Large, Tissue-regulated Domain Diversity of Heparan Sulfates Demonstrated by Phage Display Antibodies

Abstract

Heparan sulfates (HS) are long, linear polysaccharides with a high degree of variability. They bind to a vast number of proteins such as growth factors and cytokines, and these interactions are likely to be mediated by specific HS domains. To investigate the structural diversity and topological distribution of HS domains in tissues, we selected a panel of 10 unique anti-HS antibodies using phage display technology. All 10 antibodies recognize a specific HS epitope as demonstrated by enzyme-linked immunosorbent assay using defined synthetic HS oligosaccharides, modified HS/heparin molecules, and HS isolated from a variety of organs. The chemical groups involved in the epitopes could be indicated and the position of sulfate groups is of major importance. All HS epitopes have a defined tissue distribution as shown by immunohistochemistry using rat organs. Taken together, the data show that in vivo, a large number of defined HS epitopes exist that do not occur randomly but are tightly, topologically regulated. Heparan sulfates (HS) are long, linear polysaccharides with a high degree of variability. They bind to a vast number of proteins such as growth factors and cytokines, and these interactions are likely to be mediated by specific HS domains. To investigate the structural diversity and topological distribution of HS domains in tissues, we selected a panel of 10 unique anti-HS antibodies using phage display technology. All 10 antibodies recognize a specific HS epitope as demonstrated by enzyme-linked immunosorbent assay using defined synthetic HS oligosaccharides, modified HS/heparin molecules, and HS isolated from a variety of organs. The chemical groups involved in the epitopes could be indicated and the position of sulfate groups is of major importance. All HS epitopes have a defined tissue distribution as shown by immunohistochemistry using rat organs. Taken together, the data show that in vivo, a large number of defined HS epitopes exist that do not occur randomly but are tightly, topologically regulated. Heparan sulfates (HS), 1The abbreviations used are: HSheparan sulfatesPBSphosphate-buffered salineGlcNSN-sulfated glucosamineGlcNS6S6-O-, N-sulfated glucosamineIdoUAiduronic acidIdoUA2S2-O-sulfated iduronic acidUAglucuronic acid or iduronic acid6S6-O-sulfation2S2-O-sulfation3S3-O-sulfationELISAenzyme-linked immunosorbent assay a class of glycosaminoglycans, are long linear complex polysaccharides covalently bound to a protein core. They have a ubiquitous distribution in the extracellular matrix and on cell surfaces and have been implicated in many basic biological phenomena such as cell migration, adhesion, and differentiation. They play a role in such diverse processes as growth factor/cytokine handling, enzyme regulation, lipid metabolism, blood coagulation, and viral entry (1.Kjellén L. Lindahl U. Annu. Rev. Biochem. 1991; 60: 443-475Crossref PubMed Scopus (1680) Google Scholar, 2.Ruoslahti E. Yamaguchi Y. Cell. 1991; 64: 867-869Abstract Full Text PDF PubMed Scopus (1171) Google Scholar, 3.Iozzo R.V. Annu. Rev. Biochem. 1998; 67: 609-652Crossref PubMed Scopus (1349) Google Scholar, 4.Salmivirta M. Lidholt K. Lindahl U. FASEB J. 1996; 10: 1270-1279Crossref PubMed Scopus (397) Google Scholar). This involvement is mediated by the interactions of HS with a vast number of proteins such as growth factors/cytokines, enzymes, protease inhibitors, extracellular matrix molecules, and viral coat proteins (5.Conrad H.E. Heparin Binding Proteins. 1st Ed. Academic Press, San Diego, CA1998Google Scholar, 6.David G. FASEB J. 1993; 7: 1023-1030Crossref PubMed Scopus (375) Google Scholar). The large number of interactions suggests an extensive structural variation within HS. Chemical analysis of HS-derived disaccharides indeed indicates a large structural diversity (7.Maccarana M. Sakura Y. Tawada A. Yoshida K. Lindahl U. J. Biol. Chem. 1996; 271: 17804-17810Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar, 8.Lyon M. Deakin J.A. Gallagher J.T. J. Biol. Chem. 1994; 269: 11208-11215Abstract Full Text PDF PubMed Google Scholar, 9.Sanderson R.D. Turnbull J.E. Gallagher J.T. Lander A.D. J. Biol. Chem. 1994; 269: 13100-13106Abstract Full Text PDF PubMed Google Scholar), which is brought about by specific chain modifications during HS biosynthesis. The importance of defined monosaccharide sequences for specific interactions with proteins has been demonstrated for the binding and activation of antithrombin III by HS/heparin (10.Atha D.H. Stephens A.W. Rosenberg R.D. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 1030-1034Crossref PubMed Scopus (105) Google Scholar, 11.Lindahl U. Thunberg L. Backstrom G. Riesenfeld J. Nordling K. Bjork I. J. Biol. Chem. 1984; 259: 12368-12376Abstract Full Text PDF PubMed Google Scholar). In addition, specific structural requirements for binding to basic fibroblast growth factor and hepatocyte growth factor have been defined (12.Faham S. Hileman R.E. Fromm J.R. Linhardt R.J. Rees D.C. Science. 1996; 271: 1116-1120Crossref PubMed Scopus (740) Google Scholar, 13.Ashikari S. Habuch I.H. Kimata K. J. Biol. Chem. 1995; 270: 29586-29593Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 14.Nissim A. Hoogenboom H.R. Tomlinson I.M. Flynn G. Midgley C. Lane D. Winter G. EMBO J. 1994; 13: 692-698Crossref PubMed Scopus (528) Google Scholar). These observations indicate that HS modifications do not occur randomly but that a controlled expression of specific domains/sequences in HS exists. To investigate whether a large number of different HS domains indeed occur in tissues, we selected a panel of epitope-specific anti-HS antibodies using phage display technology. Using these antibodies, we chemically and topologically characterized the HS epitopes involved. We chose antibody phage display because it allows for the generation of antibodies against poorly immunogenic molecules such as HS. heparan sulfates phosphate-buffered saline N-sulfated glucosamine 6-O-, N-sulfated glucosamine iduronic acid 2-O-sulfated iduronic acid glucuronic acid or iduronic acid 6-O-sulfation 2-O-sulfation 3-O-sulfation enzyme-linked immunosorbent assay A human semisynthetic antibody phage display library (14, now officially named synthetic scFv Library No. 1) was generously provided by Dr G. Winter, Cambridge University, Cambridge, UK. This library contains 50 different VH genes with synthetic random complementarity determining region 3 segments, which are 4–12 amino acid residues in length. The heavy chains are combined with a single light chain gene (DPL 16). The library contains >108 different clones. All antibodies contain a c-Myc tag. Heparan sulfate from bovine kidney, chondroitinase ABC (Proteus vulgaris, EC 4.2.2.4), chemically modified heparan sulfate and heparin kits, antichondroitin sulfate “stub” antibody (2B6), and antiheparan sulfate stub antibody (3G10) were from Seikagaku Kogyo Co., Tokyo, Japan. Heparin from porcine intestinal mucosa, chondroitin 4-sulfate from whale cartilage, chondroitin 6-sulfate from shark cartilage, dermatan sulfate from porcine skin, keratan sulfate from bovine cornea, hyaluronate from human umbilical cord, DNA from calf thymus, dextran sulfate, bovine serum albumin (fraction V), heparinase I (from Flavobacterium heparinum, EC 4.2.2.7), heparinase II (from F. heparinum), and heparinase III (heparitinase, from F. heparinum, EC 4.2.2.8) were from Sigma. Alkaline phosphatase-conjugated rabbit anti-mouse IgG was from Dakopatts, Glostrup, Denmark. Mouse anti-c-Myc tag IgG (clone 9E10) was from Roche Molecular Biochemicals. Alexa 488-conjugated goat anti-mouse IgG was from Molecular Probes, Eugene, OR. The PCR kit was from Promega, Madison, WI. ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction Kit was from PE Applied Biosystems, Norwalk, CT. Mowiol (4–88) was from Calbiochem. Restriction enzyme BstNI was from New England Biolabs, Beverly, MA. Polystyrene Maxisorp Immunotubes were from Nunc, Roskilde, Denmark. Various HS preparations were a generous gift from Dr. Keiichi Yoshida, Seikagaku Corp., Japan. Synthetic HS oligosaccharides were all from Sanofi-Synthélabo Recherche, Toulouse, France. Phage display-derived antibodies were obtained as described (15.van Kuppevelt T.H. Dennissen M.A.B.A. van Venrooij W.J. Hoet R.M.A. Veerkamp J.H. J. Biol. Chem. 1998; 273: 12960-12966Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar) using four rounds of panning against bovine kidney HS, human lung HS (HS isolated using papaine/alkaline borohydride digestion followed by anion exchange chromatography), and mouse and human skeletal muscle HS (16.Jenniskens G.J. Oosterhof A. Brandwijk R. Veerkamp J.H. van Kuppevelt T.H. J. Neurosci. 2000; 20: 4099-4111Crossref PubMed Google Scholar). Briefly, antibody-expressing phages were added to HS-coated tubes, and bound phages were eluted at high pH to allow for the infection of Escherichia coli TG1 cells. After overnight amplification, phage were rescued by the addition of helper phage and used for further rounds of selections. After the last two rounds, single bacterial colonies were picked, and the antibodies expressed were evaluated. Screening for bacteria expressing anti-HS antibodies was performed as described (15.van Kuppevelt T.H. Dennissen M.A.B.A. van Venrooij W.J. Hoet R.M.A. Veerkamp J.H. J. Biol. Chem. 1998; 273: 12960-12966Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). Briefly, single colonies picked from the last two rounds of selection were grown in 96-well polystyrene round bottom plates until bacterial growth was visible. Antibody production was induced by adding isopropyl-β-d-thiogalactopyranoside. Plates were centrifuged, and the supernatant containing soluble antibodies was applied to wells of polystyrene microtiter plates previously coated with HS. Bound antibodies were detected using a mouse monoclonal antibody (9E10) directed against the c-Myc tag followed by incubation with alkaline phosphatase-conjugated rabbit anti-mouse IgG. Alkaline phosphatase activity was detected using p-nitrophenyl phosphate as a substrate. Absorbance was measured at 405 nm. Bacteria expressing anti-HS antibodies were analyzed for the presence of full-length inserts by 25 cycles of PCR using plasmid DNA as template (15.van Kuppevelt T.H. Dennissen M.A.B.A. van Venrooij W.J. Hoet R.M.A. Veerkamp J.H. J. Biol. Chem. 1998; 273: 12960-12966Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). Fingerprinting was performed using BstNI (CC(A/T)GG) as the restriction enzyme. PCR and fingerprinting analysis were performed using 1% (w/v) and 4% (w/v) agarose gels, respectively. To establish the complementarity determining region 3 and the germ line VHgene DNA segments, unique clones were sequenced using FOR LINK SEQ RIC (5′-GCCACCTCCGCCTGAACC) as the primer (located in the linker region between the VH and the VL gene). For this purpose, double-stranded DNA was isolated using standard procedures. To obtain optimal amounts of soluble anti-HS antibodies, phages were allowed to infect the non-suppressor E. coli strain HB2151. The periplasmic fraction of bacteria was used as the source of antibodies (15.van Kuppevelt T.H. Dennissen M.A.B.A. van Venrooij W.J. Hoet R.M.A. Veerkamp J.H. J. Biol. Chem. 1998; 273: 12960-12966Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). Briefly, bacteria were grown at 37 °C until an optical density of 0.5–0.8 was reached. Induction was obtained by the addition of isopropyl-β-d-thiogalactopyranoside. After incubation at 30 °C for 3 h, the culture was harvested, and the pellet was resuspended in 200 mm sodium borate buffer (pH 8.0) containing 160 mm NaCl and 1 mm EDTA. After centrifugation, the supernatant was filtered and dialyzed versus PBS. The preparation thus obtained is the periplasmic fraction. Reactivity of the anti-HS antibodies with various molecules was evaluated in ELISA in two ways: (a) by the application of antibodies to wells of polystyrene plates coated with the test molecules and (b) by an inhibition assay in which the antibodies were incubated with the test molecule for 16 h at 22 °C followed by transfer to wells previously coated with HS (bovine kidney, (15.van Kuppevelt T.H. Dennissen M.A.B.A. van Venrooij W.J. Hoet R.M.A. Veerkamp J.H. J. Biol. Chem. 1998; 273: 12960-12966Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar)). Test molecules included HS from bovine kidney, aorta, lung, intestine, human aorta, pig intestine, and whale lung; heparin, dermatan sulfate, chondroitin 4- and 6-sulfate, keratan sulfate, dextran sulfate, hyaluronate, and K5 polysaccharide from E. coli; bovine serum albumin; marvel (the non-fat milk preparation using as a blocking reagent during panning); and DNA. To analyze which chemical groups in HS are involved in antibody binding, a large number of modified HS preparations and synthetic HS-oligosaccharides were evaluated. They included HS from bovine kidney, which was digested with heparinase I, II, or III or with HNO2 at pH 1.5, and chemically modified HS (bovine kidney) and heparins (porcine intestine). The latter included preparations that were completely desulfated and N-acetylated, preparations that were completely O-desulfated and N-sulfated, and preparations that were N-desulfated and N-acetylated. The antibodies were also analyzed using a panel of 12 defined synthetic HS oligosaccharides. Bound antibodies were detected using anti c-Myc tag mouse monoclonal antibody 9E10 and alkaline phosphatase-conjugated rabbit anti-mouse IgG antibody. Enzyme activity was detected using p-nitrophenyl phosphate as the substrate, and absorbance was read at 405 nm. Various rat tissue specimens (Wistar, male, 3 months old) were snap-frozen in liquid isopentane cooled with liquid nitrogen and stored at −70 °C. Cryosections (5 μm) were rehydrated for 10 min in PBS, blocked with PBS containing 1% (w/v) bovine serum albumin, and incubated with anti-HS antibodies for 90 min at 22 °C. As a control, antibodies from non-related clones were used. These antibodies did not stain rat tissue were detected by incubation with mouse anti-c-Myc tag antibodies culture followed by Alexa 488-conjugated goat anti-mouse IgG in PBS containing 1% bovine serum for 90 min at 22 °C. After were in PBS for 10 were the of or antibody. To the of the antibodies, were with heparinase III and with chondroitinase ABC dermatan and chondroitin As control, were incubated in the buffer enzyme. After and the were incubated with antibodies and for as The of heparinase III was analyzed using antibodies (3G10) against HS by heparinase The of chondroitinase ABC was evaluated by incubation of with antibodies against chondroitin sulfate by chondroitinase of the anti-HS antibodies was further by overnight of the antibodies with various molecules, (bovine heparin, dermatan sulfate, chondroitin 4- and 6-sulfate, keratan sulfate, dextran sulfate, hyaluronate, and DNA as described (15.van Kuppevelt T.H. Dennissen M.A.B.A. van Venrooij W.J. Hoet R.M.A. Veerkamp J.H. J. Biol. Chem. 1998; 273: 12960-12966Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar) followed by for antibodies were selected on the of the amino acid of the complementarity determining region 3 VH gene I.M. G. Winter G. J. Biol. PubMed Scopus Google Scholar). antibodies were obtained against bovine kidney HS and and antibodies were obtained against human lung HS and The antibodies against mouse and human skeletal muscle HS and have been described previously (16.Jenniskens G.J. Oosterhof A. Brandwijk R. Veerkamp J.H. van Kuppevelt T.H. J. Neurosci. 2000; 20: 4099-4111Crossref PubMed Google of the anti-HS are the gene number to I.M. G. Winter G. J. Biol. PubMed Scopus Google Scholar), VH germ line gene and amino acid of the determining region in a are the gene number to I.M. G. Winter G. J. Biol. PubMed Scopus Google Scholar), VH germ line gene and amino acid of the determining region Using antibodies were shown to be specific for HS and heparin and not to be with such as dermatan sulfate, chondroitin hyaluronate, and keratan sulfate with molecules such as dextran sulfate and DNA. show a with HS preparations isolated from various such as bovine kidney, aorta, lung, and intestine, human aorta, and whale lung of the antibodies is with the bacterial polysaccharide which is to the HS all 10 antibodies recognize specific structural of anti-HS antibodies with various HS preparations bovine to the HS E. bovine 10 10 10 human bovine kidney (fraction A from an anion exchange between and fraction between and bovine lung 30 10 bovine bovine kidney (fraction A from an anion exchange between and fraction between and whale lung porcine of with HS from bovine kidney) The of sulfate groups to is indicated in to the HS A from an anion exchange between and fraction between and in a of with HS from bovine kidney) The of sulfate groups to is indicated in were for with modified HS/heparin preparations and with various synthetic HS oligosaccharides to which modifications in HS are involved in show that all antibodies recognize different in HS. In all sulfate groups are HS and heparin from and groups are not with the of HS with HNO2 at pH at N-sulfated glucosamine residues and the glucosamine antibody of HS with heparinase I at in the of binding activity of of 10 For a number of antibodies, structural modifications of HS for binding could be III and domains within HS it with oligosaccharides. The antibody 6-O-, N-sulfated glucosamine and iduronic acid iduronic acid The at the is and recognize containing of binding 10 and 12 and and in topological distribution indicate that antibody a different Antibody various and the be described as is not for binding This indicate the of a variety of but the topological distribution against this and are with domains containing binding 10 and For an on has and an of the of and for of importance HS epitopes by the two antibodies have a different topological distribution Antibody is not with of the synthetic HS oligosaccharides is the antibody that with completely that is of major importance. with human HS which is of 6-O-sulfation F. Lindahl U. M. 2000; PubMed Scopus Google Scholar), and which indicate that this is Taken together, these data that domains. domains containing or 2-O-sulfation of binding 10 and 3-O-sulfation of to be of importance and in a chemical by a but not by a and and 10 and The HS that could not be All antibodies, and bind to an isolated from the extracellular matrix of the mouse J.R. J. S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) not of anti-HS antibodies with modified HS and heparin molecules (bovine (porcine intestinal (pH the of test with which in a inhibition of the binding of the antibody to HS HNO2 glucosamine with the amino N-sulfated or I the heparinase II the and heparinase III the and at in a of anti-HS antibodies with synthetic HS oligosaccharides I.M. G. Winter G. J. Biol. PubMed Scopus Google the of with which in a inhibition of the binding of the antibody to HS. at for in a the of test with which in a inhibition of the binding of the antibody to HS HNO2 glucosamine with the amino N-sulfated or I the heparinase II the and heparinase III the and at the of with which in a inhibition of the binding of the antibody to HS. at for antibody that the HS it has a defined In the kidney, the anti-HS antibodies stain and have a recognize with a topological distribution as with by the These antibodies show a or tissue with in antibodies and show a topological in tissue but a distribution in tissue of defined by antibodies in rat a of is muscle blood were for as described I.M. G. Winter G. J. Biol. PubMed Scopus Google Scholar). a of is in a Cryosections were for as described I.M. G. Winter G. J. Biol. PubMed Scopus Google Scholar). of all the antibodies was by of with heparinase III but not with chondroitinase of antibodies with HS and heparin with glycosaminoglycans, with dextran sulfate, or with DNA has to proteins and polysaccharides a of This for HS, a class of linear polysaccharides involved in many basic a large of different monosaccharide sequences in HS To whether such a indeed in vivo, we selected 10 anti-HS antibodies by phage display technology. This has to be for antibodies against HS, which are (15.van Kuppevelt T.H. Dennissen M.A.B.A. van Venrooij W.J. Hoet R.M.A. Veerkamp J.H. J. Biol. Chem. 1998; 273: 12960-12966Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, G.J. Oosterhof A. Brandwijk R. Veerkamp J.H. van Kuppevelt T.H. J. Neurosci. 2000; 20: 4099-4111Crossref PubMed Google Scholar). All 10 antibodies recognize a unique epitope the position of sulfate groups of major importance. with the anti-HS antibodies described (15.van Kuppevelt T.H. Dennissen M.A.B.A. van Venrooij W.J. Hoet R.M.A. Veerkamp J.H. J. Biol. Chem. 1998; 273: 12960-12966Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, G.J. J. Biol. PubMed Scopus Google Scholar, van J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar), which are different with to the described it that at different HS epitopes are in the kidney, the of the epitopes All epitope have a defined topological tissue that in vivo, a controlled for HS exists. This also be for chondroitin sulfates a number of sulfate antibodies have been that show a specific distribution in J.R. J.E. J.R. 1984; PubMed Scopus Google Scholar, F. J. PubMed Scopus Google Scholar). The of HS allows for a large number of different The HS polysaccharide chain is of that of a glucuronic acid and a These of which be to a in HS, a number of residues are and N-sulfated, and this as a for further modifications such as of to 2-O-sulfation of and and 3-O-sulfation of in specific of the polysaccharide chain to modified domains and by modified domains (1.Kjellén L. Lindahl U. Annu. Rev. Biochem. 1991; 60: 443-475Crossref PubMed Scopus (1680) Google Scholar, 4.Salmivirta M. Lidholt K. Lindahl U. FASEB J. 1996; 10: 1270-1279Crossref PubMed Scopus (397) Google Scholar). The number of and modifications to a structural for has a monosaccharide The importance of specific modifications in have been in and the involvement of HS in cell and In in genes involved in HS and to a of specific and in in In a in the gene to gene in the genes and in and cell M. 2000; PubMed Scopus Google Scholar). In in involved in HS and to C. Y. D. K. F. 1998; PubMed Scopus Google Scholar). These indicate that specific HS modifications play a role in data indicate that indeed a large number of HS epitopes exist in and that these do not occur randomly but are tightly, regulated. This indicates the of a controlled of modifications during the of HS. in HS have been in various and bacterial and viral van J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, J. A. Lander A.D. M. J. 1998; PubMed Scopus Google Scholar, EMBO J. 1995; PubMed Scopus Google Scholar, D. J. Biol. PubMed Scopus Google Scholar, D. J. R.J. Rosenberg R.D. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, Y. Hileman Fromm J.R. Linhardt R.J. PubMed Scopus Google Scholar). The antibodies described which have a of about (15.van Kuppevelt T.H. Dennissen M.A.B.A. van Venrooij W.J. Hoet R.M.A. Veerkamp J.H. J. Biol. Chem. 1998; 273: 12960-12966Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar), be used in and cell They also be used for we did not test the HS J.E. Gallagher J.T. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, G. R. R. Science. PubMed Scopus (218) Google Scholar) and to synthetic HS oligosaccharides M. A. PubMed Scopus Google Scholar), the HS be characterized at the monosaccharide The epitope defined by anti-HS antibody was characterized C. K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). defined anti-HS antibodies it to defined structural and monosaccharide to biological processes in and We to Dr. G. Winter, Cambridge for the phage display library and to Dr. Keiichi Yoshida for heparan sulfate preparations Corp.,