Two Related but Distinct Chondroitin Sulfate Mimetope Octasaccharide Sequences Recognized by Monoclonal Antibody WF6

Abstract

Chondroitin sulfate (CS) proteoglycans are major components of cartilage and other connective tissues. The monoclonal antibody WF6, developed against embryonic shark cartilage CS, recognizes an epitope in CS chains, which is expressed in ovarian cancer and variably in joint diseases. To elucidate the structure of the epitope, we isolated oligosaccharide fractions from a partial chondroitinase ABC digest of shark cartilage CS-C and established their chain length, disaccharide composition, sulfate content, and sulfation pattern. These structurally defined oligosaccharide fractions were characterized for binding to WF6 by enzyme-linked immunosorbent assay using an oligosaccharide microarray prepared with CS oligosaccharides derivatized with a fluorescent aminolipid. The lowest molecular weight fraction recognized by WF6 contained octasaccharides, which were split into five subfractions. The most reactive subfraction contained several distinct octasaccharide sequences. Two octasaccharides, ΔD-C-C-C and ΔC-C-A-D (where A represents GlcUAβ1-3GalNAc(4-O-sulfate), C is GlcUAβ1-3Gal-NAc(6-O-sulfate), D is GlcUA(2-O-sulfate)β1-3GalNAc(6-O-sulfate), ΔCis Δ4,5HexUAα1-3GalNAc(6-O-sulfate), and ΔDis Δ4,5HexUA(2-O-sulfate)α1-3GalNAc(6-O-sulfate)), were recognized by WF6, but other related octasaccharides, ΔC-A-D-C and ΔC-C-C-C, were not. The structure and sequences of both the binding and nonbinding octasaccharides were compared by computer modeling, which revealed a remarkable similarity between the shape and distribution of the electrostatic potential in the two different octasaccharide sequences that bound to WF6 and that differed from the nonbinding octasaccharides. The strong similarity in structure predicted for the two binding CS octasaccharides (ΔD-C-C-C and ΔC-C-A-D) provided a possible explanation for their similar affinity for WF6, although they differed in sequence and thus form two specific mimetopes for the antibody. Chondroitin sulfate (CS) proteoglycans are major components of cartilage and other connective tissues. The monoclonal antibody WF6, developed against embryonic shark cartilage CS, recognizes an epitope in CS chains, which is expressed in ovarian cancer and variably in joint diseases. To elucidate the structure of the epitope, we isolated oligosaccharide fractions from a partial chondroitinase ABC digest of shark cartilage CS-C and established their chain length, disaccharide composition, sulfate content, and sulfation pattern. These structurally defined oligosaccharide fractions were characterized for binding to WF6 by enzyme-linked immunosorbent assay using an oligosaccharide microarray prepared with CS oligosaccharides derivatized with a fluorescent aminolipid. The lowest molecular weight fraction recognized by WF6 contained octasaccharides, which were split into five subfractions. The most reactive subfraction contained several distinct octasaccharide sequences. Two octasaccharides, ΔD-C-C-C and ΔC-C-A-D (where A represents GlcUAβ1-3GalNAc(4-O-sulfate), C is GlcUAβ1-3Gal-NAc(6-O-sulfate), D is GlcUA(2-O-sulfate)β1-3GalNAc(6-O-sulfate), ΔCis Δ4,5HexUAα1-3GalNAc(6-O-sulfate), and ΔDis Δ4,5HexUA(2-O-sulfate)α1-3GalNAc(6-O-sulfate)), were recognized by WF6, but other related octasaccharides, ΔC-A-D-C and ΔC-C-C-C, were not. The structure and sequences of both the binding and nonbinding octasaccharides were compared by computer modeling, which revealed a remarkable similarity between the shape and distribution of the electrostatic potential in the two different octasaccharide sequences that bound to WF6 and that differed from the nonbinding octasaccharides. The strong similarity in structure predicted for the two binding CS octasaccharides (ΔD-C-C-C and ΔC-C-A-D) provided a possible explanation for their similar affinity for WF6, although they differed in sequence and thus form two specific mimetopes for the antibody. Chondroitin sulfate proteoglycans (CS-PGs) 5The abbreviations used are: CS, chondroitin sulfate; PG, proteoglycan; GAG, glycosaminoglycan; GlcUA, d-glucuronic acid; HexUA, hexuronic acid; IdoUA, l-iduronic acid; 2S, 2-O-sulfate; 4S, 4-O-sulfate; 6S, 6-O-sulfate; ELISA, enzyme-linked immunosorbent assay; BSA, bovine serum albumin; HPLC, high performance liquid chromatography; FPLC, fast protein liquid chromatography; SAX, strong anion exchange resin; MALDI-TOF, matrix-assisted laser desorption ionization-time-of-flight; MS, mass spectrometry; 2AB, 2-aminobenzamide; ADHP, N-aminoacetyl-N-(9-anthracenylmethyl)-1,2-dihexadecyl-sn-glycero-3-phosphoethanolamine. are expressed on the surface of most cells and in extracellular matrices in vertebrates, where CS is linked to a wide range of core protein families. They are increasingly implicated as important regulators of many biological processes, contributing in various ways to the physical strength of tissues, cell adhesion, and signal transduction (1Letourneau P.C. Condic M.L. Snow D.M. Curr. Opin. Genet. Dev. 1992; 2: 625-634Crossref PubMed Scopus (54) Google Scholar, 2Reichardt L.F. Tomaselli K.J. Annu. Rev. Neurosci. 1991; 14: 531-570Crossref PubMed Scopus (657) Google Scholar, 3Hardingham 1992; PubMed Scopus Google Scholar, Curr. Opin. PubMed Scopus Google CS a the biological of which are The structure is an linked a to a in a and is of which by various where and d-glucuronic and CS is expressed in and by of and chain length, which for of the and of to which they are The sulfation of CS with in cartilage Google and which specific disaccharide the of and Curr. Opin. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, Neurosci. PubMed Scopus Google CS sequences are in binding PubMed Scopus Google is related to in chain and PubMed Scopus Google that specific of CS PubMed Scopus Google but of the characterized in PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, PubMed Scopus Google with revealed of in various and and in PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, PubMed Google The of that on CS is used in many of as and PubMed Scopus Google and in the of PubMed Scopus Google Scholar, 14: PubMed Scopus Google Scholar, PubMed Scopus Google of cartilage revealed that in were in the CS cartilage PubMed Scopus Google in the sulfation of CS were in the in joint PubMed Scopus Google CS in cartilage in of with 1991; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, Google potential for the and of the WF6 is a with using embryonic shark cartilage as is reactive with shark and the epitope in CS and from shark but the antibody is with other and enzyme-linked immunosorbent assay that an of WF6 epitope in serum from of and 14: PubMed Scopus Google and in of ovarian cancer PubMed Scopus Google The established of the antibody and distribution CS that the epitope is a specific sequence in but CS and is of A C and D thus important to structure and to sequences specific biological as in the of recognized by other and PubMed Scopus Google as as sulfate PubMed Scopus Google the structure of CS on from various tissues, but to sulfation sequences using to CS PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, PubMed Scopus Google The recognized by are sequences in CS PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, Dev. 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CS are in composition, the sulfate and sulfation as revealed by of a of oligosaccharides isolated from shark cartilage CS-C PubMed Scopus Google is to the specific CS structure for the by we and characterized CS octasaccharide sequences. the and ΔC-A-D-C PubMed Scopus Google as as their and PubMed Scopus Google isolated from shark cartilage and with chondroitinase ABC ΔD-C-C-C ΔC-C-A-D their and isolated PubMed Scopus Google two octasaccharide sequences (ΔD-C-C-C and ΔC-C-A-D) were recognized by the WF6 and as the CS for with as and sulfate which were by to with sequences from shark cartilage shark cartilage cartilage sulfate PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, PubMed Scopus Google that a chain antibody with sequences isolated from cartilage PubMed Scopus Google the octasaccharides and the and which were recognized by WF6, were isolated in and PubMed Scopus Google from shark cartilage CS chains, the are in of the by WF6 in from with 14: PubMed Scopus Google and ovarian PubMed Scopus Google of the that the WF6, with oligosaccharides similar distinct we used molecular to possible by the octasaccharides and from the octasaccharides. 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