Molecular weight characterization of PRG4 proteins using multi-angle laser light scattering (MALLS)

Abstract

Alternative splicing and variable post-translational modifications result in proteoglycan 4 (PRG4) proteins with historically reported apparent molecular weights (Ma) ranging from 150 to 400kDa. The objectives of this study were to (1) identify and determine the weight averaged molecular weights (M(W)'s) of PRG4 proteins purified from medium with transforming growth factor-beta 1 (TGF-β1) conditioned by mature bovine articular cartilage explants and (2) to examine the effect of reduction and alkylation (RA) on PRG4. Non-reduced (NR) and RA preparations of PRG4 were separated using high performance liquid chromatography-size-exclusion chromatography with an in-line multi-angle laser light scattering (MALLS) detector, which was used for absolute determination of PRG4 M(W). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting, and tandem mass spectrometry (MS/MS) analysis were used to confirm the identity of separated proteins. Three putative PRG4 monomers, one with previously uncharacterized M(W), were identified in NR and RA PRG4 preparations of 239 (223,255), 379 (369,389), and 467 (433,501) kDa. Additionally ∼1MDa putative PRG4 dimer was identified. Release of a ∼90kDa PRG4 fragment was also observed on SDS-PAGE after RA. Western Blotting with anti-PRG4 antibodies detected immunoreactive bands with Ma similar to M(W) for all species and excised bands were confirmed to be PRG4 by MS/MS. A variety of monomeric PRG4 proteins and a disulfide-bonded dimer/multimer are secreted by chondrocytes in bovine cartilage explants. The observed decrease in M(W)'s of monomeric PRG4 species upon RA may be due to the release of post-translationally cleaved fragments. Further study of these species will provide insight into the PRG4 molecular structure and function relationship.