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

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• 作者：B.L. Steele^&dagger ; ; ^{blsteele@ucalgary.ca} ; M.C. Alvarez-Veronesi^&Dagger ; ; ^{cecilia.alvarezveronesi@mail.utoronto.ca} ; T.A. Schmidt^&dagger ; ; ^&Dagger ; ; ^{tschmidt@ucalgary.ca}
• 关键词：PRG4 ; Lubricin ; MALLS ; Superficial zone protein
• 刊名：Osteoarthritis and Cartilage
• 出版年：2013
• 出版时间：March, 2013
• 年：2013
• 卷：21
• 期：3
• 页码：498-504
• 全文大小：319 K

文摘

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Summary

Objectives

Alternative splicing and variable post-translational modifications result in proteoglycan 4 (PRG4) proteins with historically reported apparent molecular weights (Ma) ranging from 150 to 400?kDa. 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-¦Â1) conditioned by mature bovine articular cartilage explants and (2) to examine the effect of reduction and alkylation (RA) on PRG4.

Methods

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.

Results

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 ¡«1?MDa putative PRG4 dimer was identified. Release of a ¡«90?kDa 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.

Conclusions

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.