Estimation of the identity of proteolytic aggrecan fragments using PAGE migration and Western immunoblot.
OBJECTIVE
To develop calculation models, using Western immunoblot, as a tool for the estimation of proteolytic human aggrecan fragment identity.


METHODS
Seven human aggrecan fragments (calibrators), purified by CsCl gradient centrifugation and identified by Western immunoblot of N- and C-terminals, were used to develop calculation models. The models were used for identification of unknown aggrecan fragments each having one of their N- or C-terminals identified.


RESULTS
The calibrator molecular weights (Mw) from sodium dodecyl sulfate (SDS)-gels (m), the Mw of amino acids (a) and the Mw of their carbohydrate substitution (g) were expressed as K = m/(a+g), or as K = 1.085m/(a+g) when compensation for the G1 domain was required. Using these models together with average K-values, 12 out of the 17 immuno-detected aggrecan fragments were calculated to a known protease cleavage site, while five were identified to domain levels.


CONCLUSIONS
With six neoepitope antibodies together with antibodies against the G1- and G3-domain it was possible to predict the identity of several proteolytic fragments from different regions within the aggrecan monomer.
Struglics
A
A
Department of Clinical Sciences, Orthopaedics, Lund University, Lund, Sweden. andre.struglics@med.lu.se
Larsson
S
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Lohmander
L S
LS
D053674:Aggrecans / Q000032:analysis*; D000911:Antibodies, Monoclonal; D015153:Blotting, Western / Q000379:methods; D002358:Cartilage, Articular / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D000939:Epitopes; D006801:Humans; D007719:Knee Joint; D008956:Models, Chemical; D008970:Molecular Weight; D020370:Osteoarthritis, Knee / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis; D013582:Synovial Fluid / Q000737:chemistry*
D053674:Aggrecans; D000911:Antibodies, Monoclonal; D000939:Epitopes; D010446:Peptide Fragments