Antibodies to Three Chondroitin Sulfate-Containing Proteoglycans in Squid Skin Recognize Hexa- or Longer Chondroitin Oligosaccharides as Major Antigenic Determinants

The reactivities of antibodies to three squid skin proteoglycans with (a) chondroitin-derived oligosaccharides and chondroitin sulfate-derived disaccharides, (b) the proteoglycans and their constituents, and (c) chondroitin, chondroitin sulfate, and hyaluronic acid were studied with enzyme-linked immunosorbant assay inhibition tests. Immunization of rabbits with two chondroitin proteoglycans (ChPG I and ChPG II) and an oversulfated chondroitin sulfate proteoglycan (CSSPG) gave rise to highly reactive antisera which were mainly reactive with the glycosaminoglycans, the oligosaccharides, and the core proteins obtained after digestion of proteoglycans with chondroitinase AC. Inhibition of binding of antibodies to ChPG I, ChPG II, and CSSPG with chondroitin-derived oligosaccharides revealed that the minimal antigenically active structure was the hexasaccharide of chondroitin and that the respective octasaccharide was more active. Sulfated Δ-disaccharides were not reactive with antibodies to ChPG I and II, whereas some reactivities (30% maximum inhibition) were obtained with antibodies to CSSPG. Chondroitin chains (80 kDa) of ChPG I and II were responsible for most of the reactivity with proteoglycans (78-95% maximum inhibition). Chemically desulfated chondroitin sulfate (12 kDa) showed considerable cross-reactivity with all antisera tested (62-68% maximum inhibition), whereas the nonsulfated molecule of hyaluronic acid and a hyaluronic acid fraction of 16 kDa were not reactive. The reactivities of antibodies with the proteoglycans′ oligosaccharides and core proteins obtained by chondroitinase AC digestion were mainly due to the presence of nonsulfated chondroitin sulfate structures, This study clearly shows that the major antigenic determinants recognized by antibodies to squid skin proteoglycans, each containing chondroitin sulfates with different sulfation patterns, involve hexa- or larger chondroitin oligosaccharides.