Cellular distribution of alpha B-crystallin in non-lenticular tissues

alpha B-Crystallin is a subunit of alpha-crystallin, a major protein component of the vertebrate lens. Recently, its expression in various extra-lenticular tissues has been demonstrated by both Western and Northern blotting. In this study, the cellular distribution of alpha B-crystallin in rat organs was examined in detail using immunohistochemistry. Positive reactions were observed in lens, iris, heart, skeletal muscle (type 1 and type 2A fibers), striated muscle in skin and esophagus, Henle's loop and medullary collecting duct of the kidney, Schwann cells of peripheral nerves, glia of the central nervous system, and decidual cells of the placenta. A close correlation with markers of oxidative activity suggests that alpha B-crystallin is expressed in cells that have high levels of oxidative function.     

Alpha B-crystallin is expressed in kidney epithelial cell lines and not in fibroblasts

We have recently shown the presence of alpha B-crystallin in non-ocular tissues of diverse embryological origins such as the heart, brain, spinal cord, kidney, retina, etc. Using an alpha B-crystallin-specific antiserum and immunofluorescence, immunoblotting, immunoprecipitation and peptide mapping with Staphylococcus aureus protease, we demonstrate differential expression of alpha B-crystallin in epithelial and fibroblast cell lines. alpha B-Crystallin was detectable only in epithelial cell lines such as MDBK, MDCK, LLCPK1 and JTC-12, and was not observed in two kidney fibroblast cell lines, one skin fibroblast cell line, and one corneal fibroblast cell line. Differential expression of the alpha B-crystallin gene was also confirmed by Northern blot analysis of the RNAs isolated from these cell lines. These data suggest a cell-type-specific role for alpha B.     

Increment of alpha B-crystallin mRNA in the brain of patient with infantile type Alexander's disease

To estimate the expression level of alpha B-crystallin in the brain of infantile type Alexander's disease, the amounts of protein and mRNA of alpha B-crystallin were measured by enzyme immunoassay (EIA) and Northern blot analysis, respectively, in the brain of patient and controls, and in the tissues from glioblastoma and astrocytoma. The alpha B-crystallin protein in the brain of patient was remarkably increased as compared with those of controls. The amount of alpha B-crystallin mRNA of patient was increased about 7-fold compared to the mean value of the control group and higher than that of glioblastoma tissue. These data suggest that increment of alpha B-crystallin mRNA in astrocytes leads to the overexpression of this protein and may be one of the main causes of infantile type Alexander's disease.     

Alpha B-crystallin mutation in dilated cardiomyopathy

Mutations in genes for sarcomeric proteins such as titin/connectin are known to cause dilated cardiomyopathy (DCM). However, disease-causing mutations can be identified only in a small proportion of the patients even in the familial cases, suggesting that there remains yet unidentified disease-causing gene(s) for DCM. To explore the novel disease gene for DCM, we examined CRYAB encoding alphaB-crystallin for mutation in the patients with DCM, since alphaB-crystallin was recently reported to associate with the heart-specific N2B domain and adjacent I26/I27 domain of titin/connectin, and we previously reported a N2B mutation, Gln4053ter, in DCM. A missense mutation of CRYAB, Arg157His, was found in a familial DCM patient and the mutation affected the evolutionary conserved amino acid residue among alpha-crystallins. Functional analysis revealed that the mutation decreased the binding to titin/connectin heart-specific N2B domain without affecting distribution of the mutant crystallin protein in cardiomyocytes. In contrast, another CRYAB mutation, Arg120Gly, reported in desmin-related myopathy decreased the binding to both N2B and striated muscle-specific I26/27 domains and showed intracellular aggregates of the mutant protein. These observations suggest that the Arg157His mutation may be involved in the pathogenesis of DCM via impaired accommodation to the heart-specific N2B domain of titin/connectin and its disease-causing mechanism is different from the mutation found in desmin-related myopathy.     

Sulfatide is expressed in neurons and astrocytes and accumulates at degradation deficiency

Few studies of lipid rafts have investigated gangliosides in brain tissue. This study focus on analyses of lipids and the major brain gangliosides (GM1, GD1a, GD1b, GT1b) in human cortex (frontal, temporal) and corresponding detergent resistant membranes (DRMs), i.e. rafts. A high proportion of the gangliosides (18–26%) as well as of cholesterol (21%) and sphingomyelin (38%) was found in rafts, while lower yields was observed for ganglioside GM2 (9%), phospholipids (8%) and in particular proteins (2%). Significant alterations in lipid composition was noticed in rafts from Alzheimer brain tissue. These results show that sphingolipids and cholesterol are major constituents of rafts also in the human brain and that the main brain gangliosides are distributed in rafts to a similar degree. Moreover, lipid rafts might be considered in the pathology of Alzheimer's disease.     

Alpha B-crystallin in skeletal muscle: purification and localization.

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.     

The apparent molecular size of native alpha-crystallin B in non-lenticular tissues

The apparent molecular size of the native alpha-crystallin B in cytosol preparations from rat heart, brain and retina was determined by gel permeation chromatography, detecting the protein by immunochemical assay (ELISA), using an alpha-crystallin specific antiserum. Native alpha-crystallin from cytosol preparations of rat lens cortex was used as a reference. alpha-Crystallin B present in all three cytosol preparations from non-lenticular tissues eluted in a single symmetrical peak, with the same elution volume as alpha-crystallin from lens cortex cytosol preparations, corresponding to an apparent average molecular size of 0.8 x 10(6) Da. No other species could be detected. The results indicate that the alpha-crystallin aggregates characterized by an apparent average molecular mass of 0.8 x 10(6) Da, and considered to be the native, physiological form of the protein in the lens, are indeed not specific to lens tissue. Furthermore, the size of these alpha-crystallin aggregates is independent of their polypeptide composition. Aggregates found in the lens, composed of alpha A and alpha B polypeptides and their respective phosphorylated forms alpha Ap and alpha Bp, are similar in size to those found in heart, brain and retina, containing the alpha B but not the alpha A polypeptide.     

alpha A-crystallin is expressed in non-ocular tissues.

alpha-Crystallin, the predominant structural protein of the ocular lens, has been considered to be composed of two subunits, alpha A-crystallin and alpha B-crystallin. Of these two, alpha B-crystallin has been previously shown to be an extralenticular protein while alpha A-crystallin has been considered to be a lens-specific polypeptide. Using an antiserum directed against an N-terminal peptide of alpha-crystallin, we have detected a 20-kDa protein in various rat tissues including the brain, liver, lung, spleen, skin, and small intestine and in a number of established epithelial and fibroblast cell lines. PCR analysis of poly(A)-enriched RNA and Southern blot analysis indicated the presence of alpha A-crystallin mRNA sequences in different non-lenticular tissues. Among the non-ocular tissues examined, spleen showed the highest levels of alpha A-crystallin protein and mRNA. The identity of alpha A-crystallin sequences in the spleen was established by cloning and sequencing a polymerase chain reaction-amplified region of alpha A-crystallin mRNA. Sequences derived from spleen and eye revealed almost 100% identity at the nucleotide level. Interestingly, alpha A-crystallin and alpha B-crystallin seem to exist in an inverse quantitative relationship in the spleen and the heart, the two non-ocular tissues where they show highest concentrations, respectively. The known conserved evolution of alpha A-crystallin and the definitive demonstration of the non-ocular expression of this polypeptide suggest important non-crystallin functions for this protein.