THE SULFATION PATTERN IN CHONDROITIN SULFATE CHAINS INVESTIGATED BY CHONDROITINASE ABC AND ACII DIGESTION AND REACTIVITY WITH MONOCLONAL-ANTIBODIES

We have used progressive chondroitinase digestion of pig aggrecan in c onjunction with ELISA assays and disaccharide analysis to derive infor mation about the pattern of 4- and 6-sulphation in chondroitin sulphat e chains. Digestion with chondroitinase ABC resulted in the release of mainly disaccharides from the nonreducing terminal of chondroitin sul phate chains but there was also the release of some tetra- and hexa-sa ccharides which were degraded to disaccharides with more extensive dig estion. Chondroitinase ACII, in contrast, released only disaccharides. Analysis of the disaccharide composition of the intact and digested p roducts at different stages of digestion showed that there was a sligh t increase in 6-sulphate content of the chains as they were shortened. Reaction of the partially digested proteoglycans with monoclonal anti bodies 3-B-3 and 3-D-5 which recognise chains terminating in 6- or 4-s ulphated disaccharides, respectively, showed major differences between chondroitinase ABC and ACII products. The results suggested that chon droitinase ABC preferentially cleaved next to I-sulphated, rather than 6-sulphated disaccharides and this resulted in some oligosaccharides as well as disaccharide being released. Chondroitinase ACII also cleav ed an additional disaccharide next to the linkage to protein of chondr oitin sulphate, which was not removed by chondroitinase ABC and this d isaccharide was mainly nonsulphated.