DETECTION OF AGGREGATABLE PROTEOGLYCAN POPULATIONS BY AFFINITY BLOTTING USING BIOTINYLATED HYALURONAN

Proteoglycans (PGs) were extracted from a range of cartilaginous ovine connective tissues using 4 M Gu-HCl and separated by composite agaros e polyacrylamide gel electrophoresis. Individual PG populations resolv ed by this electrophoretic system were identified in toluidine blue an d Stains-Ah stained gel segments and also by conventional immunoblotti ng using a range of monoclonal antibodies to defined PG epitopes. Thes e PG species were compared with aggregatable PG populations identified by affinity blotting using a biotinylated hyaluronan, and an avidin a lkaline phosphatase/nitro blue tetrazolium 5-bromo-4-chloro indolyl ph osphate detection system. Two major chondroitin sulfate-and keratan su lfate-substituted aggrecan populations were readily identified by affi nity blotting in all of the connective tissue extracts. An additional slower migrating aggrecan species was also detected by affinity and im munoblotting in fetal disc extracts. This may represent an aggrecan sp ecies containing an intact carboxyl terminal G3 domain. Link protein w as also detectable by affinity blotting; this was confirmed by immunob lotting using an anti-link protein monoclonal antibody (8-A-4). Fragme nts of aggrecan which contained a functional G1 domain were also detec table by affinity blotting. The biotinylated hyaluronan affinity blott ing technique could detect as little as 100 ng (as hexuronic acid) of aggregatable PG. Affinity blotting therefore represents a useful new d etection methodology which complements conventional immunoblotting pro tocols and yields information regarding the functional status of the G 1 domain of individual PG populations. (C) 1998 Academic Press.