Structural characterization of the epitopes of the monoclonal antibodies 473HD, CS-56, and MO-225 specific for chondroitin sulfate D-type using the oligosaccharide library.

The variation in the sulfation profile of chondroitin sulfate (CS)/dermatan sulfate (DS) chains regulates central nervous system development in vertebrates. Notably, the disulfated disaccharide D-unit, GlcUA(2-O-sulfate)-GalNAc(6-O-sulfate), correlates with the promotion of neurite outgrowth through the DSD-1 epitope that is embedded in the CS moiety of the proteoglycan DSD-1-PG/phosphacan. Monoclonal antibody (mAb) 473HD inhibits the DSD-1-dependent neuritogenesis and also recognizes shark cartilage CS-D, which is characterized by the prominent D-unit and is also recognized by two other mAbs, CS-56 and MO-225. We investigate the oligosaccharide epitope structures of these CS-D-reactive mAbs by ELISA and oligosaccharide microarrays using lipid-derivatized CS oligosaccharides. CS-56 and MO-225 recognized the octa- and larger oligosaccharides, though the latter also bound one unique hexasaccharide D-A-D, where A denotes the disaccharide A-unit GlcUA-GalNAc(4-O-sulfate). The octasaccharides reactive with CS-56 and MO-225 shared a core A-D tetrasaccharide, whereas the neighboring structural elements located on the reducing and/or nonreducing sides of the A-D gave a differential preference additionally to the recognition sequence for each antibody. In contrast, 473HD reacted with multiple hexa- and larger oligosaccharides, which also contained A-D or D-A tetrasaccharide sequences. Consistent with the distinct specificity of 473HD as compared with CS-56 and MO-225, the 473HD epitope displayed a different expression pattern in peripheral mouse organs as revealed by immunohistology, extending the previously reported CNS-restricted expression. The epitope of 473HD, but not of CS-56 or MO-225, was eliminated from DSD-1-PG by digestion with chondroitinase B, suggesting the close association of L-iduronic acid with the 473HD epitope. Despite such supplemental information, the integral epitope remains to be isolated for identification and comprehensive analytical characterisation. Thus novel information on the sugar sequences containing the A-D tetrasaccharide core was obtained for the epitopes of these three useful mAbs.

Oversulfated dermatan sulfate exhibits neurite outgrowth-promoting activity toward embryonic mouse hippocampal neurons: implications of dermatan sulfate in neuritogenesis in the brain.

Brain-specific chondroitin sulfate (CS) proteoglycan (PG) DSD-1-PG/6B4-PG/phosphacan isolated from neonatal mouse brains exhibits neurite outgrowth-promoting activity toward embryonic rat and mouse hippocampal neurons in vitro through the so-called DSD-1 epitope embedded in its glycosaminoglycan side chains. Oversulfated CS variants, CS-D from shark cartilage and CS-E from squid cartilage, also possess similar activities. We have proposed that the neuritogenic property of the DSD-1 epitope may be attributable to a distinct CS structure characterized by the disulfated D disaccharide unit [GlcUA(2S)-GalNAc(6S)]. In this study, we assessed neuritogenic potencies of various oversulfated dermatan sulfate (DS) preparations purified from hagfish notochord, the bodies of two kinds of ascidians and embryonic sea urchin, which are characterized by the predominant disulfated disaccharide units of [IdoUA-GalNAc(4S,6S)] (68%), [IdoUA(2S)-GalNAc(4S)] (66%) plus [IdoUA(2S)-GalNAc(6S)] (5%), [IdoUA(2S)-GalNAc (6S)] (>90%), and [IdoUA-GalNAc(4S,6S)] (74%), respectively. They exerted marked neurite outgrowth-promoting activities, resulting in distinct morphological features depending on the individual structural features. Such activities were not observed for a less sulfated DS preparation derived from porcine skin, which has a monosulfated disaccharide unit [IdoUA-Gal-NAc(4S)] as a predominant unit. The neurite outgrowth-promoting activities of these oversulfated DS preparations and DSD-1-PG were eliminated by the specific enzymatic cleavage of GalNAc-IdoUA linkages characteristic of DS using chondroitinase B. In addition, chemical analysis of the glycosaminoglycan side chains of DSD-1-PG revealed the DS-type structures. These observations suggest potential novel neurobiological functions of oversulfated DS structures and may reflect the physiological neuritogenesis during brain development by mammalian oversulfated DS structures exemplified by the DSD-1 epitope.