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J Biol Chem, Vol. 274, Issue 51, 36592-36600, December 17, 1999

Identification and Characterization of cvHsp
A NOVEL HUMAN SMALL STRESS PROTEIN SELECTIVELY EXPRESSED IN CARDIOVASCULAR AND INSULIN-SENSITIVE TISSUES^*

Stéphane Krief^§, Jean-François Faivre, Philippe Robert, Bertrand Le Douarin^¶, Nicole Brument-Larignon, Isabelle Lefrère, Mark M. Bouzyk^**, Karen M. Anderson, Larry D. Greller, Frank L. Tobin, Michel Souchet, and Antoine Bril

From  SmithKline Beecham Laboratoires Pharmaceutiques, 4 rue du Chesnay-Beauregard, BP 58, 35762 Saint-Grégoire, France, the  CNRS Unité Propre 041, Rennes, France, ^** SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, CM19 5AD Harlow, United Kingdom, and  SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406-0939

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Starting with computational tools that search for tissue-selective expression of assembled expressed sequenced tags, we have identified by focusing on heart libraries a novel small stress protein of 170 amino acids that we named cvHsp. cvHsp was found as being computationally selectively and highly (0.3% of the total RNA) expressed in human heart. The cvHsp gene mapped to 1p36.23-p34.3 between markers D1S434 and D1S507. The expression of cvHsp was analyzed with RNA dot, Northern blots, or reverse transcription-polymerase chain reaction: expression was high in heart, medium in skeletal muscle, and low in aorta or adipose tissues. In the heart of rat models of cardiac pathologies, cvHsp mRNA expression was either unchanged (spontaneous hypertension), up-regulated (right ventricular hypertrophy induced by monocrotaline treatment), or down-regulated (left ventricular hypertrophy following aortic banding). In obese Zucker rats, cvHsp mRNA was increased in skeletal muscle, brown, and white adipose tissues but remained unchanged in the heart. Western blot analysis using antipeptide polyclonal antibodies revealed two specific bands at 23 and 25 kDa for cvHsp in human heart. cvHsp interacted in both yeast two-hybrid and immunoprecipitation experiments with -filamin or actin-binding protein 280. Within cvHsp, amino acid residues 56-119 were shown to be important for its specific interaction with the C-terminal tail of -filamin.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Not only heat shock but also diverse stresses, including heavy metals, amino acid analogues, inflammation, and oxidative/ischemic stress, up-regulate the rapid synthesis of a multigene family of proteins, originally called heat shock proteins (Hsps)¹ (reviewed in Refs. 1 and 2). Hsps are mostly chaperones that associate with malfolded proteins, prevent their aggregation into large damaging complexes, aid their renaturation, and influence the final intracellular location of mature proteins. The Hsp superfamily comprises several subfamilies, including Hsp70, Hsp90 or Hsp110, the mitochondrial Hsp60/Hsp10 and cytosolic (t-complex polypeptide-1 (TCP-1) ring complex) chaperonin systems, and the low molecular weight heat shock or small stress proteins (smHsps), including B-crystallin, A-crystallin, Hsp20 protein, Hsp -2, Hsp-like 27, and Hsp27 (reviewed in Ref. 1). The presence of an evolutionarily conserved -crystallin domain characterizes all smHsps. This domain is preceded by an N-terminal domain, which is variable in size and sequence, and is followed by a short, poorly conserved C-terminal extension, known to undergo numerous modifications including truncations (3). The smHsp family of proteins have since been shown to play a role in stabilizing protein folding and transport and chiefly through the modulation of actin polymerization and cytoskeletal organization. B-crystallin interacts with actin, desmin, and vimentin in the heart (4, 5); Hsp27 interacts with actin and platelet factor XIII (6, 7). More recently, Hsp-2, also called MKBP, was shown to bind and activate myotonic dystrophy protein kinase in the yeast two-hybrid system (8). All of these smHsps have been shown to be highly expressed in muscular tissues, including the heart (e.g. see Ref. 8 and references therein).

Gene discovery using expressed sequence tag (EST) data bases has proved a powerful alternative to experimental cloning techniques (e.g. filter hybridization, PCR using degenerate primers, etc.) and is thought to provide a rapid route to identifying and mapping the great majority of the 65,000-80,000 genes in the human genome (9). Initial approaches for data base cloning most often rely on sequence homologies searches for paralogs or orthologs with nucleotide or protein queries using the BLAST or FASTA algorithms (10). Other strategies are based on the use of keywords queries on sequence annotations, and an example includes a text-based search to find new sequences that shared homology with ion channels (11). Similarly, a strategy based on the use of the word "prost" (for prostate) as query for EST data bases lead to the finding of three contigs (assembly of overlapping ESTs) selectively expressed in this organ (12).

In the present study, we augmented such strategies by using a novel computational approach aimed at identifying "selectively expressed" genes in chosen cDNA libraries (EST data bases) (13). This method was applied to heart cDNA libraries and allowed the identification of a novel smHsp, cvHsp, selectively expressed in cardiovascular and insulin-sensitive tissues. We report here initial characterization of cvHsp, including molecular cloning, chromosome localization, mRNA distribution in normal and pathological states, and the interaction of cvHsp with -filamin, or actin-binding protein 280 (14).

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Electronic Identification of a Tissue-selective Gene-- A computational method has been developed to identify gene products selectively expressed in a particular tissue when compared against expression levels in other tissues (13). Briefly, the algorithm identifies these exceptional levels of expression by combining a statistical test of discordancy with adjustments for the separation of the largest from the next-to-largest intensity. In addition, the algorithm estimates its own reliability in order to distinguish high confidence calculations from more ambiguous ones. The algorithm has been implemented to routinely analyze large data bases of gene abundances. The method was applied to cardiac libraries and allowed the identification of assembled ESTs highly and selectively expressed in the heart.

Cloning of cvHsp-- Several 5' clones of the assembly corresponding to cvHsp were requested and fully sequenced on both strands using an ABI automatic sequencer. 5'-Rapid amplification of cDNA ends experiments were performed using the Marathon Ready human heart cDNA (CLONTECH). The following primers were used: 5'-CCGCTCGGAAGGTGGAAGAGGTTCT-3' and 5'-CGAGGGCTGGACAGGAGAGGGTGTG-3' for the ACD transcript (see "Results" and Fig. 2, panel 3), for the initial PCR and the second nested PCR, respectively, using the protocol recommended by the manufacturer. Similarly, 5'-CCTCCTCATTCCTACAGCCCACCTT-3' and 5'-CCCCAGGGCCACAACTGTTCCTTAG-3' were used for the initial PCR and the second nested PCR, respectively, for the alternative ABCD form (see under "Results" and Fig. 2). Double stranded amplification products were then sequenced. The mouse ortholog of human cvHsp was found by homology searches in dbEST, and its sequence was further confirmed after RT-PCR amplification of the mRNA from mouse heart. The rat sequence was amplified using primers defined for the mouse. Sequences of cvHsp from human, mouse, and rat have been deposited in the GenBank^TM/EMBL data bases.

RNA Analysis-- Total RNA was extracted using the acid guanidinium thiocyanate-phenol-chloroform method (15). RT-PCR was performed using Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.) and Taq DNA polymerase (Promega) as described previously (16) in a PTC-200 Thermal Cycler (MJ Research). The sequences of the primers were as follows: 5'-CGCCCACACCCTCTCCT-3' (sense) and 5'-CTTCTCAGCCCGCACCTC-3' (antisense) for cvHsp, and 5'-TCGTGTGCACCGTGTGGGCC-3' (sense) and 5'-AGGAAACGGCGCTCGCAGCTGTCG-3' (antisense) for 1-adrenergic receptor. The expected size of the amplicons were 412 bp for cvHsp and 265 bp for 1-adrenergic receptor. Northern blot analysis was done either on 10 µg of total RNA or on 2 µg of poly(A)+ RNA using standard methods (17). A prenormalized 50-tissue poly(A)+ RNA dot blot was used to semiquantitatively assess cvHsp expression (Human RNA Master Blot, CLONTECH). Blots were exposed to x-ray films for 2-12 h. Densitometric analyses were performed for cvHsp and the ubiquitin normalization probe.

Yeast Two-hybrid Analyses-- The plasmids pHybLex/Zeo and pAS2-1 were purchased from Invitrogen and CLONTECH (Matchmaker system 2), respectively. The complete open reading frame of cvHsp (170 amino acids), and derivatives thereof (amino acids 41-170, 56-170, and 119-170) were fused to the LEXA DNA-binding domain after PCR amplification from human heart cDNA (Invitrogen) and cloning into the EcoRI and XhoI sites of pHybLex/Zeo. The 170- and 175-amino acid isoforms were also fused to the GAL4 DNA-binding domain, after cloning of the corresponding cDNAs into the EcoRI and BamHI sites of pAS2-1. The resulting fusion proteins were expressed into the yeast reporter strains PJ69-4A (18) and Y190 (CLONTECH). Plasmid pASV3, used for construction of the mouse embryo cDNA library (19), has already been described (20). For the interaction trap with LEXA-cvHsp, L40 yeast transformants were selected on plates lacking leucine, histidine, and lysine, supplemented with 300 µg of zeocin (Invitrogen) and 30 mM 3-aminotriazole (Sigma). The library inserts of clones positive in both the growth and -galactosidase assays were recovered by PCR amplification from yeast colonies and directly used for sequencing. All nucleotide sequences were verified using an ABI PRISM 377 DNA sequencer (Perkin-Elmer).

Antibody Production, Western Blotting, and Immunoprecipitation-- The deduced amino acid sequence of cvHsp was analyzed for highly antigenic regions using the Jameson-Wolf antigenic index. The following synthetic peptide QLPEDVDPTSVTSALR (amino acids 132-147) was synthesized (peptide synthesizer model 431A, Applied Biosystems), purified and conjugated to keyhole limpet hemocyanin using glutaraldehyde as the coupling agent. Fourteen-week-old New Zealand rabbits were injected every 2 weeks with peptide-carrier conjugate (150 µg/injection), and serum titers were measured by enzyme-linked immunosorbent assay on unconjugated peptide-coated plates. The immunoglobulins fraction from the cvHsp immune serum, P672, were obtained by affinity chromatography on protein A-Sepharose. For Western blotting, crude homogenates from normal human heart were subjected to a 12.5% SDS-polyacrylamide gel electrophoresis under reducing conditions (2% -mercaptoethanol) and then transferred to Biotrace polyvinylidene difluoride membranes (Gelman, Champs-sur-Marne, France). Proteins were detected using purified P672 polyclonal antibody (500 ng/ml) revealed with a donkey anti-rabbit antibody (NA934, Amersham Pharmacia Biotech), and an enhanced chemiluminescence reagent (ECL Plus, Amersham Pharmacia Biotech). Other antibodies used were monoclonal mouse anti-human -filamin (Chemicon International), polyclonal rabbit anti-human alpha-B crystallin (Serotec), and polyclonal goat anti-human Hsp27 (Santa Cruz Biotechnology). Proteins complexes containing filamin were immunoprecipitated from human heart homogenates (200 µg protein) using anti-human -filamin antibodies and protein A-agarose (Santa Cruz Biotechnology) according to the manufacturer's recommendations. The immunoprecipitates were subjected to SDS-polyacrylamide gel electrophoresis and revealed by the use of anti-cvHsp or anti-B-crystallin antibodies.

Experimental Models of Cardiovascular and Metabolic Pathologies-- The determination of the expression pattern of cvHsp was performed in tissues from different experimental models in rodents. All rats were obtained from IFFA Credo (St Germain sur l'Arbresle, France) and were maintained and used according to the National Institutes of Health guideline for the use of laboratory animals. Right ventricular hypertrophy was obtained by a single injection of monocrotaline (MCT) (60 mg/kg, subcutaneously) 3 weeks before analysis. Left ventricular hypertrophy was obtained by chronic pressure overload induced by banding the abdominal aorta (aortic banding) above the renal artery of 5 week-old Wistar male rats. Aortic stenosis was performed under pentobarbital anesthesia (30 mg/kg, intraperitoneal) using a silver clip with a clearance of 0.1 mm. Sham-operated animals underwent an identical procedure except that the clip was not tied. The magnitude of the left ventricular hypertrophy was assessed after 5 weeks by comparison of the left ventricular weight to body weight ratio from operated/treated versus sham-operated/treated animals. Other experimental models included spontaneously hypertensive rats and their age-matched Wistar-Kyoto controls, as well as Zucker obese and lean rats that were all 12 weeks old. All animals were anesthetized with pentobarbital, and tissues were withdrawn, rinsed in ice-cold RNase-free phosphate-buffered saline, and frozen in liquid nitrogen for subsequent processing.

Statistical Analysis-- Values shown are mean ± S.E. unless otherwise stated. Statistical analysis was made by using analysis of variance, and a p < 0.05 was considered as statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Electronic Identification of cvHsp as a Selectively and Highly Expressed Gene in Cardiac Libraries-- cvHsp was first identified using the "selective expression" algorithm recently reported by two of us (13) as an assembly highly "expressed" in heart libraries. This computational approach was applied to large abundance data bases (or reconstructed abundances from the dbEST and Human Genome Science data bases). In this approach, selective expression refers to a pattern of expression in one or a small number of tissues in which there is markedly high or low expression against the baseline of expression implicitly defined in the other tissues.

The assembly abundances were calculated as the number of "random" ESTs that constituted a given assembly divided by the total number of random ESTs generated from that given library. ESTs are counted as random if they were initially selected as random and not as subsequently "directed" sequencing, second walks, resequencings, the second EST of a 3'-5' pair, etc. Assembly expression was further divided into three levels of qualitative expression (high, medium, and low) such that the total amount of RNA was the same (i.e. one-third) for each expression level. According to these criteria, cvHsp was identified as selectively and highly "expressed" in heart. Indeed, cvHsp assembly represented 0.3% of the total RNA (not considering housekeeping genes; see Table I). However, because of the variability of sampling depth with different EST libraries, there can be a substantial distortion of the reconstituted abundances that has to be taken into account by the selective expression detection algorithm. To correct for this limited sampling effect, we did not use the abundances from Table I but first corrected the bias due to finite sampling (21, 22). The markedly elevated abundance in the heart libraries (mainly adult but also fetal heart) of cvHsp assembly became visually apparent (Fig. 1). According to the criteria set for detecting selective expression of genes, significance probability of 5.5 × 10⁷ was obtained for the cvHsp abundance pattern as compared with a randomly chosen pattern. Furthermore, the gap between the largest relative abundance and the next-to-largest, which is an estimator of separation of selective expression from baseline behavior and can only vary between 0 and 1, was 0.78. Overall, the algorithm assesses the cvHsp abundance pattern as a modestly strong selective expression pattern (see Ref. 13 for details). In addition to this marked expression level in adult and fetal heart libraries, cvHsp was also found to be expressed in skeletal muscle and to a lesser extent in breast, bone marrow stroma, and adipose tissue.

View larger version (5K): [in this window] [in a new window]   Fig. 1.   Computational expression pattern: selective expression of cvHsp in heart libraries. cvHsp abundances reconstituted from ESTs are plotted versus library. Abundances (see Table I) have been scaled by the maximum abundance for convenient emphasis. The marked elevation of cvHsp in the heart library (lane 7) is evident, as well as the clear separation from the levels in the other libraries, which taken together represent a baseline level of abundance. Libraries are as follows: Soares breast 2Nb HBst (lane 1), fetal heart (lane 2), Soares breast 3Nb HBst (lane 3), Soares fetal lung NbHL19W (lane 4), Soares senescent fibroblasts NbHsF (lane 5), Stratagene human skeletal muscle cDNA (lane 6), Human heart cDNA (YNakamura) (lane 7), Stratagene muscle 937209 (lane 8), synovial hypoxic fibroblasts (lane 9), bone marrow stroma (lane 10), and human adipose tissue (lane 11). (See Ref. 13 for details.)

Cloning of cvHsp-- The initial contig of cvHsp was not assigned to any known protein, and most of the ESTs that composed the initial cvHsp contig were accordingly unknown (i.e. not homologous to known genes), probably because more than 75% of the transcript consisted of the untranslated 5' and 3' regions, regions known to have much less homology than the coding block in a given family of genes. However, a few ESTs translated into an open reading frame that shared significant identity with known members of the smHsp family, i.e. Hsp27 and -crystallin. We gathered 124 ESTs by using BLAST (basic local alignment search tool) homology searches, which resolved into a contiguous sequence of about 2.2 kb.

A further analysis of the sequence suggested the presence of three different transcripts generated by alternative splicing (Fig. 2). One or two of the corresponding cDNA clone variants from these three transcripts, obtained mainly from cardiac or adipose tissue libraries, were requested and sequenced to completion. Although the ATG codon in position 75 is in a poor Kozak consensus environment (TGGATGA versus ANNATGG) and no stop codon could be found upstream of the start codon, 5'-rapid amplification of cDNA ends experiments confirmed the obtained sequence and rendered unlikely the possibility of the existence of another ATG codon upstream from that in position 75. In the three different types of transcripts, the 5' sequence was identical in bp 1-273, a region named A, and in a segment of over 1000 bp, named region D. Isoform 1 has an additional sequence of 875 bp, region E, just before the poly(A) tailing consensus signal AATAAA, thus giving a total length of 2.15 kb (in the absence of the poly(A) tail), in good agreement with the size of the major transcript determined by Northern blotting (2.3 kb; see below). The open reading frame of isoform 1, from nucleotide 75 to 584, gave a deduced amino acid sequence of 170 residues. Isoform 2 was represented in a second series of sequenced clones: these have an additional 15-bp sequence in-frame between segment A and D, named region C, which added 5 amino acids to the 170-amino acid sequence. These clones missed the E region, thus giving a transcript size of about 1.3 kb. In isoform 3, a region B of 542 bp was inserted between A and C: the corresponding sequenced clone was of 1.8 kb. Interestingly, this B segment introduced a stop codon, and the resulting deduced open reading frame (75-281) of isoform 3 was of 68 residues (Fig. 2, panel 2). Segments A, B, and C have the intronic (C/A)AG consensus sequence prior to the exon splicing donor at the end of their 3' sequences in good agreement with the AB, AC, AD, BC, and CD organization of the segments. Because the segments are at least composed of one exon, a minimal organization of the gene into five segments/exons could be envisaged (Fig. 2, panel 3). Full-length mouse and a partial rat cvHsp sequences were identified and found to share 92-95% identity with the human sequence at the amino acid level. The amino acid sequence comparison between human and mouse cvHsp is presented (Fig. 2, panel 4).

View larger version (69K): [in this window] [in a new window]   Fig. 2.   Nucleotide sequence of cvHsp cDNA, minimal organization of the cvHsp gene, and alignment of human and mouse cvHsp protein sequences. The nucleotide sequence presented in the left panel (panel 1) is that of the mainly expressed cvHsp transcript (isoform 1, ADE segments), although the additional 15 bp and 5 deduced amino acids present in the second and third transcripts only, corresponding to segment C, are represented for clarity (position 274-288, in boldface inside the rectangle). The 3 bp at the end of segment D, at the junction with the E segment, are indicated (boldface) at position 1272-1274. The sequence of segment B with its interrupting stop codon (in boldface) is indicated (panel 2). The open reading frames are of 170 amino acids (bp 75-584) for transcript 1, 175 amino acids (bp 75-599) for transcript 2, and 68 amino acids (bp 75-281) for transcript 3. The existence of the three isoforms was confirmed after sequencing of the corresponding clones. The minimal deduced organization of the cvHsp gene is presented (panel 3) (for details, see text). 1p36-p34 is the chromosome localization of the cvHsp gene. Sequence comparison of human and mouse cvHsp is presented (panel 4); these sequence shared 95% identity and 97% similarity.

Finally, seven of the components of cvHsp are NCBI ESTs with known chromosome localization: these included ESTs with GenBank^TM accession numbers R49801, H44673, AA397963, T19903, T20269, T20235, and T32631. The mapping location of these NCBI ESTs were found to be deposited in the Unigene data base, and all were in 1p36.23-p34.3 between markers D1S434 and D1S507.

Expression Pattern of cvHsp mRNA in Normal Tissue, in Heart Diseases, and in Obesity-- According to its "electronic" tissue distribution, cvHsp is expected to be principally expressed in cardiac tissue. We experimentally studied the expression pattern of cvHsp gene using several approaches summarized in Fig. 3. First, the tissue distribution of cvHsp was analyzed using a poly(A)+ RNA dot blot of 50 different tissues. The results show a high expression in adult and fetal heart and in skeletal muscle; a fainter expression was evidenced in the aorta. In contrast, cvHsp expression was virtually undetectable in other organs tested, including 15 regions of the brain, digestive tract, liver, lung, adrenal, thyroid, spleen, thymus, gonads, and placenta. Using a Northern blot analysis on 9 different tissues, a main single transcript of 2.3 kb was observed in heart and skeletal muscle only (Fig. 3, left inset). Although cvHsp mRNA could be readily detected after a 2-h exposure by Northern blot with 10 µg of total RNA from human heart, which suggested a high expression of the gene in this organ, a longer exposure revealed the presence of ~1.2- and ~1.6-kb transcripts (not shown), which are reminiscent of the sizes of transcript 2 and 3 respectively (Fig. 2, panel 3).

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Tissue distribution of cvHsp in human. A prenormalized poly(A)+ RNA dot blot of 50 different tissues was hybridized with a cvHsp probe and normalized with a cyclophilin probe. Densitometry associated with hybridization was analyzed using the Molecular Analyst software (Bio-Rad). Tissue sample number correspondence was as follows: column 1, whole brain; column 2, amygdala; column 3, caudate nuclei; column 4, cerebellum; column 5, cerebral cortex; column 6, frontal lobe; column 7, hippocampus; column 8, medulla oblongata; column 9, occipital lobe; column 10, putamen; column 11, substantia nigra; column 12, temporal lobe; column 13, thalamus; column 14, subthalamic nuclei; column 15, spinal cord; column 16, heart; column 17, aorta; column 18, skeletal muscle; column 19, colon; column 20, bladder; column 21, uterus; column 22, prostate; column 23, stomach; column 24, testis; column 25, ovary; column 26, pancreas; column 27, pituitary; column 28, adrenal; column 29, thyroid; column 30, salivary gland; column 31, mammary gland; column 32, kidney; column 33, liver; column 34, small intestine; column 35, spleen; column 36, thymus; column 37, peripheral leukocyte; column 38, lymph node; column 39, bone marrow; column 40, appendix; column 41, lung; column 42, trachea; column 43, placenta; column 44, fetal brain; column 45, fetal heart; column 46, fetal kidney; column 47, fetal liver; column 48, fetal spleen; column 49, fetal thymus; column 50, fetal lung. A main 2.3-kb transcript was observed with Northern blot analysis of cvHsp (2 µg of poly(A)+, left panel). RT-PCR on total RNA from normal heart and omental adipose tissue are displayed in the right panel: lanes 2-7 are RT-PCR using primers for cvHsp, and lane 7 is the control without reverse transcriptase. Lane 1 is the amplification of the 1-adrenergic receptor. RT-PCR was performed with the following quantities of total RNA: 10 (lane 2), 20 (lane 3), 50 (lane 4), 100 (lane 5), and 200 (lanes 1, 6, and 7) ng with 30 cycles of amplification. Lane M is the 123-bp DNA ladder molecular weight marker.

Although few ESTs were found to derive from adipose tissue libraries, we have performed RT-PCRs on RNA from human abdominal subcutaneous adipose tissue (Fig. 3, right inset). The mRNA levels of cvHsp were semiquantitatively evaluated with amplifications performed with 10-200 ng of starting total RNA from adipose tissue and heart. cvHsp could be readily amplified with 100 ng of total RNA in adipose tissue, and it appears, by comparison of signals obtained for 50 ng of RNA in heart and adipose tissue, to be expressed at lower levels in adipose tissue than in the heart.

cvHsp being mainly expressed in heart, but also in skeletal muscle and adipose tissue, we hypothesized that, if regulated, cvHsp levels of expression may be altered in cardiovascular pathologies and in metabolic disorders. Therefore, we determined the expression levels of cvHsp mRNA in right and left ventricles of hearts from rats with right ventricular hypertrophy (treated with MCT) and left ventricular hypertrophy (spontaneously hypertensive rats and aortic banding) and also in different tissues from Zucker fatty rats (Fig. 4).

View larger version (40K): [in this window] [in a new window]   Fig. 4.   cvHsp mRNA expression in rat models of heart pathologies and in insulin-sensitive tissues in genetically obese Zucker rats. Northern blot analyses of cvHsp expression using 2 µg of poly(A)+ RNA from heart left (LV) and right (RV) ventricles from spontaneously hypertensive rats (SHR) and their Wistar-Kyoto (WKY) controls and in monocrotaline-treated rats (MCT) and their sham control (SHA), chronic pressure overload in rats resulting from aortic banding (Ao. Ba.) and their sham control (SHA) (A) and in heart, skeletal muscle (from hind limb), interscapular brown adipose tissue (BAT), and epididymal white adipose tissue (WAT) from obese Zucker rats (fa/fa) and their lean (Fa/) controls (B). Densitometric values obtained after ubiquitin hybridization were used to normalize cvHsp expression and are presented in arbitrary units. Each sample consisted of a pool of RNA from three different tissues. A representative experiment is presented. Experiments were reproduced two or three times. The rat cvHsp cDNA probe was generated using the human primers described under "Experimental Procedures," sequenced, and shown to share 88% identity with the human nucleotide sequence over 280 bp. The probe hybridized to a single 2.0-kb transcript.

cvHsp mRNA expression level in heart was not changed in 12-week-old spontaneously hypertensive rats compared with age matched Wistar-Kyoto control rats. When using a model of pressure overload with aortic banding, in which the ratio of left ventricular weight to body weight increased from 1.97 ± 0.03 in sham-operated rats (n = 8) to 2.59 ± 0.07 in rats subjected to aortic stenosis for 8 weeks (n = 17, p < 0.001), there was a 2-fold decrease in cvHsp mRNA expression in both ventricles. In contrast, in MCT-treated hearts, the expression level of cvHsp was increased. In these rats, MCT induced a pulmonary hypertension, leading to an increase in the right ventricle mass (from 238 ± 17 mg in control rats receiving vehicle (n = 8) to 381 ± 39 mg in rats treated with MCT (n = 10, p < 0.001), whereas left ventricle mass remained unchanged (860 ± 18 and 841 ± 34 mg in control and MCT-treated rats, respectively)). After treatment by MCT, cvHsp mRNA expression level was increased 2.5-fold, in both the right and left ventricles, irrespectively of the site of hypertrophy (Fig. 4). It should be noticed that blots were normalized with ubiquitin, which is considered as a member of the smHsp family (23). Ubiquitin levels could possibly be modulated in the hearts from the different models of cardiac pathologies, thus rendering specious interpretation of the results. However, in MCT rats, a similar increase in cvHsp mRNA levels was obtained following normalization with glyceraldehyde-3-phosphate dehydrogenase (not shown), and, in right ventricle from aortic banded rats as compared with sham operated controls, ubiquitin levels are comparable, whereas cvHsp is clearly down-regulated (Fig. 4A).

Finally, the expression patterns of cvHsp mRNA were investigated in heart, skeletal muscle, and white and brown adipose tissues in a model of insulin-resistance associated with obesity, the Zucker fatty rat. Although there was no changes in cvHsp expression in hearts from obese rats, there was a 2-fold increase in cvHsp mRNA steady state levels in skeletal muscle (hind limb) (Fig. 4). Likewise, cvHsp expression in interscapular brown fat and epididymal white fat was increased 4-5 times in obese rats compared with their lean controls. Similar results were obtained after a 28 S ribosomal RNA normalization (not shown).

Characterization of cvHsp as a Novel Small Heat Shock Protein-- To determine the apparent molecular weight of cvHsp, we performed Western blot analyses on heart homogenates from normal human heart (Fig. 5). Antibodies raised against human B-crystallin and Hsp27 were used as controls and allowed the detection of a band at 20 and 27 kDa, respectively, as expected. The anti-cvHsp revealed a major band at 25 kDa and a less abundant band at 23 kDa, which were absent in the control preimmune antiserum. The two cvHsp bands could not be detected when the antibody was immunodepleted with the peptide that served for immunization. Because the identity of the two bands is not fully understood at the present time, and to avoid confusion with the rodent ortholog of human Hsp27, which is often called Hsp25, we suggest that this novel smHsp not be named after its molecular weight, as generally done for smHsps (1, 24). Rather, we propose that this novel smHsp be named cvHsp after its high expression in cardiovascular and insulin-sensitive tissues.

View larger version (50K): [in this window] [in a new window]   Fig. 5.   Multialignment of small heat shock proteins, their sequence comparison, and Western blots analyses of cvHsp, Hsp27, and B-crystallin. Multialignment of -crystallin A chain (CraA) (GenBankTM accession number P02489), -crystallin B chain (CraB) (P02511), Hsp20 (B53814), Hsp-2 or MKBP (Hsp beta-2 and hspB2) (U75898), Hsp27 (P04792), Hsp27-like (Hspl27) (U15590) and cvHsp (transcript 2 of 175 amino acids) is displayed in the left panel. Consensus (six identical out of seven) and conservative mutations are shown in boldface. The sequence characteristics of the small Hsp family are indicated by boxed arrows on the majority line. Length of the proteins are indicated. Homologies and identities between the small Hsps are presented in the two-entry table (determined using Bestfit algorithm). Western blot analyses (top right panel) were as follows: homogenate from human normal heart (10 µg of protein) was subjected to SDS-polyacrylamide gel electrophoresis and blotted with antibodies against cvHsp, Hsp27, or B-crystallin. Molecular weight markers and molecular size of the bands are indicated at the left and right of each image, respectively. cvHsp blots were done either in the presence (+) or absence () of the peptide that served for immunization. Preimmune serum was used as control.

The comparison of cvHsp amino acid sequence with known proteins revealed the highest level of homology with the members of the smHsp family. The alignment of cvHsp amino acid sequence with that of A-crystallin, B-crystallin, Hsp20, Hsp-2 (or MKBP), Hsp-like 27, and Hsp27 is presented in Fig. 5. All seven smHsp shown in Fig. 5 are of similar length, between 160 and 241 amino acids. Although divergent in their N-terminal region, this family is characterized structurally by the presence of a conserved C-terminal domain of about 80-100 residues (3). Motif analysis using two-dimensional visualization tools (25) suggested this C-terminal region was of about 80 amino acids (precisely 80 amino acids in the case of cvHsp), leaving further downstream a divergent region of 1-32 residues (13 for cvHsp), depending on the smHsp considered (not shown). Overall, cvHsp shared 24-30% identity and 45-54% homology with the other members of the smHsp family. These were higher when solely the C-terminal smHsp signature was taken into account: up to 36 and 58% of identity and homology, respectively. Finally, scanning of cvHsp to ProSite motifs identifies the heat shock Hsp20 protein family motif (PS00791) in the region of residues 85-158 of cvHsp. Altogether, these results clearly assign cvHsp to the small heat shock protein family.

cvHsp Interacts with -Filamin-- Because Hsps are mainly chaperones known to associate with proteins, an interaction trap designed to identify proteins that could interact with cvHsp has been carried out in yeast. The complete open reading frame of the 170-amino acid isoform of cvHsp has been cloned into the yeast expression vector pHybLex/Zeo, which allows the expression of proteins fused to the LEXA DNA-binding domain. The resulting fusion protein, thereafter referred to as LEXA-cvHsp, has been used as a bait in a two-hybrid screening carried out in the L40 yeast reporter strain, after transformation with a mouse embryo cDNA library constructed into the pASV3 plasmid, which contains the transcriptional activation domain of the VP16 viral protein (20). Given the 95% identity and 97% similarity between the human and mouse cvHsp protein sequences, the two-hybrid experiments were performed with an already reported well characterized mouse embryo library (19, 20). Screening of roughly 15 million independent yeast transformants on plates lacking histidine and supplemented with 30 mM 3-aminotriazole led to the selection of 840 clones. Among the 155 clones sequenced, 19 library plasmids contained inserts corresponding to the mouse ortholog of human actin-binding protein 280 or -filamin cloned by Gorlin et al. (14) (the mouse filamins are not fully cloned). Indeed, these sequences were assembled into three consensus sequences of 446, 598, and 699 bp. These nucleotide sequences were compared using the FrameSearch tool (GCG) to the protein sequence of human -filamin (14), -filamin (26, 27), and -filamin (28), the three known filamins. Average identities were 90, 70, and 75% with -, -, and -filamin, respectively. These data strongly suggest that isolated clones from mouse were orthologs of the human -filamin. The obtained mouse sequences covered amino acids 2181-2598 of human -filamin. The shortest mouse filamin clone isolated in the two-hybrid experiments, hip284, contained a sequence homologous to amino acids 2424-2598 of human -filamin. To further characterize cvHsp interaction with -filamin, we investigated 1) which part of cvHsp could be involved in the interaction with -filamin, and 2) whether this interaction could be observed in the human heart.

We thus generated deletion mutants of cvHsp by RT-PCR and used them in an interacting trap with hip284. Three truncated forms of cvHsp were generated and included amino acids including intervals 41-170, 56-170, and 119-170. The full-length clone of cvHsp and the two truncated clones containing segments of amino acids 41-170 and 56-170 were positive in both the growth and -galactosidase assays of this two-hybrid experiment (Table II). In contrast, the clone containing amino acids 119-170 did not interact with -filamin (hip284), suggesting that amino acids 56-119 of cvHsp were important for the interaction with -filamin.

View this table: [in this window] [in a new window]   Table II Mapping of cvHsp domains that interacted with the mouse ortholog of human -filamin (ABP-280) cvHsp deletion mutants were generated by RT-PCR and assayed for their interaction with the shortest clone found in two hybrid experiments, hip284, which corresponded to residues 2424-2598 of -filamin. Constructs positive in both the growth and -galactosidase assays are indicated (+).

Finally, to confirm whether the interaction between cvHsp and -filamin identified using the yeast two-hybrid study can take place in normal human heart, we tested the existence of cvHsp/-filamin interaction with co-immunoprecipitation experiments in human heart homogenates. In these experiments, immunodetection of cvHsp was performed after immunoprecipitation of human heart homogenates with monoclonal antibodies raised against human -filamin (Fig. 6). The coprecipitation of cvHsp, but not of B-crystallin, with human -filamin, confirmed the interaction between these proteins. Furthermore, these experiments show that the interaction between cvHsp and -filamin indeed occurs in the human heart.

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Co-immunoprecipitation of -filamin with cvHsp. Homogenate from human normal heart (200 µg of protein) was immunoprecipitated (+) with monoclonal antibody raised against -filamin, subjected to SDS-polyacrylamide gel electrophoresis, and blotted with specific antibodies raised against cvHsp or B-crystallin. Nonimmunoprecipitated () heart homogenate was used as positive controls. Molecular weight markers are indicated.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The results of the present study show that novel genes with a tissue-selective pattern of expression can be identified by computer-based alignment of ESTs combined with a selective expression algorithm applied to abundance patterns. With this approach, we identified and cloned a novel low molecular weight (25,000) heat shock protein, called cvHsp, according to its abundant expression in cardiovascular tissue. The present study provides some insight into the mechanism of action of cvHsp by providing evidence of its interaction with -filamin.

Searches in EST data bases are increasingly being used to mine for cDNA sequences from an homolog gene either in the same, i.e. paralogs (e.g. see Ref. 29) or in other species, i.e. orthologs (e.g. see Ref. 30). In effect, there are now over 2.4 million ESTs (dbEST release 052199) in the publicly available data bases, not taking into account data generated by sequencing companies contracted by pharmaceutical industries (31). Altogether, these ESTs are thought to cover the majority of the 65,000-80,000 genes in the human genome. Gene discovery using EST data bases is most often performed by homology searches using the BLAST or FASTA algorithms and their variants as nucleotide or protein queries in data bases of nucleotides, proteins, or nucleotides translated in the six reading frames (for more details, see Ref. 10). More recently, strategies based on text searching in sequence annotations were used to find novel genes, as shown with the discovery of a novel calcium channel type (11). Similarly, primary selection of ESTs from prostate libraries using keywords and further contig constructions lead to the identification of three prostate-specific cDNA contigs for which no further information was disclosed (12). We have augmented such search strategies by including expression information as well. This includes both patterns of expression (e.g. selective expression) and levels of expression in particular libraries. The selective expression pattern detection algorithm we have used (13) enabled us to determine that a particular assembly was selectively and highly abundant in heart libraries compared with that from other tissues. This has turned out to be potent combination to find new highly expressed genes with tissue-selective patterns of expression.

The computational expression pattern revealed that cvHsp was most abundant in heart libraries, with a level of expression calculated as high (Table I, Fig. 1). This high and selective expression in heart was experimentally confirmed by RNA dot-blot, Northern blot, or RT-PCR, as was the lesser expression in skeletal muscle, adipose tissue, and aorta.

It is well established that Hsps are overexpressed in pathologic situations in which a protection of the heart is evidenced and that a heat shock response is associated with enhanced postischemic recovery (32, 33). Therefore, we measured cvHsp mRNA expression in different pathologies in which the heart muscle modifies its contractile and electrophysiological activities, its mass, and its structure to adapt to a chronic stress (34, 35). cvHsp expression was unchanged in the hypertrophied heart of the spontaneous hypertensive rats. By contrast, cvHsp mRNA expression was about 2-fold decreased in both ventricles from hearts of rats with chronic pressure overload. These results suggest that no relationship can be established between developed heart hypertrophy and cvHsp expression. In addition, it appears that the overexpression of cvHsp observed in rats treated with MCT is not linked to right heart hypertrophy because it was observed in both ventricles. A similar increased in the two ventricles was observed in MCT-treated rats for Hsp72 (35). Altogether, these results suggest that cvHsp may not be directly involved in already installed cardiac pathologies. Likewise, no modification of cvHsp expression was noted in hearts from obese Zucker rats as reported for Hsp72 (36). On the other hand, our results show that cvHsp mRNA is overexpressed in the other insulin-sensitive tissues in obese Zucker rats. This suggests that cvHsp may be associated with obesity and related metabolic disorders. Interestingly, although a reduction of Hsp70 has been shown in diabetes in brown adipose tissue (37), and upon differentiation in the 3T3-L1 preadipocyte line (38), there is no report of smHsp expression profile or regulation in adipose tissue in obesity. The selective expression of a gene in heart, skeletal muscle, and white and brown adipose tissue is not unique to cvHsp, as a similar expression pattern has been reported for other proteins involved in nutritional disorders, such as the insulin-responsive glucose transporter GLUT4 (39). Because such proteins are often regulated by insulin, catecholamines, or steroids, we could speculate that similar regulation might alter cvHsp expression. Although this remains to be tested for cvHsp, altered expression of Hsp genes has been reported following treatment by insulin for Hsp72 (40), noradrenaline for Hsp27 and B-crystallin (41), and estrogen for Hsp27 (42).

cvHsp was shown to share an average of 26 and 49% identity and homology, respectively, with the six other known members of the smHsp family. Although these are lower than homologies between A-crystallin, B-crystallin, Hsp20, Hsp27, and Hsp-2, these are similar to that found for Hspl27 (Fig. 5). Phylogenically, cvHsp, Hsp-like 27, and Hsp-2 were closer to each other than to the other members of the smHsp family. In the conserved C-terminal domain of about 80 amino acid residues that characterizes the smHsp superfamily (-crystallin domain), cvHsp had a higher percentage of identity (up to 36%) and homology (58%) with the other smHsp. Importantly, this so-called -crystallin domain can be found in all known smHsp, including cvHsp, which we describe here, but has never been seen in any other protein.

Western blot analyses in human heart homogenate revealed the presence of two specific bands at 23 and 25 kDa for cvHsp. The major posttranscriptional modifications of the smHsp proteins are phosphorylations notably by cyclic AMP-dependent protein kinase and cyclic GMP-dependent protein kinase, mitogen-activated protein kinase-activated protein kinase-2, protein kinase C, and p44/42 mitogen-activated protein kinase (e.g. see Ref. 43 and references therein). Putative consensus phosphorylation functional motifs were found in the cvHsp protein (175-amino acid isoform) sequence: five protein kinase C phosphorylation sites (Ser-2, Thr-8, Thr-71, Thr-153, and Thr-168) and two casein kinase II phosphorylation sites (Thr-82 and Ser-97). However, it remains to be established whether these two 23- and 25-kDa bands corresponded to the 170- and 175-amino acid residue isoforms and/or to different phosphorylation states of cvHsp.

Structure-function studies demonstrated that the C terminus -crystallin domain of small Hsps is responsible for chaperone function, whereas the N terminus was involved in multimerization of the proteins (44). It has been reported that A- and B-crystallin could be truncated, leaving part of the N-terminal region without the -crystallin domain (reviewed in Ref. 3). Interestingly, this could also be the case for cvHsp, as transcript 3 encoded a putative protein of 68 amino acids forming solely the N-terminal region of the protein with complete exclusion of the C-terminal -crystallin domain, suggesting that cDNA transcript 3 might encode a multimerization regulatory component. Finally, the length of 170 or 175 amino acid residues compares well with the size of the prototypic member of smHsp, A-crystallin (173 residues) and with that of the other members (160-241 residues). Altogether, these data clearly assigned cvHsp to the superfamily of smHsp.

smHsps are chaperones that interact with and stabilize proteins that are damaged during biological stresses. These are mainly, but not solely, cytoskeleton proteins. B-crystallin interacts with actin, desmin, and vimentin in the heart (4, 5); Hsp27 interacts with actin and platelet factor XIII (6, 7). No data are available for Hsp20 or Hspl27. More recently Hsp-2, also called MKBP, was shown to bind and activate myotonic dystrophy protein kinase (8). We report here that cvHsp binds both in the two-hybrid and co-immunoprecipitation experiments the cytoskeleton protein -filamin (or actin-binding protein 280) and provide evidence that it occurs in the heart. Noticeably, the tissue distribution of -filamin, characterized by highest expression in heart and skeletal muscle (26), is relevant to that of cvHsp. The N-terminal region of human -filamin contains the actin-binding domain, followed by a semiflexible rod-like domain consisting of 24 homologous repeats, each of about 96 amino acids, with a total length of 2647 (14). Filamins appear to function as promoters of actin polymerization (45) and to connect cell membrane proteins to the cytoskeleton. Among these proteins are glycoprotein Ib (46), which, when complexed in the GPIa-Ib-V-IX heterotetramers, constitutes the major transmembrane receptor for von Willebrand factor. In addition to GpIb, -filamin associates with other membrane proteins, including IgG receptor FcRI (47), the 2-integrin CD18 subunit (48), presenilin-1 (49), tissue factor (50), the calcium-dependent serine proteinase furin (51), acetylcholine receptors (52), thyroid-stimulating hormone receptor (53), the cytoplasmic mitogen-activated protein kinase-4 (MKK-4) (54), and, as shown in the present study, the small stress protein cvHsp. Among the proteins known to interact with filamins, only the domains for GPIb, furin, presenilin-1, and MKK-4 have been identified. These are for GPIb repeats 17-19 (54, 55), for furin repeats 13-14 (residues 1490-1607) (51), for presenilin-1 repeats 22-24 (49), and for MKK-4 repeats 21-23 (residues 2282-2454) (55). We report here that the filamin domain for cvHsp binding covers repeats 23-24 (residues 2424-2598), including the C-terminal tail, a domain implicated in self dimerization (14), suggesting that cvHsp could be involved in the stabilization of dimerized filamin. Following back to the phosphorylation purpose, the proximity of MKK-4 and cvHsp on filamin suggests additional working hypotheses. Within cvHsp, we showed that a domain of 64 amino acids, at least corresponding to amino acids 56-119, was important for its specific interaction with filamin. This domain encompasses a majority of amino acids present in the -crystallin domain, in line with the known role for this domain in interactions with chaperoned proteins (44).

Finally, the human cvHsp gene has been mapped to chromosome 1p36.23-p34.3 between markers D1S434 and D1S507, a region representing 4 cM (~4 Mbp). It is noteworthy that several genetic diseases with a pathophysiology compatible with the expression pattern and the putative role of cvHsp have been mapped within this interval, in particular, a DNA region in 1p36 between D1S243 and D1S2660 has been associated with cardiomyopathy (56). Importantly, a missense mutation in B-crystallin was shown to cause a desmin-related myopathy, indicating that alteration in a smHsp can be responsible for a cytoskeleton-linked inherited disorder (57). This suggests that cvHsp could be a good candidate for genetic disorders mapped to 1p36-34 and characterized by cardiomyopathy or myopathy for which the defective genes are still unknown.

In conclusion, we have identified a novel smHsp primary using a novel bioinformatic strategy based on selective expression algorithms. This 25-kDa smHsp, called cvHsp, was preferentially expressed in cardiovascular and insulin-sensitive tissues. Modulation of cvHsp expression in obesity suggests that cvHsp may be associated with obesity and related metabolic disorders. In addition, the gene localization of cvHsp in 1p34-36 comprises that for several dystrophies and myopathies, including cardiomyopathy, suggesting that cvHsp could be a candidate gene for these diseases. Our results further demonstrate that cvHsp interacted with -filamin and thus could act as a chaperone protein.

	ACKNOWLEDGEMENTS

We thank Dr. Christian Carpéné (INSERM U. 317, Toulouse, France) for kindly providing human adipose tissue, Dr. Xiohua Gong (The Scripps Research Institute, La Jolla, CA) for the gift of RNA from rat lenses, Stéphane Dréano (CNRS UPR 041, Rennes, France) for sequencing on the ABI PRISM 377 DNA sequencer, and Laurence Tourtelier and Marie-Paule Laville for skillful technical assistance. Two-hybrid experiments were performed by Dr. Bertrand Le Douarin in the laboratory facilities of Prof. Francis Galibert (CNRS UPR 041, Rennes, France).

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This article is dedicated to the memory of Dr. Bertrand Le Douarin.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AF155908 (human), AF155909 (mouse), and AF155910 (rat).

^§ To whom correspondence should be addressed. Tel.: 33-299-280-04-40; Fax: 33-299-280-04-44; E-mail: Stephane_Krief@sbphrd.com.

^¶ Recipient of a SmithKline Beecham/CNRS postdoctoral fellowship.

Deceased.

	ABBREVIATIONS

The abbreviations used are: Hsp, heat shock protein; smHsp, small stress protein; EST, expressed sequence tag; kb, kilobase(s); bp, base pair(s); RT, reverse transcription; PCR, polymerase chain reaction; MCT, monocrotaline; MKK, mitogen-activated protein kinase; MKBP, myotonic dystrophy protein kinase binding protein.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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