Identification of the molecular chaperone alpha B-crystallin in demineralized bone powder and osteoblast-like cells.
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 (alphaB-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 HCI 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 alphaB-crystallins. Indirect immunofluorescence localized alphaB-crystallin to the interphase nucleus, cytoskeleton and cytoplasm of proliferating MC3T3-E1 mouse osteoblast-like cells, as well as the cytoskeleton and cytoplasm of confluent cells. In conclusion, alphaB-crystallin is present in bone and osteoblast-like cells. We hypothesize that alphaB-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.
Behnam
Keyvan
K
Department of Physiological Sciences, University of California, Los Angeles, Los Angeles, CA 90024, USA.
Murray
Samuel S
SS
Whitelegge
Julian P
JP
Brochmann
Elsa J
EJ
D000595:Amino Acid Sequence; D000818:Animals; D018775:Bone Demineralization Technique; D001842:Bone and Bones / Q000737:chemistry / Q000166:cytology; D002467:Cell Nucleus / Q000737:chemistry; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000737:chemistry; D003600:Cytosol / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D006360:Heat-Shock Proteins / Q000032:analysis; D006801:Humans; D051379:Mice; D018832:Molecular Chaperones / Q000032:analysis; D008969:Molecular Sequence Data; D010006:Osteoblasts / Q000737:chemistry* / Q000166:cytology; D011208:Powders; D040901:Proteomics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D038203:alpha-Crystallin B Chain / Q000032:analysis* / Q000737:chemistry
D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D011208:Powders; D038203:alpha-Crystallin B Chain