to-Cu(I)-reduction that is required for S-nitrosylation of glypican-1 can take place on ceruloplasmin and thereby ensure extensive glypican-1 processing in the presence of free Zn(II) ions.

The substrate specificity of heparan sulphate lyase and heparin lyase from Flavobacterium heparinum

Abstract The substrate specificity of heparan sulphate lyase and heparin lyase has been examined by using various oligosaccharides produced by deaminative cleavage or periodate oxidation—base-catalysed elimination of heparan sulphate. The saccharides were separated by gel and ion-exchange chromatography into fractions having high proportions of the following linkages: 2-acetamido-2-deoxy- d -glucosyl→ d -glucuronic acid (type I), 2-deoxy-2-sulphoamino- d -glucosyl→ d -glucuronic acid (type II), 2-deoxy-2-sulphoamino- d -glucosyl→ l -iduronic acid (type III), or 2-deoxy-2-sulphoamino- d -glucosyl→2- O -sulpho- l -iduronic acid (type IV). Heparan sulphate lyase cleaved heparan sulphate preparations containing high proportions of type I linkages. In contrast, oligosaccharides obtained after deaminative cleavage and rich in the type I linkage were poor substrates; longer saccharides were better substrates than shorter ones. Saccharides generated via periodate oxidation were cleaved when the type I linkage was present but resistant when the linkages were of types II-IV. The presence of ester sulphate on the 2-acetamido-2-deoxy- d -glucose residue in a type I linkage seems to hinder the enzyme. Heparan sulphate lyase was able to degrade intact chains to the same extent as did two cycles of periodate oxidation—base-catalysed elimination, suggesting that all the regular type I linkages were accessible to the enzyme. The heparin lyase could cleave saccharides which contained type IV linkages. When type II and III linkages were the only ones present, the saccharides were resistant. This enzyme also appears to require ester-sulphation of the 2-deoxy-2-sulphoamino- d -glucose residue.

Sequence analysis of p-hydroxyphenyl-O-beta-D-xyloside initiated and radio-iodinated dermatan sulfate from skin fibroblasts.

To generate xyloside-primed dermatan sulfate suitable for sequence analysis, skin fibroblasts were incubated withp-hydroxyphenyl-β-d-xylopyranoside and [3H]galactose, and free [3H]glycosaminoglycan chains were isolated from the culture medium by ion exchange and gel chromatography. After125I labelling of their reducing-terminal hydroxyphenyl groups, chains were subjected to various chemical and enzymatic degradations, both partial and complete, followed by gradient polyacrylamide gel electrophoresis and autoradiographic identification of fragments extending from the labelled reducing-end to the point of cleavage. Results of periodate oxidation-alkaline scission indicated that the xylose moiety remained unsubstituted at C-2/C-3; exhaustive treatment with chondroitin AC-I lyase afforded the fragment ΔHexA-Gal-Gal-Xyl-R (R = radio-iodinated hydroxyphenyl group), and complete degradations with chondroitin ABC lyase as well as testicular hyaluronidase yielded the fragments ΔHexA/HexA-GalNAc-GlcA-Gal-Gal-Xyl-R with or without sulfate on theN-acetylgalactosamine. Partial digestions with testicular hyaluronidase or chondroitin B lyase indicated that glucuronic acid was common in the first three repeats after the linkage region and that iduronic acid could occupy any position thereafter. Hence, there were no indications of a repeated, periodic appearance of the clustered GlcA-GalNAc repeats which was previously observed in proteoglycan derived dermatan sulfate [Fransson L-Å, Havsmark B, Silverberg I (1990)Biochem J269:381–8], suggesting a role for the protein part in controlling the formation of particular copolymeric features during glycosaminoglycan assembly.

Structural requirements for heparan sulphate self-association

To investigate heparan sulphate self-association, various sub-fractions of beef-lung heparan sulphate have been subjected to affinity chromatography on heparan sulphate-agarose. A particular variant of heparan sulphate was chiefly bound to matrices substituted with the same or cognate heparan sulphates. N-desulphation and N-acetylation abolished the chain-chain interaction. Also, dermatan sulphates and chondroitin sulphates showed affinity for heparan sulphate-agarose. [3H]Heparan sulphates that were bound to a heparan sulphate-agarose were desorbed by elution with the corresponding heparan sulphate chains and also with unrelated heparan sulphates, heparin, and the galactosaminoglycans to various degrees. However, the corresponding heparan sulphate species was the most efficient at low concentrations. Dextran sulphate was unable to desorb bound heparan sulphate. When the corresponding heparan sulphate was N-desulphated/N-acetylated, carboxyl-reduced, or periodate-oxidised (D-glucuronate), the modified polymer was unable to displace [3H]heparan sulphate from heparan sulphate-agarose. The displacing ability of heparin was also destroyed by periodate oxidation. It is concluded that self-interaction between heparan sulphate chains is strongly dependent on the overall molecular conformation. The N-sulphate and carboxylate groups as well as the integrity of the D-glucuronate residue are all essential for maintaining the proper secondary structure.

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

Abstract 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. 13 C-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.-A., 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.

Structural differences between beef-lung heparan sulphates with specific self-associations

Three, specifically self-associating variants of heparan sulphate (HS2-A, HS3-A, and HS4-A) from beef lung were subjected to (a) deaminative cleavage of bonds between 2-deoxy-2-sulphoaminoglucose and uronic acid and (b) periodate oxidation of glucuronic acid residues in fully N-acetylated block-regions. In addition, the periodate-oxidised and alkali-cleaved chains were re-oxidised with periodate to identify the glucuronic acid residues in the N-sulphate-containing regions. The results showed that HS2-A was distinguished by much longer (GlcA-GlcNAc)n-segments than HS3-A and HS4-A. The latter two species were characterised by the structure of the variously N-acetyl- and N-sulphate-containing regions. In HS3-A, there was a significant contribution from segments composed of both N-acetylated and N-sulphated 2-amino-2-deoxyglucose residues. The N-sulphate-rich regions contained chiefly iduronic acid. In contrast, HS4-A had mixed or alternating arrangements of the two epimeric uronic acids in the N-sulphate-rich regions. These differences may be the basis for specific self-associations between heparan sulphate chains.

Effects of primer-concentration on uronosyl-epimerization and sulfation patterns in p-hydroxyphenyl-O-β-D-xylopyranoside-primed galactosaminoglycans produced by skin fibroblasts

By supplying skin fibroblasts with different concentrations of the galactosaminoglycan chain-primer p-hydroxyphenyl-O-β-D-xylopyranoside we have produced and recovered glycan-chains that were subsequently radio-iodinated in the hydroxyphenyl group and subjected to sequence analysis by using graded enzymic treatment followed by a combination of gel chromatography and electrophoresis. Fragments extending from the tagged reducing end to the cleavage-point were identified and quantified. Degradation by chondroitin B lyase of chains primed at 0.1 or 0.5 mM xyloside gave profiles indicating a periodic and wave-like distribution of iduronate-containing repeats, with high incidence around positions 2, 5 and onwards, whereas in chains produced at 1.0 mM xyloside the incidence of iduronate was similar in positions 1–4 and then declined. Degradation by chondroitin AC lyase indicated a high incidence of glucuronate in or near the linkage-region. There was a relatively uniform degree of sulfation in chains primed at low xyloside concentration, whereas chains primed at 1.0 mM xyloside gave very heterogeneous charge-patterns in all segments of the chain, including the linkage-region, giving the impression that adequate sulfation, probably at C-4 and at the first opportunity, is necessary to obtain an ordered and periodic epimerization pattern. Abbreviations:CS, chondroitin sulfate; DS, dermatan sulfate; GAG, glycosaminoglycan; Gal, D-galactose, GaINAc, N-acetyl-D-galactosamine; GlcUA, D-glucuronic acid; GlyUA, glycuronic acid; ΔGlyUA, 4,5-unsaturated glycuronic acid; IdoUA, L-iduronic acid; Xyl-Phe-OH, p-hydroxyphenyl-O-β-D-xylopyranoside; Xyl, D-xylose

Structural features of heparin variants having high anti-Xa clotting-activity

Abstract Two heparin-related preparations from beef lung and pig mucosa are able to inhibit the enzymic activity of the clotting factor X a . These preparations were subjected to deaminative cleavage and periodate oxidation-alkaline elimination. The following structural features were observed: ( a ) N -acetylated and glucuronate-rich regions are short and frequently intercalated between N -sulphated and iduronate-rich segments of deca- to hexadeca-saccharide size; ( b ) in the latter segments, sulphated iduronate occurs together with non-sulphated iduronate and glucuronate in a random fashion. These characteristics are distinctly different from those of regular heparan sulphate and of archetypal heparin.