Molecular weight characterization of PRG4 proteins using multi-angle laser light scattering (MALLS)
Molecular weight characterization of PRG4 proteins using multi-angle laser light scattering (MALLS)
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DOI：
10.1016/j.joca.2012.12.002
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发表时间：
2013-03-01
影响因子：
7
通讯作者：
Schmidt, T. A.
中科院分区：
文献类型：
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作者：
Steele, B. L.;Alvarez-Veronesi, M. C.;Schmidt, T. A.
目的：选择性剪接和可变的翻译后修饰导致蛋白多糖4 （PRG4）蛋白的表观分子量（Ma）从150到400 kDa不等。本研究的目的是：(1)鉴定和确定从成熟牛关节软骨外植体调节的转化生长因子- β 1 （tgf - β 1）培养基中纯化的PRG4蛋白的重量平均分子量（M-W）；(2)检测还原和烷基化（RA）对PRG4的影响。方法：采用高效液相色谱-排阻色谱-直列多角度激光散射（MALLS）检测器分离PRG4的未还原（NR）制剂和RA制剂，并对PRG4 M-W进行绝对含量测定。采用十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）、免疫印迹和串联质谱（MS/MS）分析确认分离蛋白的身份。结果：在NR和RA PRG4的239(223,255),379（369,389）和467 (433,501)kDa的制剂中鉴定出三个推定的PRG4单体，其中一个具有先前未表征的M-W。此外，还鉴定了类似于1mda的推定PRG4二聚体。在SDS-PAGE上也观察到RA后释放了一个类似于90 kDa的PRG4片段。抗PRG4抗体的Western Blotting检测到所有物种的Ma与M-W相似的免疫反应条带，MS/MS证实切除的条带为PRG4。结论：牛软骨外植体软骨细胞分泌多种PRG4单体蛋白和二硫键二聚体/多聚体。观察到单体PRG4在RA上的分子量下降可能是由于翻译后裂解片段的释放。对这些物种的进一步研究将有助于深入了解PRG4的分子结构和功能关系。(C) 2012国际骨关节炎学会。Elsevier Ltd.出版。版权所有。
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-beta 1) 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 similar to 1 MDa putative PRG4 dimer was identified. Release of a similar to 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 Mw'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. (C) 2012 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.