10E4 Antigen of Scrapie Lesions Contains an Unusual Nonsulfated Heparan Motif

The carbohydrate antigen on heparan sulfate recognized by monoclonal antibody 10E4 is uniquely codistributed with the abnormal prion protein, PrPSc, even in the earliest detectable brain lesions of scrapie-infected mice. Determining the chemical structure of 10E4 antigen is, therefore, an important aspect of structure elucidation of scrapie lesions, and a prerequisite for designing experiments to understand its role in scrapie pathogenesis. Toward this aim, we have examined preparations of heparan sulfate, with differing sulfate contents, for binding by 10E4 antibody. The highest antigenicity was observed in a preparation (HS-1) with the lowest sulfate content. HS-1 was partially depolymerized with heparin lyase III, and oligosaccharide fragments examined for 10E4 antigen expression by the neoglycolipid technology. An antigen-positive and two antigen-negative tetrasaccharides were isolated and examined by electrospray mass spectrometry. The antigen-positive tetrasaccharide sequence on heparan sulfate was thus deduced to contain a unique unsulfated motif that includes an N-unsubstituted glucosamine in the sequence, UA-GlcN-UA-GlcNAc. Antibody binding experiments with neoglycolipids prepared from a series of heparin/heparan sulfate disaccharides, and the trisaccharide derived from the antigen-positive tetrasaccharide after removal of the terminal hexuronic acid, show that both the penultimate glucosamine and the outer nonsulfated hexuronic acid are important for 10E4 antigenicity. The carbohydrate antigen on heparan sulfate recognized by monoclonal antibody 10E4 is uniquely codistributed with the abnormal prion protein, PrPSc, even in the earliest detectable brain lesions of scrapie-infected mice. Determining the chemical structure of 10E4 antigen is, therefore, an important aspect of structure elucidation of scrapie lesions, and a prerequisite for designing experiments to understand its role in scrapie pathogenesis. Toward this aim, we have examined preparations of heparan sulfate, with differing sulfate contents, for binding by 10E4 antibody. The highest antigenicity was observed in a preparation (HS-1) with the lowest sulfate content. HS-1 was partially depolymerized with heparin lyase III, and oligosaccharide fragments examined for 10E4 antigen expression by the neoglycolipid technology. An antigen-positive and two antigen-negative tetrasaccharides were isolated and examined by electrospray mass spectrometry. The antigen-positive tetrasaccharide sequence on heparan sulfate was thus deduced to contain a unique unsulfated motif that includes an N-unsubstituted glucosamine in the sequence, UA-GlcN-UA-GlcNAc. Antibody binding experiments with neoglycolipids prepared from a series of heparin/heparan sulfate disaccharides, and the trisaccharide derived from the antigen-positive tetrasaccharide after removal of the terminal hexuronic acid, show that both the penultimate glucosamine and the outer nonsulfated hexuronic acid are important for 10E4 antigenicity. Scrapie is the prototype of an unusual group of transmissible neurodegenerative diseases characterized by deposition of an alternatively folded, abnormally protease-resistant, isoform of the host prion protein, PrPC 1The abbreviations used are:HSPGsheparan sulfate proteoglycansADHPN-aminoacetyl-N-(9-anthracenylmethyl)-1,2-dihexadecyl-sn-glycero-3-phosphoethanolamineCIDcollision-induced dissociationES-MSelectrospray-mass spectrometryGlcAglucuronic acidGlcNglucosamineGlcNAcN-acetylglucosamineGlcNSN-sulfated glucosamineHPLChigh performance liquid chromatographyHPTLChigh performance thin layer chromatographyHSheparan sulfateNGLneoglycolipidPBSphosphate-buffered salinePrPCnormal cellular prion proteinPrPScabnormal scrapie prion proteinSAXstrong anion exchangeTBSTris-buffered salineUAhexuronic acidΔUA4,5-unsaturated hexuronic acid, which is proposed to constitute the infective agent (1Prusiner S.B. Science (Wash. D. C.). 1982; 216: 136-144Crossref PubMed Scopus (4205) Google Scholar). There is considerable progress in knowledge of the molecular and cellular biology of prions, but precise details of the mechanism of conversion to the disease-associated form, PrPSc, remain to be elucidated (2Harris D.A. Prions. Molecular and Cellular Biology. Horizon Scientific Press, Wymondham, UK1999Google Scholar). Much interest has been stimulated in the interactions of PrP with the glycosaminoglycans heparan sulfate and heparin. This has arisen as a result of the finding (3Snow A.D. Wight T.N. Nochlin D. Koike Y. Kimata K. DeArmond S.J. Prusiner S.B. Lab. Invest. 1990; 63: 601-611PubMed Google Scholar) that amyloid plaques, which occur in the clinically manifest stage of experimental scrapie (as in the human transmissible encephalopathies and Alzheimer's disease) are rich in heparan sulfate proteoglycans (HSPGs).1 The effects of administering heparin and heparan sulfate and other highly acidic materials have been investigated on the course of experimental scrapie infections (4Gabizon R. Meiner Z. Halimi M. Ben-Sasson S.A. J. Cell. Physiol. 1993; 157: 319-325Crossref PubMed Scopus (122) Google Scholar, 5Caughey B. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 1994; 343: 399-404Crossref PubMed Scopus (19) Google Scholar). It has been shown that such acidic compounds can “cure” infected cells or lengthen the survival of scrapie-infected animals. It is not yet known, however, whether these effects are mediated by direct binding to PrPC and PrPSc, or by indirect mechanisms. Nor is it known whether endogenous glycosaminoglycans or proteoglycans are involved in the normal physiological interactions with prion protein or in pathological interactions as has been suggested (5Caughey B. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 1994; 343: 399-404Crossref PubMed Scopus (19) Google Scholar). It will be important to elucidate at the molecular level any such interactions as a lead to rational therapeutic designs. heparan sulfate proteoglycans N-aminoacetyl-N-(9-anthracenylmethyl)-1,2-dihexadecyl-sn-glycero-3-phosphoethanolamine collision-induced dissociation electrospray-mass spectrometry glucuronic acid glucosamine N-acetylglucosamine N-sulfated glucosamine high performance liquid chromatography high performance thin layer chromatography heparan sulfate neoglycolipid phosphate-buffered saline normal cellular prion protein abnormal scrapie prion protein strong anion exchange Tris-buffered saline hexuronic acid 4,5-unsaturated hexuronic acid Heparan sulfates are major components of the proteoglycans in nervous tissue occurring at the surface of cells and in the extracellular matrix (6Ruoslahti E. J. Biol. Chem. 1989; 264: 13369-13372Abstract Full Text PDF PubMed Google Scholar, 7Lander A.D. Stipp C.S. Ivins J.K. Perspect. Dev. Neurobiol. 1996; 3: 347-358PubMed Google Scholar, 8Lyon M. Gallagher J.T. Matrix Biol. 1998; 17: 485-493Crossref PubMed Scopus (114) Google Scholar). The carbohydrate chains are negatively charged polymers containing disaccharide units of hexuronic acid andN-acetylated or N-sulfated glucosamine. There are diverse patterns of sulfation along the oligosaccharide chains, there being less sulfation overall in heparan sulfates relative to heparin (9Kjellén L. Lindahl U. Annu. Rev. Biochem. 1991; 60: 443-475Crossref PubMed Scopus (1714) Google Scholar). Evidence has been forthcoming for the possible involvement of a specific carbohydrate sequence of heparan sulfate in scrapie pathogenesis. In an immunohistochemical study aimed at examining the temporal relationship between HSPGs and the known sites of PrP pathology, a mouse model of scrapie was selected, which is characterized by both fibrillar amyloid and nonamyloid PrPSc (10McBride P.A. Wilson M.I. Eikenlenboom P. Tunstall A. Bruce M.E. Exp. Neurol. 1998; 149: 447-454Crossref PubMed Scopus (65) Google Scholar). Six monoclonal antibodies to HSPGs were investigated of which three were directed to core proteins and three to the carbohydrate chains. The presence of one of the carbohydrate antigens, detected with 10E4 antibody (but not with the other antibodies), correlated strikingly with the areas of abnormal PrP deposition, not only in the amyloid plaques, but also in the earliest detectable lesions in the brain of the infected mice. In particular, the presence of the 10E4 antigen in proximity to the diffuse forms of PrPSc in neuro-anatomically defined target sites, suggests a close relationship between this determinant on heparan sulfate and the formation of the scrapie lesions. We are investigating the carbohydrate sequence of the 10E4 determinant by the neoglycolipid (NGL) technology (11Feizi T. Stoll M.S. Yuen C.-T. Chai W. Lawson A.M. Methods Enzymol. 1994; 230: 484-519Crossref PubMed Scopus (119) Google Scholar) in conjunction with electrospray mass spectrometry (ES-MS). A recent advance involving the generation of oligosaccharide probes in the form of fluorescent NGLs (12Stoll M.S. Feizi T. Loveless R.W. Chai W. Lawson A.M. Yuen C.-T. Eur. J. Biochem. 2000; 267: 1795-1804Crossref PubMed Scopus (41) Google Scholar) has enhanced the power of the technology in pinpointing and isolating bioactive oligosaccharides among highly heterogeneous populations. Here we describe the identification by this means and the assignment of an unusual carbohydrate sequence in the 10E4 antigen. Mouse monoclonal antibody 10E4, IgMκ (13David G. Bai X.M. Van Der, Schueren B. Cassiman J.-J. Van Der, Berghe H. J. Cell Biol. 1992; 119: 961-975Crossref PubMed Scopus (414) Google Scholar) was from Seikagaku (Japan). Rabbit anti-mouse Ig-peroxidase was from Dako (Denmark). Protein LA-peroxidase was from Actigen (Cambridge, UK). FASTTM 3,3′-diaminobenzidine and 2,2′-azino-bis(3-ethylbenzthiazoline-sulfonic acid) were from Sigma (Poole, UK). Heparan sulfate fractions, HS-1, -2, -3, and -4 with sulfur contents of around 5, 8, 9, and 10%, respectively, from porcine intestinal mucosa (14Griffin C.C. Lindhardt R.J. Van Gorp C.L. Toida T. Hileman R.E. Schubert R.L. Brown S.E. Carbohydr. Res. 1995; 276: 183-197Crossref PubMed Scopus (116) Google Scholar) were from Celsus Laboratories (Cincinnati, OH). Recombinant heparin lyase III (heparinase III, E.C. 4.2.2.8) was from IBEX Technologies (Montreal, Quebec, Canada). Heparin/heparan sulfate disaccharides (I-, II-, III-, and IV-A; I-, II-, III-, and IV-S; I-, II-, III-, and IV-H), chondroitin sulfate A and poly-l-lysine (150–300 kDa) were from Sigma. HS-1 was partially depolymerized by limited digestion with heparin lyase III essentially as described (15Chai W. Luo J. Lim C.K. Lawson A.M. Anal. Chem. 1998; 70: 2060-2066Crossref PubMed Scopus (102) Google Scholar), and the reaction stopped at around 40% completion. Briefly, the heparan sulfate (150 mg) was incubated with heparin lyase III (300 mU) in 50 mm sodium phosphate buffer (pH 7.1, 6 ml) containing 0.1m NaCl at 30 °C for 8.5 h. The digestion mixture was desalted on a Sephadex G-10 column (1.6 × 36 cm) eluted with water at a flow rate of 20 ml/h, lyophilized, and fractionated on a gel filtration column of Bio-Gel P-6 (1.6 × 90 cm) eluting with 0.2m NH4Cl (pH 3.5) at a flow rate of 15 ml/h. The eluate was monitored on-line by refractive index and UV at 232 nm. The pooled fractions were lyophilized and desalted on a Sephadex G-10 column (1.6 × 36 cm). HPLC of HS fragments was carried out on an analytical strong anion exchange (SAX) column, S5-SAX (4.6 × 250 mm, Phase Separations Ltd., Clwyd, UK) using a titanium-lined Gilson liquid chromatograph system fitted with a variable wavelength UV detector monitor at 232 nm. Elution was carried out with a gradient of NaCl (solvent A: 0.2m NaCl and solvent B: 1.5 m NaCl; pH 3.5): 0–5% B in 15 min followed by 5–10% B in 15 min at a flow rate of 1 ml/min. The major fractions were collected, desalted on a Sephadex G10 column, and lyophilized. Fluorescent NGLs were prepared and resolved by high performance (HP) TLC essentially as described previously (12Stoll M.S. Feizi T. Loveless R.W. Chai W. Lawson A.M. Yuen C.-T. Eur. J. Biochem. 2000; 267: 1795-1804Crossref PubMed Scopus (41) Google Scholar). In brief, the following were added to lyophilized HS fragments, typically 50 nmol: of 50 and of prepared of The mixture was incubated at °C for 90 h. were resolved by using a solvent mixture of The fluorescent NGLs were UV at nm. The fluorescent NGLs were fractionated on an column × 250 using a gradient of to B in solvent A (solvent A: and solvent B: both containing acid) in 30 min and a flow rate of ml/min. The were monitored with a detector at and The 4,5-unsaturated hexuronic at the of the HS-1 tetrasaccharides was by W. H. Lawson A.M. Anal. Biochem. 1996; PubMed Scopus Google Scholar). In brief, to a of of tetrasaccharide in was added of mm in mm sodium pH The reaction mixture was at for 30 min and lyophilized. of the lyophilized reaction to was essentially as described but the was limited to h. TLC (11Feizi T. Stoll M.S. Yuen C.-T. Chai W. Lawson A.M. Methods Enzymol. 1994; 230: 484-519Crossref PubMed Scopus (119) Google Scholar) was to the major from the two reaction and to the and reaction NGLs were resolved by using as solvent by of monoclonal antibody 10E4 to NGLs on was essentially as described previously M.S. T. Feizi T. Biochem. J. PubMed Scopus Google Scholar) that was not In brief, after binding sites with in Tris-buffered mm mm pH the was with 10E4 antibody in antibody binding was detected using of in followed by FASTTM 3,3′-diaminobenzidine of antibody were and the derived areas using a with a at nm. binding were essentially as described R.W. G. Yuen C.-T. Feizi T. J. 1992; PubMed Scopus Google Scholar) that UK) were by to 50 of a 50 of poly-l-lysine in phosphate-buffered mm phosphate mm pH and for at in were added at the and to at °C for h. with in binding of 10E4 was detected using antibodies to mouse followed by 2,2′-azino-bis(3-ethylbenzthiazoline-sulfonic acid) was at nm. and collision-induced dissociation were carried out with a Ltd., UK). NGLs were in by typically at a of to 20 of which was mm was by a at a flow rate of was at 30 was at were using as the at a of The was at The was for between and of NGLs resolved on TLC was carried out in by liquid mass spectrometry on a mass as described previously W. Lawson A.M. Carbohydr. Res. 1995; PubMed Scopus Google Scholar). binding experiments that the preparation of heparan sulfate, HS-1, that the lowest sulfate the binding with 10E4 antibody binding were detected with chondroitin sulfate which has a sulfate to that of The HS-1 preparation was for of HS-1 was carried out using heparin lyase III, and this was followed by gel filtration fractions containing the fragments, fractions 1 to were to and to be to with and sulfate by of the contents and with and sulfate in oligosaccharides the pooled HS-1 oligosaccharide fractions in a HS-1 oligosaccharide fractions to 6 were to fluorescent resolved by and examined for 10E4 antibody binding A or be detected in the oligosaccharides at the highest not antigen-positive components were detected among the NGLs of fractions 5, and but not in in fractions and 6 were components as were detectable by which tetrasaccharides and in which the antigen-positive components was for The oligosaccharides were resolved by HPLC and the -2, -3, and were fluorescent NGLs and examined for 10E4 antibody binding B A of components was in but was detected in the other fractions The mixture of NGLs in was resolved by HPLC and and were by binding to to the and in B of HS-1 fragments and of NGLs derived from The oligosaccharides in HS-1 were resolved on a S5-SAX column and the fluorescent NGLs derived from were resolved on a column as described The mass of the a the molecular mass as being and not This to the of a nonsulfated tetrasaccharide containing a as was detected also among the oligosaccharides in The was deduced to be a form of a mass of The a in the of oligosaccharides N-acetylglucosamine at the (11Feizi T. Stoll M.S. Yuen C.-T. Chai W. Lawson A.M. Methods Enzymol. 1994; 230: 484-519Crossref PubMed Scopus (119) Google Scholar) Stoll and W. that the tetrasaccharide a at the of the charged from a in the mass and B. J. Scopus Google Scholar) from the of the oligosaccharide the sequence of the tetrasaccharide be deduced as The a at the The at a mass of to glucosamine The at a mass of to a hexuronic acid and to the sequence The mass of is in with the presence of a to the the sequence of the antigen-positive is that the of the highly heparin lyase III is at a glucuronic acid R.J. D. Gallagher J.T. 1990; PubMed Scopus Google Scholar, T. H. H. T. Hileman R.E. R.J. Biochem. J. PubMed Scopus Google Scholar, G. Z. R. R. Science (Wash. D. C.). PubMed Scopus Google Scholar), the antigen-positive oligosaccharide in the HS-1 is sequence and molecular mass of the antigen-positive tetrasaccharide and of the derived fluorescent The sequence of the tetrasaccharide was deduced from the that from of the The with a molecular mass of was deduced to be a in which the of water are from the or (11Feizi T. Stoll M.S. Yuen C.-T. Chai W. Lawson A.M. Methods Enzymol. 1994; 230: 484-519Crossref PubMed Scopus (119) Google Scholar) neoglycolipid The components in the antigen-negative fractions and were by not The in molecular mass of and is deduced to contain a nonsulfated tetrasaccharide with two Here the carbohydrate sequence be from the by of the charged The at a at the The mass of to a to the and that mass of to an The three the sequence The mass a at the terminal to the these the sequence of this antigen-negative tetrasaccharide is deduced to in molecular mass and is deduced to be a tetrasaccharide with one sulfate and one The sequence can be from the by of at a mass of to and the a mass of to The mass of a to the The of sulfate in can be to the as the at to a mass of sequence of this antigen-negative tetrasaccharide is deduced to There was in of and 6 to NGLs of these were we examined for 10E4 the NGLs of a series of defined heparin/heparan sulfate disaccharides and the derived from by the binding binding less in this was to and It be in the the is in a The in with the of the sequence in 10E4 antigenicity. of these are that or N-sulfated but not a in the penultimate with the and the antibody an outer hexuronic sulfate disaccharides investigated for 10E4 antibody binding binding was by the at are as areas relative to that for as or binding in a Antibody binding was by the at are as areas relative to that for as or binding The major after to the terminal hexuronic acid in the 10E4 antigen-positive tetrasaccharide 6 by a at and two at to the sequence, The liquid mass of the major of the antigen-negative tetrasaccharide to that for at and and The derived from the antigen-positive and antigen-negative with the tetrasaccharides were examined for 10E4 antigenicity by TLC was as in the tetrasaccharide but not in the the hexuronic acid in the tetrasaccharide is for 10E4 antigenicity. The 10E4 tetrasaccharide is, to the carbohydrate antigen sequence to have been on heparan the sequence sulfate and a unique motif with an N-unsubstituted glucosamine. This is important for antigenicity of the tetrasaccharide as shown by the of antibody binding to the two tetrasaccharide one of which has a and the other a N-sulfated glucosamine at this The of N-unsubstituted glucosamine for strong 10E4 antibody binding is by the binding experiments with the series of NGLs derived from heparin/heparan sulfate only the disaccharide containing glucosamine strong The 10E4 binding to the NGLs of the three disaccharides, and the in the heparan sulfate of the glucosamine to antigenicity. the binding with the and disaccharides, a is that a hexuronic acid on the of the glucosamine is for 10E4 antigenicity and that it be are in with the binding of 10E4 to heparan sulfate that is It is that N-unsubstituted glucosamine was by J. K. L. M. M. Lindahl U. J. Biol. Chem. 1995; PubMed Scopus Google Scholar) to be a of the heparan sulfate antigen recognized by monoclonal This antibody been by P.A. Wilson M.I. Eikenlenboom P. Tunstall A. Bruce M.E. Exp. Neurol. 1998; 149: 447-454Crossref PubMed Scopus (65) Google Scholar) in the immunohistochemical of scrapie lesions but was not to at with 10E4 The antigen sequence is from that of the 10E4 and the of the two will be important for the heparan sulfate The of N-unsubstituted glucosamine as a on HSPGs was This is the in the of the chains the formation of a of this is followed by the of a that of N-acetylglucosamine U. K. D. L. Res. 1994; Full Text PDF PubMed Scopus Google Scholar). This to be a for of the carbohydrate chains, which the of glucuronic to acid, and of the at U. K. D. L. Res. 1994; Full Text PDF PubMed Scopus Google Scholar). A that the glucosamine has not yet been glucosamine has been detected by chemical in preparations of heparan A preparation of HSPGs that was by was to contain such N-unsubstituted glucosamine K. A. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). In T. H. H. T. Hileman R.E. R.J. Biochem. J. PubMed Scopus Google Scholar), N-unsubstituted glucosamine was detected as a variable in 50 in heparan sulfates isolated from with oligosaccharide fragments isolated from heparan sulfates have suggested that such glucosamine be by a glucuronic acid on the and and in the between and high T. H. H. T. Hileman R.E. R.J. Biochem. J. PubMed Scopus Google Scholar). It has been suggested that also be in highly that contain acid J. Z. P. K. R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The glucosamine on being an of or of the glycosaminoglycans has been by the finding that oligosaccharides containing N-unsubstituted glucosamine can as for a characterized heparan J. Z. P. K. R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, D. J. P. Bai R.J. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). the for the glucosamine is that it has been as being a of the recognized by two monoclonal 10E4 and of which with a (10McBride P.A. Wilson M.I. Eikenlenboom P. Tunstall A. Bruce M.E. Exp. Neurol. 1998; 149: 447-454Crossref PubMed Scopus (65) Google Scholar, G. Bai X.M. Van Der, Schueren B. Cassiman J.-J. Van Der, Berghe H. J. Cell Biol. 1992; 119: 961-975Crossref PubMed Scopus (414) Google Scholar, J. K. L. M. M. Lindahl U. J. Biol. Chem. 1995; PubMed Scopus Google Scholar, J. 1992; Full Text PDF PubMed Scopus Google Scholar). of and that the expression of 10E4 antigen and (13David G. Bai X.M. Van Der, Schueren B. Cassiman J.-J. Van Der, Berghe H. J. Cell Biol. 1992; 119: 961-975Crossref PubMed Scopus (414) Google Scholar). 10E4 a of monoclonal carbohydrate antigens, is a which by with of the group and series T. PubMed Scopus Google Scholar, S.J. Cell. 1990; 63: Full Text PDF PubMed Scopus Google Scholar, J. Invest. 1993; PubMed Scopus Google Scholar, T. Biol. 1993; 3: Scopus Google Scholar) can be to have There is strong for of to heparan for as and of the of J.T. 1998; Scopus Google Scholar). as added can PrPSc from scrapie-infected cells Prusiner S.B. Sci. U. A. PubMed Scopus Google Scholar), we that the 10E4 with its of a in on heparan is a endogenous for a protein or proteins in the of PrPC to PrPSc it will be important to the and sequence of the 10E4 and its role in the normal and pathological of prion In particular, the as components in binding experiments with fragments of HS-1 be as the of (13David G. Bai X.M. Van Der, Schueren B. Cassiman J.-J. Van Der, Berghe H. J. Cell Biol. 1992; 119: 961-975Crossref PubMed Scopus (414) Google Scholar) the 10E4 antigen glucosamine that It can be therefore, that the 10E4 antigen the tetrasaccharide sequence we have on one or other by N-sulfated glucosamine. The sulfation in to 10E4 antigen sequence will be important to as it has been suggested (5Caughey B. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 1994; 343: 399-404Crossref PubMed Scopus (19) Google Scholar) that endogenous glycosaminoglycans or proteoglycans the in of experiments have that of PrP is a prerequisite for the formation of PrPSc (2Harris D.A. Prions. Molecular and Cellular Biology. Horizon Scientific Press, Wymondham, UK1999Google Scholar) and that negatively charged the rate of of PrPC D.A. J. Biol. Chem. 1995; PubMed Scopus Google Scholar). is to and oligosaccharides for 10E4 antigen We for on and IBEX Technologies for heparin lyase III used for the

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