αB-Crystallin in Skeletal Muscle: Purification and Localization1

Abstract

Atrophy of rat soleus muscles by hindlimb suspension is characterized by an early dramatic decrease in a soluble 22-kDa protein. The 22-kDa protein was purified from rat red skeletal muscle and rat lens by three different methods of chromatography. The partial amino acid sequence (65% of total amino acids) determined for muscle 22-kDa protein was identical with that of rat lens crystallin. The HPLC elution patterns of lysylendopeptidase fragments of 22-kDa protein from the two sources were identical. Polyclonal antibodies to rat muscle and bovine lens alpha B-crystallin with the two proteins on immunoblotting. alpha B-Crystallin protein was expressed and synthesized efficiently in slow skeletal muscle and poorly in fast muscle. Thus, the decreased 22-kDa protein of slow muscle in the suspension treatment was confirmed to be alpha B-crystallin. Immunoblotting confirmed that most of the alpha B-crystallin was solubilized, though some was tightly bound to myofibrils. This bound portion was localized in Z-bands of isolated myofibrils by immunocytochemical light and electron microscopy. Muscle alpha B-crystallin is tentatively proposed to be a myofibril-stabilizing protein, based upon its extraction characteristics, localization, and amino acid sequence.

Identification of O-linked N-Acetylglucosamine Proteins in Rat Skeletal Muscle Using Two-dimensional Gel Electrophoresis and Mass Spectrometry

“…These specific interactions between glycolytic enzyme complexes and the contractile apparatus, which could be modulated by phosphorylation and/or O-GlcNAc, could allow the formation of ATP at its site of utilization. Similarly, muscular creatine kinase, an important enzyme catalyzing the reversible transfer of a phosphate moiety between ATP and creatine, has been described in myofibrils as a structural protein of M-band (43) as well as ␣B-crystallin that interacted with actin and desmin intermediate filaments to increase the stability of Z-bands (44).…”

The expanding small heat-shock protein family, and structure predictions of the conserved “α-crystallin domain”

“…Another structural function may be found for the HR-29 protein of Halocynthia roretzi, which is localized in body-wall muscle (Shirakata et al 1986). It may be a component of myofibrils and may act as a stabilizing protein like aB-crystallin does in skeletal muscle (Atomi et al 1991). This brief and necessarily fragmentary account makes it clear that gene duplications and divergent evolution have produced a broad array of structural and functional properties in the shsp family, using the common "~-crystallin domain" as an essential building block.…”

Influences of temperature, oxidative stress, and phosphorylation on binding of heat shock proteins in skeletal muscle fibers

“…A great deal of work in skeletal muscle has focused on the increase in HSP protein and/or mRNA expression levels occurring in hours to days after various stressful stimuli, such as eccentric exercise (11,17,26,27,39), oxidative stress (50), heat (4, 35), and glycogen depletion (12). The present study focuses instead on the acute responses of HSPs to stresses, examining the effects of heat, contraction, and oxidative stress on the distribution and binding of three important HSPs present in rat skeletal muscle fibers, namely ␣B-crystallin, HSP25 (also known as HSP27), and HSP72 (also known as HSP70).In nonstressed muscle fibers the majority (ϳ80% to 95%) of each of these HSPs is readily diffusible within the cytoplasmic space (2,16,20,21,26,39). Following various stress interventions the two small HSPs, HSP25 and ␣B-crystallin, have been found to bind to a wide variety of different cytoskeletal/ myofibrillar proteins (2, 16), including desmin (15,19,20,38), titin (15), actin (28, 43), myosin (24), and possibly also Z-disk proteins.…”

“…In nonstressed muscle fibers the majority (ϳ80% to 95%) of each of these HSPs is readily diffusible within the cytoplasmic space (2,16,20,21,26,39). Following various stress interventions the two small HSPs, HSP25 and ␣B-crystallin, have been found to bind to a wide variety of different cytoskeletal/ myofibrillar proteins (2, 16), including desmin (15,19,20,38), titin (15), actin (28, 43), myosin (24), and possibly also Z-disk proteins.…”