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 GuHCl and separated by composite agarose polyacrylamide gel electrophoresis. Individual PG populations resolved by this electrophoretic system were identified in toluidine blue and Stains-All stained gel segments and also by conventional immunoblotting using a range of monoclonal antibodies to defined PG epitopes. These PG species were compared with aggregatable PG populations identified by affinity blotting using a biotinylated hyaluronan, and an avidin alkaline phosphatase/nitro blue tetrazolium 5-bromo-4-chloro indolyl phosphate detection system. Two major chondroitin sulfate- and keratan sulfate-substituted aggrecan populations were readily identified by affinity blotting in all of the connective tissue extracts. An additional slower migrating aggrecan species was also detected by affinity and immunoblotting in fetal disc extracts. This may represent an aggrecan species containing an intact carboxyl terminal G3 domain. Link protein was also detectable by affinity blotting; this was confirmed by immunoblotting using an anti-link protein monoclonal antibody (8-A-4). Fragments of aggrecan which contained a functional G1 domain were also detectable by affinity blotting. The biotinylated hyaluronan affinity blotting technique could detect as little as 100 ng (as hexuronic acid) of aggregatable PG. Affinity blotting therefore represents a useful new detection methodology which complements conventional immunoblotting protocols and yields information regarding the functional status of the G1 domain of individual PG populations.