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| 3. |
- Carlsson, Pernilla, et al.
(författare)
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Heparin/heparan sulfate biosynthesis : Processive formation of N-sulfated domains
- 2008
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 283:29, s. 20008-20014
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Tidskriftsartikel (refereegranskat)abstract
- Heparan sulfate (HS) proteoglycans influence embryonic development as well as adult physiology through interactions with various proteins, including growth factors/morphogens and their receptors. The interactions depend on HS structure, which is largely determined during biosynthesis by Golgi enzymes. A key step is the initial generation of N-sulfated domains, primary sites for further polymer modification and ultimately for functional interactions with protein ligands. Such domains, generated through action of a bifunctional GlcNAc N-deacetylase/N-sulfotransferase (NDST) on a [GlcUA-GlcNAc](n) substrate, are of variable size due to regulatory mechanisms that remain poorly understood. We have studied the action of recombinant NDSTs on the [GlcUA-GlcNAc](n) precursor in the presence and absence of the sulfate donor, 3'-phosphoadenosine 5'-phosphosulfate (PAPS). In the absence of PAPS, NDST catalyzes limited and seemingly random N-deacetylation of GlcNAc residues. By contrast, access to PAPS shifts the NDST toward generation of extended N-sulfated domains that are formed through coupled N-deacetylation/N-sulfation in an apparent processive mode. Variations in N-substitution pattern could be obtained by varying PAPS concentration or by experimentally segregating the N-deacetylation and N-sulfation steps. We speculate that similar mechanisms may apply also to the regulation of HS biosynthesis in the living cell.
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| 4. |
- Feyzi, Emadoldin, et al.
(författare)
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Characterization of heparin and heparan sulfate domains binding to the long splice variant of platelet-derived growth factor A chain
- 1997
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Ingår i: Journal of Biological Chemistry. - : Elsevier BV. - 0021-9258 .- 1083-351X. ; 272:9, s. 5518-5524
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Tidskriftsartikel (refereegranskat)abstract
- Platelet-derived growth factors (PDGFs) are homo- or heterodimers of two related polypeptides, known as A and B chains. The A chain exists as two splice variants due to the alternative usage of exons 6 (PDGF-AL, longer) and 7 (PDGF-AS, shorter). Exon 6 encodes an 18-amino acid sequence rich in basic amino acid residues, which has been implicated as a cell retention signal. Several lines of evidence indicate that the retention is due to binding of PDGF-AL to glycosaminoglycans, especially to heparan sulfate. We have analyzed the saccharide domains of smooth muscle cell-derived heparan sulfate involved in this interaction. Furthermore, we have employed selectively modified heparin oligosaccharides to elucidate the dependence of the binding on different sulfate groups and on fragment length. The shortest PDGF-AL binding domain consists of 6-8 monosaccharide units. Studies using selectively desulfated heparins and heparin fragments suggest that N-, 2-O-, and 6-O-sulfate groups all contribute to the interaction. Structural comparison of heparan sulfate oligosaccharides separated by affinity chromatography on immobilized PDGF-AL showed that the bound pool was enriched in -IdceA(2-OSO3)-GlcNSO3(6-OSO3)- disaccharide units. Furthermore, analogous separation of a partially O-desulfated heparin decamer preparation, using a highly selective nitrocellulose filter-trapping system, yielded a PDGF-AL-bound fraction in which more than half of the disaccharide units had the structure -IdceA(2-OSO3)-GlcNSO3(6-OSO3)-. Our results suggest that the interaction between PDGF-AL and heparin/heparan sulfate is mediated via N-sulfated saccharide domains containing both 2-O- and 6-O-sulfate groups.
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| 5. |
- Feyzi, Emadoldin, et al.
(författare)
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Structural requirement of heparan sulfate for interaction with herpes simplex virus type 1 virions and isolated glycoprotein C
- 1997
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Ingår i: Journal of Biological Chemistry. - : Elsevier BV. - 0021-9258 .- 1083-351X. ; 272:40, s. 24850-24857
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Tidskriftsartikel (refereegranskat)abstract
- Cell surface heparan sulfates mediate primary attachment of herpes simplex virus type 1, the first step in virus invasion of the cells. Removal of the host cell heparan sulfate results in a significantly diminished susceptibility of the cell to virus infection. On the virus envelope, glycoprotein C has been identified as the major binding site for heparan sulfate in the primary attachment of the virus to host cells. Using selectively desulfated heparins and metabolically labeled host cell heparan sulfate, we have analyzed the structural requirements of heparan sulfate to provide binding sites for glycoprotein C and the whole virus. Employing glycoprotein C affinity chromatography and a virus binding assay, we subfractionated oligosaccharides derived from heparan sulfate and partially desulfated heparin into selectively bound and unbound pools. These were chemically depolymerized and analyzed at the disaccharide level. The shortest glycoprotein C-binding fragment consisted of 10-12 monosaccharide units containing at least one 2-O- and one 6-O-sulfate group that have to be localized in a sequence-specific way, based on the finding that bound and unbound HS fragments do not differ in charge or composition. The binding sequence is found within N-sulfated blocks of heparan sulfate, although several N-acetyl groups can be tolerated within the minimal binding sequence. These minimal requirements for herpes simplex virus type 1 binding to heparan sulfate are clearly distinct from other identified protein binding sites.
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| 6. |
- Gong, Feng, et al.
(författare)
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Processing of macromolecular heparin by heparanase
- 2003
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 278:37, s. 35152-35158
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Tidskriftsartikel (refereegranskat)abstract
- Heparanase is an endo-glucuronidase expressed in a variety of tissues and cells that selectively cleaves extracellular and cell-surface heparan sulfate. Here we propose that this enzyme is involved also in the processing of serglycin heparin proteoglycan in mouse mast cells. In this process, newly synthesized heparin chains (60-100 kDa) are degraded to fragments (10-20 kDa) similar in size to commercially available heparin (Jacobsson, K. G., and Lindahl, U. (1987) Biochem. J. 246, 409-415). A fraction of these fragments contains the specific pentasaccharide sequence required for high affinity binding to antithrombin implicated with anticoagulant activity. Rat skin heparin, which escapes processing in vivo, was used as a substrate in reaction with recombinant human heparanase. An incubation product of commercial heparin size retained the specific pentasaccharide sequence, although oligosaccharides (3-4 kDa) containing this sequence could be degraded by the same enzyme. Commercial heparin was found to be a powerful inhibitor (I50 approximately 20 nM expressed as disaccharide unit, approximately 0.7 nM polysaccharide) of heparanase action toward antithrombin-binding oligosaccharides. Cells derived from a serglycin-processing mouse mastocytoma expressed a protein highly similar to other mammalian heparanases. These findings strongly suggest that the intracellular processing of the heparin proteoglycan polysaccharide chains is catalyzed by heparanase, which primarily cleaves target structures distinct from the antithrombin-binding sequence.
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| 8. |
- Jastrebova, Nadja, et al.
(författare)
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Heparan Sulfate Domain Organization and Sulfation Modulate FGF-induced Cell Signaling
- 2010
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 285:35, s. 26842-26851
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Tidskriftsartikel (refereegranskat)abstract
- Heparan sulfates (HSs) modulate various developmental and homeostatic processes by binding to protein ligands. We have evaluated the structural characteristics of porcine HS in cellular signaling induced by basic fibroblast growth factor (FGF2), using CHO745 cells devoid of endogenous glycosaminoglycans as target. Markedly enhanced stimulation of cell signaling, measured as phosphorylation of ERK1/2 and protein kinase B, was only observed with the shortest HS chains isolated from liver, whereas the longer chains from either liver or intestine essentially prolonged duration of signals induced by FGF2 in the absence of polysaccharide. Structural analysis showed that contiguous sulfated domains were most abundant in the shortest HS chains and were more heavily sulfated in HS from liver than in HS from intestine. Moreover, the shortest chains from either source entered into ternary complexes with FGF2 and FGF receptor-1c more efficiently than the corresponding longer chains. In addition to authentic HSs, decasaccharide libraries generated by chemo-enzymatic modification of heparin were probed for effect on FGF2 signaling. Only the most highly sulfated decamers, previously found most efficient in ternary complex formation (Jastrebova, N., Vanwildemeersch, M., Rapraeger, A. C., Gimenez-Gallego, G., Lindahl, U., and Spillmann, D. (2006) J. Biol. Chem. 281, 26884-26892), promoted FGF2 cellular signaling as efficiently as short HS chains from liver. Together these results suggest that the effects of HS on FGF2 signaling are determined by both the structure of the highly sulfated domains and by the organization/availability of such domains within the HS chain. These findings underpin the need for regulation of HS biosynthesis in relation to control of growth factor-induced signaling pathways.
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| 9. |
- Jastrebova, Nadja, et al.
(författare)
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Heparan sulfate-related oligosaccharides in ternary complex formation with fibroblast growth factors 1 and 2 and their receptors
- 2006
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 281:37, s. 26884-26892
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Tidskriftsartikel (refereegranskat)abstract
- Biosynthesis of heparan sulfate (HS) is strictly regulated to yield products with cell/tissue-specific composition. Interactions between HS and a variety of proteins, including growth factors and morphogens, are essential for embryonic development and for homeostasis in the adult. Fibroblast growth factors (FGFs) and their various receptors (FRs) form ternary complexes with HS, as required for receptor signaling. Libraries of HS-related, radiolabeled oligosaccharides were generated by chemo-enzymatic modification of heparin and tested for affinity to immobilized FR ectodomains in the presence of FGF1 or FGF2. Experiments were designed to enable assessment of N-sulfated 8- and 10-mers with defined numbers of iduronic acid 2-O-sulfate and glucosamine 6-O-sulfate groups. FGF1 and FGF2 were found to require similar oligosaccharides in complex formation with FR1c-3c, FGF2 affording somewhat more efficient oligosaccharide recruitment than FGF1. FR4, contrary to FR1c-3c, bound oligosaccharides at physiological ionic conditions even in the absence of FGFs, and this interaction was further promoted by FGF1 but not by FGF2. In all systems studied, the stability of FGF-oligosaccharide-FR complexes correlated with the overall level of saccharide O-sulfation rather than on the precise distribution of sulfate groups.
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| 10. |
- Jemth, Per, et al.
(författare)
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Biosynthetic oligosaccharide libraries for identification of protein-binding heparan sulfate motifs : exploring the structural diversity by screening for fibroblast growth factor (FGF)1 and FGF2 binding.
- 2002
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 277:34, s. 30567-30573
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Tidskriftsartikel (refereegranskat)abstract
- Heparan sulfate is crucial for vital reactions in the body because of its ability to bind various proteins. The identification of protein-binding heparan sulfate sequences is essential to our understanding of heparan sulfate biology and raises the possibility to develop drugs against diseases such as cancer and inflammatory conditions. We present proof-of-principle that in vitro generated heparan sulfate oligosaccharide libraries can be used to explore interactions between heparan sulfate and proteins, and that the libraries expand the available heparan sulfate sequence space. Oligosaccharide libraries mimicking highly 6-O-sulfated domains of heparan sulfate were constructed by enzymatic O-sulfation of O-desulfated, end-group (3)H-labeled heparin octasaccharides. Acceptor oligosaccharides that were 6-O-desulfated but only partially 2-O-desulfated yielded oligosaccharide arrays with increased ratio of iduronyl 2-O-sulfate/glucosaminyl 6-O-sulfate. The products were probed by affinity chromatography on immobilized growth factors, fibroblast growth factor-1 (FGF1) and FGF2, followed by sequence analysis of trapped oligosaccharides. An N-sulfated octasaccharide, devoid of 2-O-sulfate but with three 6-O-sulfate groups, was unexpectedly found to bind FGF1 as well as FGF2 at physiological ionic strength. However, a single 2-O-sulfate group in the absence of 6-O-sulfation gave higher affinity for FGF2. FGF1 binding was also augmented by 2-O-sulfation, preferentially in combination with an adjacent upstream 6-O-sulfate group. These results demonstrate the potential of the enzymatically generated oligosaccharide libraries.
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