Characterization of Anti-heparan Sulfate Phage Display Antibodies AO4B08 and HS4E4

Heparan sulfates (HS) are linear carbohydrate chains, covalently attached to proteins, that occur on essentially all cell surfaces and in extracellular matrices. HS chains show extensive structural heterogeneity and are functionally important during embryogenesis and in homeostasis due to their interactions with various proteins. Phage display antibodies have been developed to probe HS structures, assess the availability of protein-binding sites, and monitor structural changes during development and disease. Here we have characterized two such antibodies, AO4B08 and HS4E4, previously noted for partly differential tissue staining. AO4B08 recognized both HS and heparin, and was found to interact with an ubiquitouys, N-, 2-O-, and 6-O-sulfated saccharide motif, including an internal 2-O-sulfate group. HS4E4 turned out to preferentially recognize low-sulfated HS motifs containing iduronic acid, and N-sulfated as well as N-acetylated glucosamine residues. Contrary to AO4B08, HS4E4 did not bind highly O-sulfated structures such as found in heparin. Heparan sulfates (HS) are linear carbohydrate chains, covalently attached to proteins, that occur on essentially all cell surfaces and in extracellular matrices. HS chains show extensive structural heterogeneity and are functionally important during embryogenesis and in homeostasis due to their interactions with various proteins. Phage display antibodies have been developed to probe HS structures, assess the availability of protein-binding sites, and monitor structural changes during development and disease. Here we have characterized two such antibodies, AO4B08 and HS4E4, previously noted for partly differential tissue staining. AO4B08 recognized both HS and heparin, and was found to interact with an ubiquitouys, N-, 2-O-, and 6-O-sulfated saccharide motif, including an internal 2-O-sulfate group. HS4E4 turned out to preferentially recognize low-sulfated HS motifs containing iduronic acid, and N-sulfated as well as N-acetylated glucosamine residues. Contrary to AO4B08, HS4E4 did not bind highly O-sulfated structures such as found in heparin. Heparan sulfates (HS) 3The abbreviations used are: HS, heparan sulfate; GlcNAc, N-acetyl-d-glucosamine; GlcA, d-glucuronic acid; IdoA, l-iduronic acid; RPIP-HPLC, reversed phase-ion pair high performance liquid chromatography; PAPS, adenosine 3′-phosphate 5′-phosphosulfate; CHO, Chinese hamster ovary; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; BSA, bovine serum albumin; VSV, vesicular stomatitis virus; dpc, days post coitus. 3The abbreviations used are: HS, heparan sulfate; GlcNAc, N-acetyl-d-glucosamine; GlcA, d-glucuronic acid; IdoA, l-iduronic acid; RPIP-HPLC, reversed phase-ion pair high performance liquid chromatography; PAPS, adenosine 3′-phosphate 5′-phosphosulfate; CHO, Chinese hamster ovary; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; BSA, bovine serum albumin; VSV, vesicular stomatitis virus; dpc, days post coitus. are linear, sulfated polysaccharides that occur covalently bound to core proteins in proteoglycan structures at cell surfaces and in extracellular matrices. Due to their negative charge, HS chains interact with a variety of proteins, and thus modulate important processes in embryogenesis and tissue homeostasis (1Esko J.D. Selleck S.B. Annu. Rev. Biochem. 2002; 71: 435-471Crossref PubMed Scopus (1231) Google Scholar, 2Lin X. Development. 2004; 131: 6009-6021Crossref PubMed Scopus (526) Google Scholar). Biosynthesis of HS involves formation of a precursor polysaccharide composed of alternating N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcA) residues, which is subsequently modified through a series of enzymatic reactions. The modifications include N-deacetylation and N-sulfation of GlcNAc residues, C5-epimerization of GlcA to iduronic acid (IdoA) units, and finally O-sulfation at C2 of hexuronic acid and C6, more rarely C3, of glucosamine units. The reactions are generally incomplete, yielding products of extensive structural heterogeneity (3Esko J.D. Lindahl U. J. Clin. Investig. 2001; 108: 169-173Crossref PubMed Scopus (783) Google Scholar). The modification process is not under template control, yet tightly regulated, such that differences in structure of HS generated in different tissues are strikingly conserved (4Maccarana M. Sakura Y. Tawada A. Yoshida K. Lindahl U. J. Biol. Chem. 1996; 271: 17804-17810Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 5Ledin J. Staatz W. Li J.P. Goötte M. Selleck S. Kjellén L. Spillmann D. J. Biol. Chem. 2004; 279: 42732-42741Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). Although aspects of specificity of HS-protein interactions remain unclear, these differences are presumably of functional significance (6Kreuger J. Spillmann D. Li J.-p. Lindahl U. J. Cell Biol. 2006; 174: 323-327Crossref PubMed Scopus (399) Google Scholar). Elucidation of these problems is hampered by the lack of high-throughput tools for detailed structural characterization of HS, which remains a tedious task. Phage display antibodies have been developed to overcome the limited immunogenicity of HS, thereby making it possible to highlight tissue-specific HS structures and to follow changes in development and disease. Using such antibodies, the differential expression of HS motifs in various tissues has been demonstrated (7van 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 (216) Google Scholar, 8ten Dam G.B. Hafmans T. Veerkamp J.H. van Kuppevelt T.H. J. Histochem. Cytochem. 2003; 51: 727-739Crossref PubMed Scopus (32) Google Scholar, 9Lensen J.F.M. Rops A.L.W.M.M. Wijnhoven T.J.M. Hafmans T. Feitz W.F.J. Oosterwijk E. Banas B. Bindels R.J.M. van den Heuvel L.P.W.J. van der Vlag J. Berden J.H.M. van Kuppevelt T.H. J. Am. Soc. Nephrol. 2005; 16: 1279-1288Crossref PubMed Scopus (43) Google Scholar). Yet, few of these antibodies have so far been characterized for their epitope specificity, which hampers further use of these tools and interpretation of results. We have selected two of these phage display antibodies, AO4B08 and HS4E4, based on their tissue staining properties (8ten Dam G.B. Hafmans T. Veerkamp J.H. van Kuppevelt T.H. J. Histochem. Cytochem. 2003; 51: 727-739Crossref PubMed Scopus (32) Google Scholar, 9Lensen J.F.M. Rops A.L.W.M.M. Wijnhoven T.J.M. Hafmans T. Feitz W.F.J. Oosterwijk E. Banas B. Bindels R.J.M. van den Heuvel L.P.W.J. van der Vlag J. Berden J.H.M. van Kuppevelt T.H. J. Am. Soc. Nephrol. 2005; 16: 1279-1288Crossref PubMed Scopus (43) Google Scholar) and their selective interaction with HS from different sources (10Dennissen M.A.B.A. Jenniskens G.J. Pieffers M. Versteeg E.M.M. Petitou M. Veerkamp J.H. van Kuppevelt T.H. J. Biol. Chem. 2002; 277: 10982-10986Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). The epitopes recognized by these two antibodies were further defined in the present study. AO4B08 thus preferentially binds a N-sulfated octasaccharide epitope with three consecutive 6-O-sulfates and an internally located 2-O-sulfate group, and interacts efficiently with fully sulfated heparin. HS4E4 binds preferentially to different structures present in HS with a moderate degree of sulfation and N-acetylated in addition to N-sulfated glucosamine residues and epimerized iduronic acid. Materials—Protein A-Sepharose and Sephadex G-15 beads, a prepacked Superdex 30 (1.6 × 60 cm) and prepacked PD-10 columns were purchased from GE Healthcare. A Propac PA1 column was purchased from Dionex (Surrey, United Kingdom), a Partisil-10 SAX column from Whatman Inc., and Bio-Gel P-10, fine, was obtained from Bio-Rad. Molecular porous membrane dialysis tubing was from Spectrum (Gardena, CA). NaB3H4 (50–75 Ci/mmol) and [3H]acetic anhydride (500 mCi/mmol) were purchased from GE Healthcare. Carrier-free Na 352SO4 (1500 Ci/mmol) was purchased from PerkinElmer Life Sciences. Ham’s F-12 medium was from Invitrogen and fetal calf serum from PAA Laboratories (Pasching, Austria). The mouse anti-VSV tag IgG antibody P5D4 was obtained from Roche Applied Science and the rabbit anti-VSV tag IgG antibody was from Sigma. The Alexa 488-conjugated goat antirabbit IgG and Alexa 488-conjugated goat anti-mouse IgG were from Molecular Probes (Eugene, OR). Alkaline phosphatase-conjugated rabbit anti-mouse IgG was from Dakopatts (Glostrup, Denmark). The phosphatase substrate p-nitrophenyl phosphate was from ICN (Aurora, OH). Mowiol was from Calbiochem CA). and cell J.D. U. S. A. PubMed Scopus Google Scholar) were by J. and were in Ham’s F-12 medium with fetal calf and mouse were as J.P. E. M. X. Lindahl U. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). and mouse M. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar) were obtained from polysaccharide from epimerized of hexuronic acid O-sulfated and high of were by from in heparin, heparin, heparin, and N-sulfated degree and were by A. and were from was from HS in was purchased from The of HS, various and are in from and A. PubMed Scopus Google Scholar) and from bovine and bovine U. Lindahl B. L. J. Biol. Chem. Full Text PDF PubMed Google Scholar) were as was used for The was a by and PAPS, and were purchased from Sigma. was by M. was generated by and with Na 352SO4 J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). The was by on of a linear from to were of of and polysaccharide used in polysaccharides of heparin, HS and E. polysaccharides used of as from of by the The of the are as of as and for and different of in a polysaccharides of heparin, HS and E. polysaccharides used of as from by the The of the are as of as and for and HS different of in a in a and of Phage display antibodies HS4E4 HS (7van 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 (216) Google and AO4B08 mouse HS G.J. A. Veerkamp J.H. van Kuppevelt T.H. J. PubMed Google to the and have the and The for the antibodies were M. of of antibodies, of were as Dam G.B. S. van Versteeg Lindahl U. Spillmann D. van Kuppevelt T. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). HS4E4 AO4B08 antibodies were and by to The containing antibody was PBS, and AO4B08 and HS4E4 were Dam G.B. S. van Versteeg Lindahl U. Spillmann D. van Kuppevelt T. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). The antibody was at and bovine serum and were for and The of was by and as Dam G.B. S. van Versteeg Lindahl U. Spillmann D. van Kuppevelt T. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). from HS and and were as previously M. J. Lindahl U. Biochem. PubMed Scopus Google glucosamine residues. was in of in and of [3H]acetic anhydride was in a of The was from on PD-10 columns with in from the were and by extensive dialysis dialysis tubing with a The for the various N-sulfated N-sulfated epimerized N-sulfated two of HS from and HS from and from bovine polysaccharide were modified with as of and N-sulfated and were with from a in the of of of polysaccharide of PAPS, and of in of at the reactions were by at for The were and the was through a column × cm) of Sephadex G-15 with in the of the was and by The for the various N-sulfated N-sulfated N-sulfated from bovine were generated by at N-sulfated glucosamine residues, by of products with and by as E. Spillmann D. Lindahl U. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The products a of × octasaccharide was further by on as Dam G.B. S. van Versteeg Lindahl U. Spillmann D. van Kuppevelt T. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). were generated from bovine as Dam G.B. S. van Versteeg Lindahl U. Spillmann D. van Kuppevelt T. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). was to all and of the 2-O-sulfate and was to by NaB3H4 × was and with different of 2-O-sulfate was with in the of PAPS, 6-O-sulfated was essentially as Dam G.B. S. van Versteeg Lindahl U. Spillmann D. van Kuppevelt T. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar) with of antibody to of in polysaccharide were with a of column of and in polysaccharide for antibody were in based on by Biochem. PubMed Scopus Google Scholar). ELISA, of were with polysaccharide and as (7van 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 (216) Google Scholar) by with PBS, BSA, for containing AO4B08 HS4E4 in PBS, BSA, were and for antibodies were mouse anti-VSV tag IgG antibody P5D4 by with phosphatase-conjugated rabbit anti-mouse IgG both for was of p-nitrophenyl phosphate in as a was at of were obtained from and mouse and mouse and and mouse were for with PBS, BSA, were with antibodies AO4B08 HS4E4 in for antibodies were by with rabbit anti-VSV tag IgG antibody by Alexa 488-conjugated goat IgG for were in for and in of were with antibodies AO4B08 HS4E4 in the of to the of antibody to HS Dam G.B. S. van Versteeg Lindahl U. Spillmann D. van Kuppevelt T. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). antibodies were by with mouse anti-VSV tag IgG antibody P5D4 by Alexa 488-conjugated goat anti-mouse IgG on and cell was by antibodies AO4B08 HS4E4 in medium to the for antibodies were essentially as of of epitope selected were on an AO4B08 The and were in a and through a of and enzymatic as J. M. L. Lindahl U. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). were to by with in at were for and and the was by addition of The were and a of the by on a Propac column as J. M. L. Lindahl U. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). of the were to enzymatic of in a of of at for were on the Propac was obtained by in the of the HS was to at PubMed Scopus Google Scholar) to N-deacetylation by at Y. Biochem. PubMed Scopus Google Scholar) by at The products were to on HS4E4 and HS were with of and in bovine serum and for at were and in for by reversed phase-ion pair high performance liquid as J. Staatz W. Li J.P. Goötte M. Selleck S. Kjellén L. Spillmann D. J. Biol. Chem. 2004; 279: 42732-42741Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). the degree of were obtained by of the chains at PubMed Scopus Google by NaB3H4 and by on a Sephadex G-15 column E. Spillmann D. Lindahl U. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The were by on a Partisil-10 SAX column as E. Spillmann D. Lindahl U. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) and the by on Whatman in M. Lindahl U. Biochem. J. PubMed Scopus Google Scholar). were by of hexuronic acid the Biochem. PubMed Scopus Google Scholar) with GlcA as a of was to to saccharide of and HS to AO4B08 and two antibodies, AO4B08 and HS4E4, been selected by to mouse and HS, and as well as differential staining of tissue (8ten Dam G.B. Hafmans T. Veerkamp J.H. van Kuppevelt T.H. J. Histochem. Cytochem. 2003; 51: 727-739Crossref PubMed Scopus (32) Google Scholar). in staining were were with AO4B08 and HS4E4 J.F.M. Rops A.L.W.M.M. Wijnhoven T.J.M. Hafmans T. Feitz W.F.J. Oosterwijk E. Banas B. Bindels R.J.M. van den Heuvel L.P.W.J. van der Vlag J. Berden J.H.M. van Kuppevelt T.H. J. Am. Soc. Nephrol. 2005; 16: 1279-1288Crossref PubMed Scopus (43) Google Scholar). we with antibodies at to highlight differences in for different epitopes in the antibodies and at staining of the and the was the in a AO4B08 staining of as well as and HS4E4, the in staining of the at a of was staining of and that the two antibodies preferentially bind to different We the antibodies the to bind various defined polysaccharides in AO4B08 and HS to both of polysaccharides to HS from bound with HS and was at The of antibody HS4E4 from that of AO4B08, in that both and HS were and of the HS high for of by two antibodies more in their interactions a of in AO4B08 with to a with HS from of from antibody that all of to not to the as by the of HS with for of the modified AO4B08 sulfation residues of thus hexuronic acid residues in epitope HS4E4 a different interaction Contrary to AO4B08, which was to bind heparin, HS4E4 a strikingly to bind as well as with the fully sulfated of both of the of heparin, to that of of all in the that are for antibody of in polysaccharide HS4E4 that were of the in in these that HS4E4 an epitope containing and residues, and that O-sulfation antibody of in HS functional of the HS have been to with structural J.P. E. M. X. Lindahl U. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, M. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). K. J. Biol. Chem. J. M. J. and T. van AO4B08 was to tissue from of the in with the that both and were for antibody The to tissues from the the that such tissues lack 2-O-sulfate with a for in antibody The for 2-O-sulfate was by the of antibody AO4B08 to recognize in HS4E4 was of staining tissues from as well as bound to did not tissues from the the that residues are for HS4E4 2-O-sulfate are in AO4B08 of and of for AO4B08 was further by a of fully N-sulfated defined of in different Dam G.B. S. van Versteeg Lindahl U. Spillmann D. van Kuppevelt T. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). The was such that octasaccharide in with to the of for antibody containing three were essentially to bind AO4B08 at A fully was from the antibody column with to an octasaccharide selected for to that did not to AO4B08 the of was not the an with three and in was to bind the the in with interaction was bound and on the AO4B08 The of bound for with 2-O-sulfate of and of two the was in of differences of for antibody these were all fully 6-O-sulfated for the residues, that were not recognized as substrate by the was limited to the of the group. we the to on the AO4B08 by of and J. M. L. Lindahl U. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). The were to yielding of all the of the of thus from the two and three that by with previously structures J. M. L. Lindahl U. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, J. M. L. Lindahl U. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google to a at a with at a with two at a with three at and the with at the of the of products was with and the was to in of by a for of 2-O-sulfate in the octasaccharide The octasaccharide was not by a of structures in The was in the due to of 2-O-sulfate at The was by the the of at The was not by the are in of the two structures a and a 2-O-sulfate on of the two internal residues. of the octasaccharide and products A of the was at a at was The was not by of the octasaccharide and the with of 2-O-sulfate these two both fully their 2-O-sulfate located at the of at the of the structures in and is that the 2-O-sulfate for AO4B08 on of the two hexuronic acid residues. of to at for interaction with HS4E4 presumably of the structural for epitope polysaccharide chains were on HS4E4 columns that of HS bound well to the antibodies such was used to the of epitopes in the was by in the HS with [3H]acetic The was to on HS4E4, yielding a with and two bound with and of the and of were to at thus all N-sulfated N-acetylated B. Lindahl U. Chem. Biochem. 2001; PubMed Scopus Google Scholar). The products were all to bind the antibody not a for more for with N-sulfation (3Esko J.D. Lindahl U. J. Clin. Investig. 2001; 108: 169-173Crossref PubMed Scopus (783) Google Scholar). were by by at a process N-sulfated structures B. Lindahl U. Chem. Biochem. 2001; PubMed Scopus Google Scholar). the in N-deacetylation these products were by with of the N-sulfated to the HS4E4 column to and the that epitopes recognized by HS4E4 both N-acetylated and N-sulfated structural further the of modification in HS4E4 we a series of various N-sulfated polysaccharide as for GlcA and in the of The polysaccharide has the structure as the precursor in and is modified through C5-epimerization of GlcA to units, and O-sulfation in various M. Lindahl U. Biochem. J. PubMed Scopus Google Scholar). We three that been such that and of the glucosamine residues were of the were all by HS4E4 of the N-sulfated was on the antibody column enzymatic and for the N-sulfated enzymatic modification the N-sulfated was a precursor to epitopes The at and at of the polysaccharides were for by the obtained from modified and N-sulfated in the were of the glucosamine residues the from both of precursor of O-sulfation with their the of a epitope structure recognized by HS4E4, the structural including both and as well as The of by N-sulfated the lack of N-acetylated units. with these fully N-sulfated of the degree of were to interact with the was the high for recognized not were to modification as by of the N-sulfated The from to of the hexuronic acid in these we have the degree of of the and N-sulfated with an degree of O-sulfation to the HS4E4 column to a not the of to HS4E4 modified polysaccharides used in polysaccharides of the of N-sulfation were modified by with as under and used in of as from of of chains by of of of N-sulfated by and of by and E. polysaccharides of the of N-sulfation were modified by with as under and used in of as from of chains by of of of N-sulfated by and of by and in a Here we the characterization of epitopes recognized by two different phage display antibodies, AO4B08 and antibodies to a of antibodies, selected for to HS to tools for HS structural changes in development and (7van 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 (216) Google Scholar). was to AO4B08 and HS4E4, which show differences in epitope to HS involves of proteins to the various sulfated of the polysaccharide are of essentially two composed of N-sulfated that both and and composed of alternating N-acetylated and N-sulfated units. The as well as from in 2-O-sulfate residues (4Maccarana M. Sakura Y. Tawada A. Yoshida K. Lindahl U. J. Biol. Chem. 1996; 271: 17804-17810Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). The structural are fully N-acetylated and essentially lack and AO4B08 was selected mouse G.J. A. Veerkamp J.H. van Kuppevelt T.H. J. PubMed Google Scholar) and with both and HS from various sources and not a variety of tissue structures as it with a N-sulfated containing both 6-O-sulfated glucosamine and hexuronic acid. N-, 2-O-, and are all for antibody as from with sulfated polysaccharides and modified as well as from of tissues in selected in HS of O-sulfated of AO4B08 a for three consecutive N-, 6-O-sulfated with an internally hexuronic acid and 2-O-sulfate in of M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Lindahl U. M. PubMed Scopus Google and the of in addition to the did not epitope that the epitope in tissues by staining with AO4B08 the of O-sulfated composed of at three units. to in HS J. T. M. T. L. Spillmann D. Lindahl U. J. 2002; PubMed Scopus Google Scholar, M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). mouse were not by antibody AO4B08 that the epitope was not generated through the of and the differential expression of the J. K. 2004; PubMed Scopus Google a for in consecutive 6-O-sulfated units. HS4E4, selected by to bovine HS, more tissue staining (8ten Dam G.B. Hafmans T. Veerkamp J.H. van Kuppevelt T.H. J. Histochem. Cytochem. 2003; 51: 727-739Crossref PubMed Scopus (32) Google Scholar). of to an HS epitope structure more the recognized by Contrary to AO4B08, HS4E4 was thus to bind to tissue from to chains is as in the a from in the antibody A for was from the with modified polysaccharide and HS4E4 did not bind to of the fully N-sulfated with defined of selective of from HS4E4 of HS chains at N-acetylated sites, antibody that the more group. The that the N-sulfated in from HS are to did not bind to HS4E4 not important for HS4E4 is by the that HS4E4 not bind to tissue from the a of GlcA residues, to the HS from such lack 2-O-sulfate with the of J.P. E. M. X. Lindahl U. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). HS4E4 recognize epitopes in and in not to the these to a epitope N-acetylated and N-sulfated glucosamine residues, and GlcA polysaccharides a and O-sulfated group. O-sulfated structures are fully O-sulfated The various phage display antibodies from the and their which at a acid (10Dennissen M.A.B.A. Jenniskens G.J. Pieffers M. Versteeg E.M.M. Petitou M. Veerkamp J.H. van Kuppevelt T.H. J. Biol. Chem. 2002; 277: 10982-10986Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). these are to for the not of the two antibodies in of antibody that preferentially binds motifs Dam G.B. S. van Versteeg Lindahl U. Spillmann D. van Kuppevelt T. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). structural motifs in proteins HS of specificity in HS-protein interactions HS is regulated, and we that the different of in a are generated for functional HS chains, as of at the cell surfaces in the extracellular as extracellular for in proteins for interactions with their and in formation of and has on the of HS-protein are a few of highly to more on the of HS on the of sulfation of such on their structure (6Kreuger J. Spillmann D. Li J.-p. Lindahl U. J. Cell Biol. 2006; 174: 323-327Crossref PubMed Scopus (399) Google Scholar). The AO4B08 epitope a of a to interact with a variety of proteins. The functional properties of such are presumably by in the of The structural recognized by antibody HS4E4 on the of the HS We that characterization of antibodies of both important tools to HS in and disease. We for and J. for cell and tissue M. for and J. for is for the of and A. modified polysaccharide and modified