Biosynthesis of heparin. ULF LINDAHL. Depurtmtwt of Veteritiuty Medical C'hemistty, Swedish. Utiitwsity of'Agriculturul Sciences. l'he Riornedicul ('enter,.
803
PROTEOGLYCANS
Biosynthesis of heparin ULF LINDAHL Depurtmtwt of Veteritiuty Medical C'hemistty, Swedish Utiitwsity of'Agriculturul Sciences. l'he Riornedicul ('enter, S- 7.51 2.3 U[)psulu,Sweden
overall extent of polymer modification, and is therefore a factor of prime importance in the regulation of the process toward the formation of either extensively modified heparin or less modified heparan sulphate. Polymer modification is not controlled by substrate specificity alone, since most of the reactions involved will engage only a fraction of the available target residues. This property greatly increases the potential for generating polysaccharide sequences of different structure, and is therefore of importance with regard to the elaboration of defined regions with specific functional properties. One such region is the AT-binding pentasaccharide sequence, the formation of which is outlined in Fig. 2. The distinguishing structural feature of this region is the 3-0-sulphate group, which substitutes the internal GlcN unit of the pentasaccharide sequence and which is either absent or very rare in heparin molecules with low affinity for AT. High-affinity binding to AT requires the presence of the 3-0-sulphate group, the appropriate sequence of monosaccharide units, with one GlcA unit (which will be secured in 11-glucoconfiguration by the adjacent N-acetyl group) and one IdoA unit, both Nsulphate groups, and, in addition, the 6-O-sulphate group o n the GlcNAc residue at the non-reducing extreme of the pentasaccharide sequence.
The concept of heparin as a proteoglycan evolved gradually and can in fact be traced back to the initial discovery by Helen Muir [ 11 of serine constituting the point of linkage between the protein and polysaccharide constituents of the chondroitin sulphate proteoglycan of cartilage. The subsequent demonstration of residual serine units in commercially available heparin preparations suggested that native heparin may occur in a proteoglycan of similar type, and this proposal was substantiated by the finding that chondroitin sulphate and heparin/heparan sulphate are linked to protein via the same galactosyl-galactosyl-xylosyl-serine sequence (Fig 1; for a review, see 121). The heparin proteoglycan is n o w recognized as a macromolecule composed of - 10- 15 polysaccharide chains of 60- 100 kDa, densely substituted on t o a I-Ser-Gly],, repeat sequence in a - 17 kDa core protein [ 3 Specific pentasaccharide sequences (see below) that bind antithrombin (AT) with high affinity, and thus are essential to the blood anticoagulant activity of the polysaccharide, are accumulated within a limited fraction of the polysaccharide chains (Fig. 1; see also [ 2 ] ) After . completed biosynthesis, in the connective-tissue type mast cell, the polysaccharide chains are degraded by a specific endo-p-Dglucuronidase into fragments, 5-25 kDa, commonly recognized as 'heparin'. Depending on whether these fragments contain or lack the AT-binding region, they will show high or low affinity for AT, respectively, hence high or low anticoagulant activity. Heparan sulphate proteoglycans appear to be variable in / HEPARIN PROTEOGLYCAN \ type, but generally display a more conventional structure with fewer, shorter and more sparsely distributed polysaccharide chains (Fig. 1). The heparan sulphate chains d o not differ qualitatively from those of heparin, but typically contain more D-glucuronic acid (GlcA) and N-acetylated glucosamine (GlcNAc) units and less L-iduronic acid (IdoA) Heparan sulphate proteoglycan and N- and 0-sulphate residues [ 2 ] . The biosynthesis of the polysaccharide chains is initiated by formation of the linkage region on the preformed protein , I core. presumably in the Golgi system, and continues by alternating addition of GlcNAc and GlcA units at the non' I reducing termini of the nascent polymers. The polysacchaOH H N C O C H ~ AH OH OH bH I rides are subsequently modified, in a stepwise manner, I I ,il 4 1.4 11 1.3 j 1.3 81 4 I through a series of reactions involving N-deacetylation and +GlcA + GlcNAc +GlcA + Gal + Gal + Xyl + Serlne I L-------------------------------------------~ N-sulphation of GlcNAc residues, C-5 epimerization of GlcA t o ldoA units and 0-sulphation in various positions (see [2, Fig. 1. Models of u hepuriri proteoglycun wid of u (srnull-size 41, for reviews). The reactions are shown in Fig. 2, which also y p e ) hepuruti siilphute proteoglycuti illustrates the outstanding characteristic feature of the process, i.e. the incomplete course of the reactions that leads to The heparin chains are densely substituted on to a [-Serthe well-known microheterogeneity expressed by the final Gly-I,, repeat sequence o f the 17 kDa core protein (dashed products, heparin o r heparan sulphate. The selection of tar- line). The AT-binding regions (thicker segments) are accumuget residues in individual reactions depends on the substrate lated in a fraction o f the chains; most of the proteoglycan specificity of the corresponding enzymes, as illustrated in molecules (rat-skin heparin preparation; see [ 81) are comFig. 2 for the hexuronosyl C-5 epimerase. This enzyme pletely devoid of this region (and thus of AT-dependent antirequires the presence of an N-sulphate group on one of the coagulant activity). The arrows indicate the specific cleavage GlcN residues adjacent to a target GlcA unit, but not on the sites for the endo-P-D-ghcuronidase which converts the other, and the occurrence of residual N-acetyl groups will heparin proteoglycan, in the mast cell, to polysaccharide therefore have a profound influence on the distribution of fragments (conventional 'heparin', having either high or low ldoA units in the product. Also the various 0- affinity for AT). Both types of proteoglycan contain the same sulphotransferases depend on the presence of preformed N- polysaccharide-protein linkage region (indicated for one of sulphate groups. In fact, the extent of the initial the heparan sulphate chains), composed of a galactosylN-deacetylationlN-sulphationreaction will determine the galactosyl-xylosyl trisaccharide sequence linked to a serine residue in the peptide core. (Reproduced from [ 2 ] , with Abbreviations used: AT, antithrombin; PAPS, 3'-phosphopermission from the publishers.) adenosyl-5'-phosphosulphate.
I.
y
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804
BIOCHEMICAL SOCIETY TRANSACTIONS
HNAc
N-deacetylation; N-sulphation
' 0 HNS03
C5-epimerization 2-0-sulphation
I I
HNSOS
@
I
6-0-sulphation
HNAc
HNSOj
Fig. 2. Iblymer-rnod~icutiotireucrions iti the hiosytithesis ofhepuriri, Ieuditig to the fortmtioti of'rheA T-binding regioti (cotitiriiioiis bracket) A pentasaccharide sequence (dashed bracket) which satisfies the substrate requirements of the glucosaminyl 3-0-sulphotransferasc is converted into a functional binding region by 3-0-sulphation of the internal GlcNSO, unit; this reaction concludes the overall modification process. The scheme also illustrates the substrate spccificity of the GlcA C-5 epimerase, by indicating hexuronic acid residues that are susceptible to attack ( + ) by the enzyme and those that resist epimerization ( - ) due t o inhibitory structural elements in the vicinity. For additional information see [ 2 I. (Reproduced from [ 21, with permission from the publishers.) breakthrough fraction that did not bind t o the lectin column. Further purification of the N-deacetylase (where all assays were performed in the presence of breakthrough material from the lectin column) by chromatography on Blue Sepharose and 3',5'-ADP/agarose yielded a protein fraction C'o-piirifcutiori of ghtcosumiriyl N-deucetyluse urid N-sirlpho- which showed a single band on SDS/PAGE, corresponding to an apparent molecular mass of - 1 1 0 kDa. trutisjeruse The N-sulphotransferase had been purified previously The non-sulphated heparin-precursor polysaccharide from rat liver by Brandan & Hirschberg [5],who concluded formed during incubation of a murine mastocytoma micro- that the enzyme is a glycoprotein with affinity for wheat-germ soma1 fraction with UDP-GlcA and UDP-GlcNAc is agglutinin and an apparent molecular mass of - 9 7 kDa. partially N-deacetylated, owing to the action of a specific Analysis of the purified mastocytoma N-deacetylase deacetylase (see 121 for references to previous work). Addi- preparation showed that it contained also N-sulphotion of the sulphate donor 3'-phosphoadenosyl-5'-phospho- transferase; however, contrary to the N-deacetylase, the Nsulphate (PAPS) to microsomes containing such preformed, sulphotransferase activity was expressed in the absence of partially N-deacetylated polysaccharide, resulted in sulpha- mastocytoma proteins lacking affinity for wheat-germ tion of the free amino groups, and at the same time stimu- agglutinin. These results implied that the expression of lated further release of the residual N-acetyl groups. These glucosaminyl N-deacetylase activity depends o n the conand other results indicated that N-deacetylation and N- certed action of (at least) two proteins, one of which is identsulphation may occur separately, yet seem to act in a con- ical to the glucosaminyl N-sulphotransferase. The further certed manner in the intact biosynthetic system. Novel characterization of these proteins and their interaction will aspects on these relations emerged from recent attempts to be fundamental to the understanding of the N-sulphation purify the N-deacetylase from mouse mastocytoma tissue (I. process and its regulation. Pettersson, M. Kusche, H. Wlad, L. Nylund, U. Lindahl & L. Kjellen, unpublished work). Chromatography of a detergent Formutioti of the A T-binding regioti extract on immobilized wheat-germ agglutinin yielded a The mechanism behind the generation of thc AT-binding protein fraction (eluted with 0.3 M-GIcNAc)which expressed N-deacetylase activity, but only after recombination with the pentasaccharide sequence has been investigated with parThe following discussion will focus o n two areas of recent development in the studies of polymer modification, i t . the concerted N-dcacetylation/N-sulphation reaction and the formation of the AT-binding region.
PROTEOGLYCANS
805
ticular regard to the incorporation of the glucosaminyl 3 - 0 sulphate marker group. A capsular polysaccharide derived from Escherichia coli K5, having the same [-GlcA-GlcNAc-I,, structure as the initial polymerization product in heparin biosynthesis, was chemically N-deacetylated and N-sulphated and used as a substrate for detergent-solubilized mastocytoma microsomal enzymes, in the presence of ["SIPAPS (M. Kusche & U. Lindahl, unpublished work). Analysis of the products showed "S-labelled components representative of all the major disaccharide units recognized in heparin. including IdoA-containing species formed through the action of the GlcA C-5 epimerase. A small proportion, - 1-2% of the total labelled material, had acquired high affinity for AT, thus suggesting that formation of the AT-binding pentasaccharide sequence did not require any as yet unidentified 'committing' structural element, in addition to the simple alternating -GlcA-GlcNAc- sequence. Likewise, a heparinderived pentasaceharide with the structure, GlcNS0,-GlcAGlcNS0,-ldoA-GlcNSO,, yielded a "S-labelled AT-binding component during similar incubation, whereas isomeric pentasaceharides with IdoA and GlcA in reversed sequence, or with two ldoA or two GlcA units, did not; nor did they incorporate any glucosaminyl 3- 0-sulphate group (M. Kusche, R. Reynertson, L. Roden & U. Lindahl, unpublished work). These findings emphasize the importance of the appropriate monosaccharide sequence in formation of the binding region. Further experiments, involving an analogous octasaccharide substrate and detailed structural characterization of subfractions obtained by separation of the "Slabelled products on AT-Sepharose, led to the proposal illustrated in Fig. 2. The incorporation of the 3-0-sulphate group concludes polymer modification, and requires an acceptor structure which contains all other structural components needed for AT-binding [6].These findings raised the question as to the structural difference between heparin with high and low affinity for AT. Detailed structural analysis between corresponding high-affinity and low-affinity fractions showed that the difference was essentially restricted to the presence or absence of the glycosaminyl 3-0-sulphate group 171. On the other hand, the latter fractions were found
to contain appreciable quantities of the -1doA-GlcNAc(6OS0,)-GlcA-GlcNS0,- tetrasaccharide sequence, the three reducing-terminal units of which form part of the postulated acceptor sequence for the 3-0-sulphate group (Fig. 2). The two remaining positions of the acceptor regions were not examined in the low-affinity heparin; however, since these positions are occupied by the most abundant disaccharide unit [-IdoA(2-OSO,)-GlcNS0,(6-OSO3)-] in heparin, it would seem reasonable to assume that low-affinity heparin contains the acceptor sequence for 3-0-sulphation. Yet this material did not yield any labelled high-affinity components on incubation with [ "S]PAPS, in the presence of the solubilized 3-0-sulphotransferase. While the reason for this failure is unknown, 3-0-sulphation may conceivably be restricted by as yet unidentified structural elements that are preferentially expressed in polysaccharide sequences selected for the generation of low-affinity heparin. The mechanism behind such inhibition and its release appears particularly intriguing in view of the markedly non-random distribution of functional AT-binding regions in the heparin proteoglycan (Fig. 1). I . Muir. H. ( 1958)Hiochem.J . 69. 195-204 2. Lindahl. U. ( 1989) in llt,poriri: Chemical and Biologic~aII'roperlies, C'linicul App1icdori.s (Lane. D. A. & Lindahl, U.. eds.). pp. 159-189, Edward Arnold, London 3. Kjellen, L., Pettersson. I., Lillhager, P., Steen, M.-L., Pettersson, U.. Lehtonen, P.. Karlsson, T.. Ruoslahti, E. & Hellman, L. (1989)Biochem. J. 263, 105-1 13 4. Lindahl, U., Feingold, D. S. & Roden, L. (1986) Trends. Biochem. Sci. 1 1 . 2 2 1-225 5. Brandan, E. & Hirschberg. C. B. (1988) J . Hiol. C'hcm. 263, 2417-2422 6 . Kusche, M., Backstriim, G., Riesenfeld, J., Petitou, M., Choay. J. & Lindahl, U. ( 1 088) J . Hiol. C'hem. 263, 15474- I5484 7. Kusche. M.. Torri. (3.. Caw, B. & Lindahl, U. ( 1990) J . Hiol. C'hem. in the press 8. Jacobsson. K. G.. Lindahl, U., Horner, A. A. (1986) Hiochern. J . 240.625-632 Received I9 April 1990
Heparan sulphate proteoglycans of human fibroblasts GUlDO DAVID C'etiire filr I liitnurI Get 1 etics. University OJ Leii vet 1, C i i tnpiis Gusihiiisberg, Iterestruut 49, H-.10W Leuven, Belgiim
abundance and function. Studies on the heparan sulphatc proteoglycans of human lung fibroblasts support these concepts.
Hcparan sulphate proteoglycans occur in close association with cells. They accumulate at cell-cell and cell-matrix interfaces, both as plasma-membrane-associated molecules and as integral components of the pericellular matrix. By binding cell surface and matrix components, they seem to function as part of the 'glue' that sticks cells and tissues together. However, by activating cellular receptors, growth factors and proteinase inhibitors, these proteoglycans apparently also act as cell-surface- and matrix-associated 'catalysts' that influence cell growth, modulate the assembly and remodelling o f the matrix, and control the activity of coagulation factors and other extracellular proteinases. These functions are primarily based on the binding interactions and allosteric effects of the heparan sulphate chains, but also depend on the interactions and associations of the proteins which carry these glycosaminoglycans. These core proteins come in a variety of forms, seem to specify the accumulation of the proteoglycans at their sites of occurrence, and may be subject to regulatory events that modulate proteoglycan
Mem hrane-associated proteoglycutis
Vol. 18
Cell-surface-associated heparan sulphate proteoglycans have now been isolated from a large number of different tissues and cells in culture [ 1, 21. Several of these heparan sulphate proteoglycans have tentatively been identified as membrane proteins, based on the ability to solubilize these components with detergent, or to extract the proteoglycans as detergent-susceptible aggregates by using strong chaotropes. Furthermore, these proteoglycans are able to interact with hydrophobic gels ( e g octyl-Sepharose) and can be intercalated into liposomcs. In cultured human lung fibroblasts, the membrane heparan sulphate proteoglycans represent 20-30"/0 o f the total cellular proteoglycan [3]. Their unique hydrophobic properties and their isolation from plasma membranes that were washed in hypertonic salt solutions suggest that the core proteins of these proteoglycans are intercalated into the lipid bilayers of the membranes. In lung fibroblasts, highly
