Identification of the molecular chaperone alpha B‐crystallin in demineralized bone powder and osteoblast‐like cells

Abstract

Bone is subjected to a variety of physiological, as well as cell‐deforming biomechanical stresses, including hydrostatic compression and fluid flow. However, little is known about the molecular mechanisms that protect bone cells from mechanical, ischemic, or oxidative damage. Crystallins are 20 kD heat shock proteins that function as molecular chaperones. We tested the hypothesis that alpha B‐crystallin (αB‐crystallin), the most widely expressed vertebrate crystallin, is present in bone and osteoblast‐like cells. Noncollagenous proteins (NCPs) were extracted from human demineralized bone matrix with 4 M guanidine HCl containing 0.5 M CaCl2 and protease inhibitors, defatted, dialyzed against 0.2% (v/v) Triton X‐100 in 100 mM Tris‐HCI (pH 7.2) and water, centrifuged, and lyophilized. The NCPs were separated by 2D IEF/SDS‐PAGE. The two most abundant 20 kD spots, with apparent pIs of 7.85 and 7.42 in urea gels, were excised, subjected to matrix‐assisted laser desorption ionization/time‐of‐flight mass spectrometry, and identified as αB‐crystallins. Indirect immunofluorescence localized αB‐crystallin to the interphase nucleus, cytoskeleton and cytoplasm of proliferating MC3T3‐El mouse osteoblast‐like cells, as well as the cytoskeleton and cytoplasm of confluent cells. In conclusion, αB‐crystallin is present in bone and osteoblast‐like cells. We hypothesize that αB‐crystallin may play a role in protecting the osteoblast cytoskeleton from mechanical stress and may be important in modulating nuclear or cellular functions, such as transcription or apoptosis, as observed in other tissues. Published by Elsevier Science Ltd. On behalf of Orthopaedic Research Society.

A proteomic analysis of adult rat bone reveals the presence of cartilage/chondrocyte markers

“…Proteins extracted from human demineralized bone matrix have been separated using 2D-gel electrophoresis and individual proteins identified by MALDI-TOF. From this analysis, alpha B-crystallin was found to be present in bone matrix [Behnam et al, 2002]. Separation of proteins by 2D-gel electrophoresis followed by MALDI-TOF analyses have also been used to identify proteins involved in the mechanism of action of glucocorticoids on osteoblast cell lines [Olkku et al, 2004] as well as identifying proteins associated with TRAF6 regulating osteoclast differentiation in vitro [Ryu et al, 2005].…”

Recombinant Expression, Isolation, and Proteolysis of Extracellular Matrix-Secreted Phosphoprotein-24 kDa

“…One-dimension SDS 4% to 20% polyacrylamide gradient gel electrophoresis, Coomassie brilliant blue staining of gels, protein transblotting and Western development were conducted using standard equipment and commercial reagents as previously outlined in detail [7,[9][10][11]. Visualization of His 6 -tagged spp24 was accomplished by Western blotting using a primary antibody and colorimetric kit from Novagen (EMD Biosciences).…”

“…The proteins in representative fractions containing His 6 -spp24 and its degradation products were separated by 2D SDS-PAGE by the method of O'Farrell [12] (Nancy Kendrick, Ph.D., Kendrick Laboratories, Madison, WI, USA) [7,[9][10][11]. These fractions had been stored at −20 • C for 3 months in urea buffer plus protease inhibitors, resulting in the slow accumulation of degradation products similar to those previously reported [8] that are not abundant in freshly prepared fractions.…”

“…These fractions had been stored at −20 • C for 3 months in urea buffer plus protease inhibitors, resulting in the slow accumulation of degradation products similar to those previously reported [8] that are not abundant in freshly prepared fractions. The gel was stained with Coomassie brilliant blue, and the three major spots of the appropriate molecular weight (<24 kDa) were selected, excised, eluted, reduced in 5% β-mercaptoethanol, alkylated, trypsin digested, and subjected to peptide mass fingerprint analysis by MALDI/ToF MS (Dr. Gawinowicz) to verify their identities and structures [7,[9][10][11]. Methionines were not oxidized.…”

“…Peptides with a mass [M + H + ] > 1000 Da were analyzed. The peptide fingerprint was compared with those in the SWISS-PRO and NCBI databanks [7,[9][10][11]. The spots in replicate gels were subjected to N-terminal sequencing by automated Edman degradation.…”