Search PubMed⌕ Search

Biomedical subjects

U Lindahl

Publications and source records attributed to U Lindahl.

At least 73 records · Page 4Linked to original sources
PubMed1994

Heparin-like compounds prepared by chemical modification of capsular polysaccharide from E. coli K5.

O-Sulfation of sulfaminoheparosan SAH, a glycosaminoglucuronan with the structure-->4)-beta-D-GlcA(1-->4)-beta-D-GlcNSO3(-)-(1-->, obtained by N-deacetylation and N-sulfation of the capsular polysaccharide from E. coli K5, was investigated in order to characterize the sulfation pattern eliciting heparin-like activities. SAH was reacted (as the tributylammonium salt in N,N-dimethylformamide) with pyridine-sulfur trioxide under systematically different experimental conditions. The structure of O-sulfated products (SAHS), as determined by mono- and two-dimensional 1H and 13C NMR, varied with variation of reaction parameters. Sulfation of SAH preferentially occurred at O-6 of the GlcNSO3- residues. Further sulfation occurred either at O-3 or at O-2 of the GlcA residues, depending on the experimental conditions. Products with significantly high affinity for antithrombin and antifactor Xa activity were obtained under well-defined conditions. These products contained the trisulfated aminosugar GlcNSO3-3,6SO3-, which is a marker component of the pentasaccharide sequence through which heparin binds to antithrombin.

Carbohydrate Conformation↗
PubMed1994

Biosynthesis of heparin. Different molecular forms of O-sulfotransferases.

O-Sulfotransferases involved in heparin biosynthesis were purified > or = 10,000-fold from detergent extracts of mouse mastocytoma tissue by sequential chromatographies on DEAE-Sephacel, heparin-agarose, blue Sepharose, and 3',5'-ADP-Sepharose. The resultant preparation catalyzed the transfer of 35S from 3'-phosphoadenosyl-5'-phospho-[35S]sulfate into N,O-desulfated, re-N-sulfated heparin. Anion-exchange high performance liquid chromatography of disaccharides obtained by deaminative cleavage of the 35S-labeled polysaccharide product revealed O-35S-sulfation at C-2 of L-iduronic acid and at C-6 of D-glucosamine units. SDS-polyacrylamide gel electrophoresis of semipurified enzyme followed by extraction of gel segments and renaturation of proteins consistently showed two distinct fractions of O-sulfotransferase activity, corresponding to proteins of approximately 20 and approximately 60 kDa. The approximately 60-kDa enzyme(s) catalyzed both the 2-O- and 6-O-sulfotransferase reactions, whereas the approximately 20-kDa fraction promoted iduronosyl 2-O-sulfation only. These results are discussed in relation to previous findings, indicating that some of the enzymes involved in heparin biosynthesis catalyze more than one reaction.

Animals↗
PubMed1994

cDNA cloning and sequencing of mouse mastocytoma glucosaminyl N-deacetylase/N-sulfotransferase, an enzyme involved in the biosynthesis of heparin.

A 110-kDa protein involved in heparin biosynthesis in mouse mastocytoma cells was previously shown to express both glucosaminyl N-deacetylase and N-sulfotransferase activity. In this study, the complete nucleotide sequence corresponding to this protein is reported. The mRNA, estimated to contain 3.9 kilobases encodes a protein with an M(r) of 101,092. The predicted domain structure of the protein resembles those of previously characterized Golgi proteins with an N-terminal cytoplasmic tail, a single membrane-spanning domain, and a large catalytic domain linked to the transmembrane domain through a "stem region." Comparison of the deduced amino acid sequence of the mouse mastocytoma protein and a previously cloned similar enzyme from rat liver demonstrated that while large portions of the proteins, corresponding essentially to the putative catalytic domains, were closely related, other portions, in particular in the N-terminal parts, were markedly different. The divergence was not due to species differences since two separate mouse transcripts could be identified that hybridized with probes specific for the two proteins. Also, functional differences were noted since the mastocytoma enzyme, contrary to the liver enzyme, requires a polycation cofactor for expression of N-deacetylase activity. The results are discussed in relation to the structural properties of heparin and heparan sulfate.

Amidohydrolases↗
PubMed1994

Substrate specificities of glycosyltransferases involved in formation of heparin precursor and E. coli K5 capsular polysaccharides.

The E. coli K5 capsular polysaccharide is composed of 4)GlcpA(beta 1-4)GlcpNAc(alpha 1-disaccharide units. A partially N-deacetylated/N-sulfated heptasaccharide, derived from this polymer and having a nonreducing terminal GlcNAc unit, was used as acceptor for a mastocytoma microsomal GlcA-transferase involved in heparin biosynthesis. An octasaccharide with nonreducing-terminal GlcA similarly served as acceptor for the microsomal GlcNAc-transferase. Analysis of the labeled octa- and nona-saccharides formed by transfer of monosaccharide units from UDP-[14C]GlcA and UDP-[3H]GlcNAc, respectively, showed that both glycosyltransferases could utilize partially N-sulfated acceptors. The GlcA-transferase showed a marked preference for a terminal GlcNAc-GlcA-GlcNSO3-sequence, particularly when this sequence was followed by an additional N-sulfated disaccharide unit. Enzymes catalyzing the same GlcA and GlcNAc transfer reactions were solubilized from E. coli K5 membranes. The K5 capsular polysaccharide, like the heparin/heparan sulfate precursor polysaccharide, thus probably grows by stepwise, alternating addition of the two constituent monosaccharide units, from the corresponding UDP-sugars, to the nonreducing ends of the chains. Moreover, the bacterial glycosyltransferases utilized the same partially N-sulfated oligosaccharide substrates as the mammalian enzymes, and with similar preference for N-sulfate groups in certain positions.

Animals↗
PubMed1994

[The great Scandinavian Jahre Prize 1993. What is the function of heparan sulfate?].

The polysaccharide heparin, produced by mast cells, had been used in the prophylaxis and treatment of thrombotic disease long before the molecular mechanism responsible for its anticoagulant activity was elucidated. The anticoagulant effect is particularly dependent upon the presence of a specific pentasaccharide sequence, which binds to the proteinase inhibitor, antithrombin. Detailed knowledge of this mechanism enabled the development of low-molecular weight heparin with improved therapeutic properties. Studies on the biosynthesis of heparin have revealed the essential features of the generation of such complex saccharide sequences, the initial formation of a simple, repetitive polymer is followed by the action of a number of modifying enzymes. Though the mode of regulation of these enzymes is still only partly understood, heparan sulphate, structurally related to heparin, has been found to be generated by virtually all the mammalian cells studied, and is typically exposed as a proteoglycan at cell surfaces. Recent findings suggest specific heparan sulphate sequences to be crucially involved in the action of certain growth factors.

Awards and Prizes↗
PubMed1993

Activating and inhibitory heparin sequences for FGF-2 (basic FGF). Distinct requirements for FGF-1, FGF-2, and FGF-4.

Chlorate-treated Swiss 3T3 fibroblasts, with impaired synthesis of heparan sulfate proteoglycan, were used as target cells in assessing the ability of exogenous heparin-derived saccharides to promote the mitogenic activity of basic fibroblast growth factor 2 (FGF-2). Full-size native heparin (carrying iduronosyl 2-O-sulfate and glucosaminyl 6-O-sulfate groups), as well as a dodecasaccharide fraction isolated after limited deaminative cleavage of heparin, were efficient promoters, whereas the corresponding decasaccharides, or smaller oligosaccharides, were inactive. Neither selectively 2-O-desulfated nor preferentially 6-O-desulfated heparin were active. However, the latter derivative competed with native heparin for binding to FGF-2 and thus blocked the ability of native heparin to promote the mitogenic activity of FGF-2. The 6-O-desulfated heparin also prevented the ability of FGF-2 to suppress myogenic differentiation in MM14 mouse myoblasts. The binding region for FGF-2 has been identified as a pentasaccharide sequence containing a single essential O-sulfate group, at C2 of iduronic acid (1). It is proposed that the dodecasaccharide sequence required to promote receptor signaling by FGF-2 encompasses this pentasaccharide region, which binds the growth factor, and a site interacting with the receptor that contains essential 2-O- and 6-O-sulfate groups. Similar studies involving the related growth factors, FGF-1 and FGF-4, revealed differential effects of saccharides. The mitogenic effect induced by FGF-1 thus was not blocked by either the 2-O- or the 6-O-desulfated heparins. However, both of these derivatives, at high concentrations, promote mitogenic activity of FGF-4. It is concluded that specific saccharide sequences within heparan sulfate glycosaminoglycan chains favor the signaling by distinct members of the FGF family.

3T3 Cells↗
PubMed1993

Minimal sequence in heparin/heparan sulfate required for binding of basic fibroblast growth factor.

Experiments based on interaction in free solution between basic fibroblast growth factor (FGF-2) and saccharides related to heparin/heparan sulfate showed that the growth factor binds to heparin and to selectively glucosaminyl 6-O-desulfated heparin but poorly to iduronosyl 2-O-desulfated heparin. 2-O-sulfate groups thus are essential to the interaction, whereas 6-O-sulfates are not required nor do they interfere with FGF-2 binding. Comparison of various bound/nonbound oligosaccharides implicated a minimal pentasaccharide sequence for FGF-2 binding, with the structure: -hexuronic acid-glucosamine N-sulfate-hexuronic acid-glucosamine N-sulfate-iduronic acid 2-O-sulfate- (reducing terminus to the right). Such (overlapping) sequences are abundant in heparin, albeit heavily obscured by irrelevant O-sulfate groups, and occur also in heparan sulfate, with or without additional O-sulfates.

Animals↗
PubMed1993

Biosynthesis of heparin/heparan sulfate. Identification of a 70-kDa protein catalyzing both the D-glucuronosyl- and the N-acetyl-D-glucosaminyltransferase reactions.

The D-glucuronosyl- (GlcA) and N-acetyl-D-glucosaminyl- (GlcNAc) transferase reactions involved in heparin/heparan sulfate biosynthesis were assayed, measuring transfer of radiolabeled GlcA or GlcNAc monosaccharide units from the corresponding UDP-sugars to the appropriate oligosaccharide acceptors. The assays were applied to enzyme purification from bovine serum. The two activities remained inseparable through a series of different chromatographic steps, resulting in approximately -2000-fold purification. Further purification was achieved by chromatofocusing, which showed an isoelectric point of pH approximately -7.0, similar for both activities. SDS-polyacrylamide gel electrophoresis (PAGE) of subfractions from the chromatofocusing procedure revealed an approximately 70-kDa protein in amounts reflecting enzyme activity. SDS-PAGE followed by extraction of gel segments and renaturation of proteins showed that the GlcA- and GlcNAc-transferase activities were both recovered from the same single segment, corresponding to the 70-kDa component. It is proposed that the two glycosyltransferase reactions are catalyzed by the same Golgi enzyme (see also Lidholt, K., Weinke, J. L., Kiser, C. S., Lugemwa, F. N., Bame, K. J., Cheifetz, S., Massagué, J., Lindahl, U., and Esko, J. D. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 2267-2271).

Animals↗
PubMed1993

FABMS/derivatisation strategies for the analysis of heparin-derived oligosaccharides.

Derivatisation/FABMS strategies applicable to the structure analysis of low microgram quantities of heparin-derived oligosaccharides are described. Negative and positive FAB data from permethyl derivatives and positive FAB data from the products of subsequent methanolysis are reported for sulfated tetrasaccharides prepared by nitrous acid degradation of heparin. The preparation and FAB behaviour of acetylated derivatives of sulfated oligosaccharides are described for the first time, and the stability of the sulfate groups to base-catalysed acetylation is demonstrated. The acetylation/FABMS methodology, which yields high quality data, shows promise for the characterisation of a wide range of sulfated glycoconjugates.

Acetylation↗
PubMed1993

Mode of interaction between platelet factor 4 and heparin.

Platelet factor 4 (PF4) is a platelet-derived protein capable of binding to, and thus neutralizing, the biological activities of heparin and heparan sulphate. The mode of binding of PF4 to heparin was investigated in a comparative study also involving antithrombin (AT; previously shown to selectively bind a specific oligosaccharide sequence) and fibronectin (FN; non-specific electrostatic interaction). Heparin-derived saccharides were incubated with each of the three proteins, followed by separation of free and protein-bound carbohydrate on a nitrocellulose filter. The interaction systems involved either (i) competition for the protein ligand between 3H-labelled heparin and unlabelled, size-fractionated heparin oligosaccharides (isolated after deaminative cleavage with HNO2) or (ii) direct binding of 3H-labelled oligosaccharides. Species smaller than octasaccharides were unable to bind AT, whereas binding to FN and PF4 increased continuously throughout the series, with increasing size of the oligosaccharides. Further separation by anion-exchange chromatography showed that the PF4-binding and FN-binding octasaccharides represented essentially all components present in the initial octasaccharide fraction, the proportion of binding species increasing with charge (hence with the degree of sulphation). The AT-binding octasaccharides, on the other hand, selectively represented only a few of the total octasaccharide components, without any correlation to overall charge. These results indicate that the binding of PF4 to heparin occurs by relatively non-specific electrostatic interactions. The methodology delineated here may be generally useful in assessing specificity in glycosaminoglycan-protein interactions.

Animals↗
PubMed1993

Low-sulphated oligosaccharides derived from heparan sulphate inhibit normal angiogenesis.

Heparin, with or without the addition of an adrenocorticosteroid, can inhibit normal angiogenesis in the chick embryo chorioallantoic membrane. Low- or non-sulphated heparin fragments also have anti-angiogenic effect. Attempts to define a saccharide structure responsible for the anti-angiogenic effect implicated a -[GlcA beta 1,4-GlcNAc alpha 1,4]n-sequence. This structure represents the product of the initial polymerization reaction in heparin/heparan sulphate biosynthesis. It persists in the non-sulphated regions of heparan sulphate and also occurs in the Escherichia coli K5 capsular polysaccharide. The K5 polysaccharide, fragments thereof down to octasaccharide size and analogous N-acetylated fragments of heparan sulphate, all showed anti-angiogenic activity. Hyaluronan, however, with the isomeric -[GlcA beta 1,3-GlcNAc beta 1,4]n-structure was inactive. The anti-angiogenic activity of -[GlcA beta 1,4-GlcNAc alpha 1,4]n-containing saccharides was potentiated by the presence of L-iduronic acid and one or two O-sulphate groups in the non-reducing-terminal disaccharide unit. The anti-angiogenic effect of these non- or low-sulphated saccharides was unaffected by the addition of hydrocortisone. Endothelial cell surface-bound heparan sulphate proteoglycans may represent a pool of precursors of anti-angiogenic oligosaccharides which may be of primary importance in the regulation of angiogenesis.

Animals↗
PubMed1992

Stable heparin-producing cell lines derived from the Furth murine mastocytoma.

Stable cell lines that synthesize heparin have been established from the Furth murine mastocytoma. The parental line (MST) divides in suspension every 14-18 h in growth medium supplemented with fetal bovine serum or defined growth factors. Adherent subclones were selected by adhesion to plastic culture vessels. Both adherent and nonadherent cells contain about 0.4 micrograms of glycosaminoglycan hexuronic acid per 10(6) cells, composed of 80% heparin and 20% chondroitin sulfate E. Deaminative cleavage of MST heparin by HNO2 at pH 1.5 released disaccharides that were similar in composition to those obtained from commercial heparin, except that disaccharides containing 3,6-O-desulfated GlcN units were not found. Greater than 90% of the glycosaminoglycans were stored in cytoplasmic granules, and challenge of the cells with dinitrophenylated bovine serum albumin and anti-dinitrophenyl IgE released a portion of the stored material. Growth studies of subclones showed that MST cells tolerate a 10-fold variation in glycosaminoglycan content. Incubation of cells with sodium chlorate reduced glycosaminoglycan sulfation by > 95% without affecting cell growth. Thus, granule glycosaminoglycans appear to be nonessential for growth of MST cells.

Animals↗
PubMed1992

Biosynthesis of heparin. The D-glucuronosyl- and N-acetyl-D-glucosaminyltransferase reactions and their relation to polymer modification.

Oligosaccharides with the general structure [GlcA-GlcNAc]n-GlcA-aMan (aMan is 2,5-anhydro-D-mannose), derived from the Escherichia coli K5 capsular polysaccharide, were found to serve as monosaccharide acceptors for a GlcNAc-transferase, solubilized from a mouse mastocytoma microsomal fraction and implicated in heparin biosynthesis. Digestion of these oligosaccharides with beta-D-glucuronidase yielded acceptors for the GlcA-transferase that acts in concert with the GlcNAc-transferase. Assays based on the oligosaccharide acceptors showed broad pH optima for the two enzymes, centred around pH 6.5 for the GlcNAc-transferase and around pH 7.0 for the GlcA-transferase. The GlcNAc-transferase showed an absolute requirement for Mn2+, whereas the GlcA-transferase was stimulated by Ca2+ and Mg2+ but not by Mn2+. The GlcNAc acceptor ability of the [GlcA-GlcNAc]n-GlcA-aMan oligosaccharides increased with increasing chain length, as reflected by the apparent Km, which was 60 microM for a hexasaccharide but 6 microM for a hexadecasaccharide. By contrast, the Km for [GlcNAc-GlcA]n-aMan oligosaccharides in the GlcA-transferase reaction was higher, approximately 0.5 mM, and unaffected by acceptor size. After chemical modification of the oligosaccharides to obtain mixed N-substituents (N-unsubstituted, N-acetylated or N-sulphated GlcN residues), GlcNAc transfer was found to be virtually independent of the N-substituent pattern of the acceptor sequence. The GlcA-transferase, on the other hand, showed marked preference for an acceptor with a non-reducing-terminal GlcNAc-GlcA-GlcNSO3- sequence, which would thus have a lower Km for the enzyme than the corresponding fully N-acetylated structure. These results, along with our previous finding that chain elongation in a mastocytoma microsomal system is strongly promoted by concomitant N-sulphation of the nascent chain [Lidholt, Kjellén & Lindahl (1989) Biochem. J. 261, 999-1007], raise the possibility that the glycosyltransferases and the N-deacetylase/N-sulphotransferase act in concert during chain elongation, assembled into an enzyme complex.

Animals↗
PubMed1992

A single mutation affects both N-acetylglucosaminyltransferase and glucuronosyltransferase activities in a Chinese hamster ovary cell mutant defective in heparan sulfate biosynthesis.

Mutants of Chinese hamster ovary cells have been found that no longer produce heparan sulfate. Characterization of one of the mutants, pgsD-677, showed that it lacks both N-acetylglucosaminyl- and glucuronosyltransferase, enzymes required for the polymerization of heparan sulfate chains. pgsD-677 also accumulates 3- to 4-fold more chondroitin sulfate than the wild type. Cell hybrids derived from pgsD-677 and wild type regained both transferase activities and the capacity to synthesize heparan sulfate. Two segregants from one of the hybrids reexpressed the dual enzyme deficiency, the lack of heparan sulfate synthesis, and the enhanced accumulation of chondroitin sulfate, suggesting that all of the traits were genetically linked. These findings indicate that the pgsD locus may represent a gene involved in the coordinate control of glycosaminoglycan formation.

Animals↗