Alternative RNA splicing of the murine alpha A-crystallin gene: protein-coding information within an intron

The eye lens contains a structural protein (alpha-crystallin), composed of two homologous primary gene products, alpha A2 and alpha B2. In certain rodents, there is another minor alpha-crystallin polypeptide, alpha Ains, which is identical to alpha A2 except for a 22 amino acid insert between residues 63 and 64 of the alpha A2 chain. Here we show that the mouse contains a single alpha A-crystallin gene, which has a 1376 bp intron separating codons 63 and 64 of the alpha A2-crystallin mRNA. A sequence encoding a 23 amino acid insert peptide was found 266 bp into the intron. The nucleotide borders of this sequence deviate from the AGGT consensus sequence. The DNA sequence encoding the insert peptide hybridizes to a cytoplasmic 14S RNA, demonstrating that it is transcribed in the lens. We propose that the murine alpha A2-crystallin gene generates both the alpha A2 and the alpha Ains mRNAs by alternative splicing.     

An alternative splice variant of human αA-crystallin modulates the oligomer ensemble and the chaperone activity of α-crystallins

In humans, ten genes encode small heat shock proteins with lens αA-crystallin and αB-crystallin representing two of the most prominent members. The canonical isoforms of αA-crystallin and αB-crystallin collaborate in the eye lens to prevent irreversible protein aggregation and preserve visual acuity. α-Crystallins form large polydisperse homo-oligomers and hetero-oligomers and as part of the proteostasis system bind substrate proteins in non-native conformations, thereby stabilizing them. Here, we analyzed a previously uncharacterized, alternative splice variant (isoform 2) of human αA-crystallin with an exchanged N-terminal sequence. This variant shows the characteristic α-crystallin secondary structure, exists on its own predominantly in a monomer–dimer equilibrium, and displays only low chaperone activity. However, the variant is able to integrate into higher order oligomers of canonical αA-crystallin and αB-crystallin as well as their hetero-oligomer. The presence of the variant leads to the formation of new types of higher order hetero-oligomers with an overall decreased number of subunits and enhanced chaperone activity. Thus, alternative mRNA splicing of human αA-crystallin leads to an additional, formerly not characterized αA-crystallin species which is able to modulate the properties of the canonical ensemble of α-crystallin oligomers.     

Affinity chromatographic isolation of the melanoma adhesion receptor for the IIICS region of fibronectin and its identification as the integrin alpha 4 beta 1

Eukaryotic cells adhere to at least two different regions of the fibronectin molecule: a central domain present in all fibronectin isoforms, and the type III connecting segment domain (IIICS), the expression of which is controlled by complex alternative splicing of precursor mRNA. Using affinity chromatography on a matrix containing a synthetic peptide ligand (CS1) representing the strongest active site within the IIICS, we have isolated the human melanoma cell receptor recognizing this region of fibronectin. The receptor is a complex of two polypeptides with subunit molecular masses of 145 and 125 kDa. This heterodimeric structure resembles that of receptors for other extracellular matrix proteins. Immunological analysis with specific antibodies identified these polypeptides as the integrin subunits alpha 4 and beta 1. In addition, antifunctional monoclonal antibodies directed against either alpha 4 or beta 1, but not against other integrin subunits, were potent inhibitors of CS1-mediated melanoma cell spreading. Furthermore, when the function of the central cell-binding domain was blocked, anti-alpha 4 and anti-beta 1 antibodies abolished spreading of A375-M cells on fibronectin, indicating that alpha 4 beta 1 is an authentic fibronectin receptor. Taken together, these results identify the human fibronectin IIICS receptor as the integrin heterodimer alpha 4 beta 1.     

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.     

The virtues of gaps: xenarthran (Edentate) monophyly supported by a unique deletion in alpha A-crystallin

Shared insertions or deletions (indels) in protein-coding DNA can be strong indicators of the monophyly of a taxon. A three-amino acid deletion had previously been noted in the eye lens protein alpha A-crystallin of two species of sloths and two species of anteaters, which represent the Pilosa, one of the two infraorders of Xenarthra (Edentata). This deletion has not been observed in 55 species from 16 other eutherian orders, or in 2 species of marsupials, or in 34 nonmammalian vertebrates, from birds to shark. At the genomic level, we have now detected this deletion in two species of armadillos of the second xenarthran infraorder, Cingulata, as well as in an additional species of anteater. Phylogenetic trees were constructed from a 145-bp sequence of the alpha A-crystallin gene of 39 tetrapod species, supporting xenarthran monophyly with values from 76% to 90%. To quantify the additional support for xenarthran monophyly, as given by the three-residue deletion, we computed the probabilities for the occurrence of this deletion per evolutionary time unit for alternative hypothetical tree topologies. In the estimates obtained, the six trees in which the xenarthran subgroups are unresolved or paraphyletic give an increasingly lower likelihood than do the two trees that assume xenarthran monophyly. For the monophyletic trees, the probability that the deletion observed in the xenarthrans is due to a single event is > 0.99. Thus, this deletion in alpha A-crystallin gives strong molecular support for the monophyly of this old and diverse order.     

Comparison of d-aspartic acid contents in alpha A-crystallin from normal and age-matched cataractous human lenses

We have previously shown that biologically uncommon d-beta-aspartic acids (Asp) were localized with very high contents at Asp-151 and Asp-58 of alpha A-crystallin from aged human lenses. The amounts increased with age, and we have proposed the mechanism of this reaction. In the present study, in order to elucidate the possible relationship between the formation of d-beta-aspartic acids in alpha A-crystallin and cataract formation, we measured the d/l ratio of beta-Asp-151 of alpha A-crystallin from both cataractous and age-matched normal human lenses. alpha A-crystallin from total proteins of cataractous and age-matched normal lenses was prepared, followed by tryptic digestion and quantification of d/l ratios for tryptic fragments containing the alpha- and beta-aspartate forms of Asp-151 residues. The results demonstrate that the d/l ratio of beta-Asp-151 of alpha A-crystallin from normal lenses is not statistically significant from that of alpha A-crystallin from cataractous lenses, suggesting that formation of this biologically uncommon amino acid may not play a role in human cataractogenesis.     

Human oestrogen receptors: differential expression of ER alpha and beta and the identification of ER beta variants

Two structurally related subtypes of oestrogen receptor (ER), known as alpha (ER alpha, NR3A1) and beta (ER beta, NR3A2) have been identified. ER beta mRNA and protein have been detected in a wide range of tissues including the vasculature, bone, and gonads in both males and females, as well as in cancers of the breast and prostate. In many tissues the pattern of expression of ER beta is distinct from that of ER alpha. A number of variant isoforms of the wild type beta receptor (ER beta 1), have been identified. In the human these include: (1). use of alternative start sites within the mRNA leading to translation of either a long (530 amino acids, hER beta 1L) or a truncated form (487aa hER beta 1s); (2). deletion of exons by alternative splicing; (3). formation of several isoforms (ER beta 2-beta 5) due to alternative splicing of exons encoding the carboxy terminus (F domain). We have raised monoclonal antibodies specific for hER beta1 as well as to three of the C terminal isoforms (beta2, beta 4 and beta 5). Using these antibodies we have found that ER beta 2, beta 4 and beta 5 proteins are expressed in nuclei of human tissues including the ovary, placenta, testis and vas deferens.In conclusion, in addition to the differential expression of full length ER alpha and ER beta a number of ER variant isoforms have been identified. The impact of the expression of these isoforms on cell responsiveness to oestrogens may add additional complexity to the ways in which oestrogenic ligands influence cell function.     

Study of subunit interactions of alpha A- and alpha B-crystallins and the effects of gamma-irradiation on their interactions by surface plasmon resonance

Alpha-crystallin, a major protein of mammalian lens, consists of two subunits, alpha A-crystallin and alpha B-crystallin. They interact to form an aggregate and play a prominent role in the maintenance of lens transparency. We evaluated the interaction between these subunits via surface plasmon resonance (SPR) using four combinations of immobilized protein and analyte: 1) AA: alpha A-crystallin was ligand immobilized onto the sensor and alpha A-crystallin was passed over the ligand, 2) AB: ligand - alpha A-crystallin, analyte - alpha B-crystallin, 3) BB: ligand - alpha B-crystallin, analyte- alpha B-crystallin, 4) BA: ligand - alpha B-crystallin, analyte - alpha A-crystallin. The order of rate of dissociation was AA approximately BA>BB approximately AB. We also examined the dissociation of gamma irradiated alpha A- and alpha B-crystallins. As radiation dose increased, so did the dissociation rate of all of the crystallins. The order of rate of dissociation of irradiated crystallins was BB>AB approximately BA>AA. The results indicate that BB is the most susceptible to gamma-irradiation and that alpha B-crystallin forms a more stable aggregate than alpha A-crystallin under normal conditions. However, when alpha B is irradiated the aggregate becomes unstable.     

The L-type calcium channel alpha 1C subunit gene undergoes extensive, uncoordinated alternative splicing

The alpha1C subunit is the pore-forming protein for the L-type calcium channel. Previous studies indicate that there is possible tissue-specific alternative splicing of this gene. In this study we cloned the entire open reading frame of the alpha1C subunit cDNA from adult rat cardiac myocytes in a single piece (6.64 kb). Using 75 positive clones that were identified by restriction enzyme mapping, we tested the alternative splicing patterns of the Ca(v) 1.2 gene that encodes the alpha1C subunit protein and focused on five loci: IS6, post-IS6, IIIS2, IVS3, and the c-terminus. The results indicate that: (1) alternative splicing occurs in most of the loci, giving rise to two or three different isoforms at those sites; (2) there is a predominant form for each splicing site, (3) there does not appear to be consistent coordination of splicing at multiple loci of this gene. Alternative splicing is not tissue-specific in most regions.     

Structural domains of agrin required for clustering of nicotinic acetylcholine receptors.

Agrin is an extracellular matrix component which promotes the clustering of nicotinic acetylcholine receptors (nAChRs) and other proteins at the neuromuscular junction. This aggregation process is one of the earliest steps in synapse formation. Expression of highly active isoforms of agrin, generated by alternative splicing, is restricted to neurons in the central nervous system (CNS) including motoneurons. In the experiments reported here we investigate the regions of agrin necessary for nAChR clustering activity using two different methods. First, we expressed truncated soluble forms of the agrin protein in mammalian cells and assessed their clustering activity. Second, we generated a panel of monoclonal antibodies (mAbs) against agrin and mapped their epitopes. Several mAbs block agrin-induced aggregation of nAChRs. One of the mAbs, Agr86, binds exclusively to the CNS-specific splicing variants and thus identifies an epitope common only to these more active isoforms. Mapping of the Agr86 epitope suggests that alternative splicing results in a distributed conformational change in the agrin protein. Taken together our data suggest that four domains in the C-terminal 55 kDa of agrin contribute to its nAChR clustering activity.     

Expression and aggregation of recombinant alpha A-crystallin and its two domains.

The 20 kDa alpha A and alpha B subunits of alpha-crystallin from mammalian eye lenses form large aggregates with an average molecular weight of 800,000. To get insight into the interactions responsible for aggregate formation, we expressed in Escherichia coli the putative N- and C-terminal domains of alpha A-crystallin, as well as the intact alpha A-crystallin chain. The proteins are expressed in a stable form and in relatively high amounts (20-60% of total protein). Recombinant alpha A-crystallin and the C-terminal domain are expressed in a water-soluble form. Recombinant alpha A-crystallin forms aggregates comparable with alpha-crystallin aggregates from calf lenses, whereas the C-terminal domain forms dimers or tetramers. The N-terminal domain is expressed in an initially water-insoluble form. After solubilization, denaturation and reaggregation the N-terminal domain exists in a high molecular weight multimeric form. These observations suggest that the interactions leading to aggregation of alpha A-crystallin subunits are mainly located in the N-terminal half of the chain.     

Complete nucleotide sequence of the chicken alpha A-crystallin gene and its 5' flanking region

The chicken alpha A-crystallin gene and 2.6 kb of its 5' flanking sequence have been isolated and characterized by electron microscopy and sequencing. The structural gene is 4.5 kb long and contains two introns, each approx. 1 kb in length. The first intron divides codons 63 and 64, and the second intron divides codons 104 and 105, as in rodents. There is little indication that the insert exon of rodents (an alternatively spliced sequence) is present in complete form in the chicken alpha A-crystallin gene; small stretches of similarity to this sequence were found throughout the gene. The 5' flanking sequence of the chicken alpha A-crystallin gene shows considerable sequence similarity with other mammalian alpha B-crystallin genes. In addition, one consensus sequence (GCAGCATGCCCTCCTAG) present in the 5' flanking region of the chicken alpha A-crystallin gene was found in the 5' flanking region of most reported crystallin genes.     

Differential susceptibility of alpha A- and alpha B-crystallin to gamma-ray irradiation

Alpha-crystallin, a major protein of all vertebrate lenses, consists of two different subunits, alpha A and alpha B, which form polymeric aggregates with an average molecular mass of 300-800 kDa. Both the alpha A and alpha B subunit have a molecular mass of about 20 kDa. It is not known why alpha crystallin aggregates comprise two different subunits, given that the physicochemical properties of these proteins are very similar. The present study compares the susceptibility of the alpha A and alpha B subunits to gamma-rays. We prepared a recombinant form of human alpha A- and alpha B-crystallin and then irradiated the proteins with gamma-rays. Based on far-UV CD spectra, alpha A-crystallin retained beta-sheet conformation after gamma irradiation up to 3.0 kGy, whereas alpha B-crystallin lost beta-sheet conformation upon exposure to gamma irradiation at >1.0 kGy. Size exclusion chromatography showed that the aggregation and polydispersity of recombinant alpha A-crystallin increased slightly after >1.0 kGy irradiation. In contrast, irradiation of alpha B-crystallin at 1.0 kGy resulted in the formation of huge aggregates and a marked increase in heterogeneity. We have also compared the chaperone activities of gamma-irradiated alpha A- and alpha B-crystallin aggregates. The activity of irradiated alpha A-crystallin was retained while that of the irradiated alpha B-crystallin was became inactive after irradiation of >0.5 kGy. Our results indicate that alpha A-crystallin is more stable to gamma irradiation than alpha B-crystallin.     

Monoclonal antibodies in the analysis of fibronectin isoforms generated by alternative splicing of mRNA precursors in normal and transformed human cells

Recent results showing that a single fibronectin gene can give rise to several different mRNAs by alternative splicing have offered an explanation for fibronectin polymorphism. Here we report on monoclonal antibodies that show specificity for a fibronectin segment (ED) that can be included or omitted from the molecule depending on the pattern of splicing of the mRNA precursors. Using these monoclonals, we have quantitatively analyzed the expression of the ED sequence in human fibronectin from different sources. The results demonstrated that, at the protein level, the ED segment is not expressed in plasma fibronectin and that, in fibronectin from the tissue culture medium of tumor-derived or simian virus-40-transformed human cells, the percentage of fibronectin molecules containing the ED segment is about 10 times higher than in fibronectin from normal human fibroblasts. These results suggest that in malignant cells the mechanisms that regulate the splicing of mRNA precursors are altered.     

Differential expression of two exons of the alpha1(XI) collagen gene (Col11a1) in the mouse embryo.

The amino terminal domain of collagen XI has a unique structure, which is believed to participate in the regulation of matrix assembly. Interestingly, several distinct isoforms of the amino terminal domain of alpha1(XI) and alpha2(XI) collagen chains exist as a result of alternative splicing. Here we report the analysis of the alternative splicing pattern of the mouse alpha1(XI) collagen gene (Col11a1). Like other vertebrate species, the mutually exclusive expression of exons 6A and 6B of Col11a1 results in the inclusion in the alpha1 chain of either an acidic peptide (pI 3.14) or a basic peptide (pI 11.66). Expression of these two exons was monitored in several tissues of the 16.5-day mouse embryo by in situ hybridization and immunohistochemistry, with exon-specific cDNA probes and peptide-specific antibodies, respectively. The results documented that isoforms containing the exon 6B-encoded peptide accumulate predominantly in the vertebrae, skeletal muscles and intestinal epithelium. By contrast, exon 6A products were found to be most abundant in the smooth muscle cells of the intestine, aorta and lung. The results using in situ hybridization confirmed those using immunohistochemistry. Albeit correlative, the evidence suggests distinct contributions of the two peptides to the differential assembly of tissue-specific matrices.     

Expression and immunochemical analysis of rat and human fibroblast growth factor receptor (flg) isoforms.

Potentially 96 splice variants among four genes that code for the human heparin-binding fibroblast growth factor receptor family complicate study of structure, metabolism, and function of single isoforms in mammalian cells. As an alternative, we expressed structural subdomains and isoforms of the flg receptor gene in bacteria and baculoviral-infected insect cells. We developed and characterized a panel of 16 isoform and domain-specific polyclonal and monoclonal antibodies. The panel of antibodies was used to distinguish mature glycosylated ligand-binding and kinase-active and -inactive recombinant isoforms in baculoviral insect cells and transfected mammalian cells and natural isoforms in rat prostate and human liver cells. The results revealed a cell type-specific expression of the flg gene and isoforms that result from combinations of splice variations. Reactive epitopes of monoclonal antibodies against both the three (alpha) and two (beta) immunoglobulin-like disulfide loop extracellular domain isoforms were mapped by cross-reactivity with synthetic polypeptide sequences and deletion mutants expressed in bacteria. The native alpha and beta receptor isoforms differed in display of shared epitopes and suggested that the NH2-terminal Loop I and COOH-terminal Loops II and III of the alpha isoform are interactive. Although the common Loops II and III appear qualitatively sufficient for ligand binding, the results suggest that tertiary relationships among loops in the three and two loop isoforms are distinct and, therefore, the two isoforms may have distinct activities. Spatial models for arrangement of immunoglobulin-like loops in the extracellular domain of the two isoforms are presented.