Molecular Cloning and Characterization of ZASP mRNA
Many novel genes have been discovered from the systematic sequencing of human skeletal muscle ESTs carried out in one of our laboratories (Lanfranchi et al. 1996
), including telethonin (Valle et al. 1997
), which is bound, as well as phosphorylated, by titin and located at the level of the Z-band (Mayans et al. 1998
; Mues et al. 1998
). Currently, >30,000 ESTs have been identified, representing well over 4,000 different independent transcripts. Amongst these, transcript HSPD00686, hereafter named ZASP, appeared to be particularly interesting, as it was found at a moderately high frequency (0.06%) and, from a preliminary study based on reverse transcriptase-PCR, seemed to be expressed only in heart and skeletal muscle.
ZASP was initially identified in our laboratory as a cluster of muscle ESTs corresponding to the 3′ terminus of the mRNA; then we isolated and sequenced the entire human and mouse ZASP transcripts by screening full-length muscle cDNA libraries and performing 5′ RACE experiments on muscle mRNA. The total length of the nucleotide sequence shown in is 1,607 bases for human and 1,469 bases for mouse. The translation of the human sequence reveals an open reading frame of 849 bases, encoding a putative protein of 283 amino acids with a molecular weight of 30,998 D, whereas the open reading frame of mouse ZASP encodes 288 amino acids and has a molecular weight of 31,426 D. The human and mouse coding sequences are very similar; there is a single insertion of 15 bases (five amino acid residues: Ala, Ser, Pro, Leu, and Ala) in mouse, and 69 base substitutions between mouse and human, resulting in eight changes in amino acid residues, as can be seen in (Val→Ile, Thr→Ser, Val→Ala, Ala→Val, Ile→Val, Ser→Thr, Asn→Ser, Phe→Tyr). Thus, the identity of human and mouse ZASP is 92% at the nucleotide level and 97% at the amino acid level.
Figure 1 cDNA and amino acid sequences of human and mouse ZASP. The start and stop codons are in bold characters and the polyadenylation sites are underlined. In the coding part of the mouse sequence, the conserved nucleotides and amino acids are represented by (more ...)
An extensive similarity search that was done using the coding sequences (nucleotide and amino acid) of human ZASP revealed a significant similarity to KIAA0613, both at the nucleotide and amino acid level. The KIAA0613 sequence (GenBank/EMBL/DDBJ accession number AB014513) was found as a cDNA clone from brain as part of a systematic sequencing project (Ishikawa et al. 1998
). A PDZ domain was detected at the NH2
-terminal of ZASP, from amino acid 1 to 85 by the ProfileScan, SMART, and Pfam programs as described in Materials and Methods. PredictProtein (Rost et al. 1994
), Psort (Nakai and Kanehisa 1992
), and SMART programs did not detect any transmembrane domain in the ZASP protein.
Genomic mapping was done using the radiation hybrid technique, as described in Materials and Methods, revealing that the ZASP gene maps near the locus for infantile-onset spinocerebellar ataxia (OMIM 271245) on the human chromosome region 10q22.3-23.2, with a significant lod score of 17.
ZASP Is Primarily Expressed in Heart and Skeletal Muscle
Northern blot analysis of different tissues using the 3′ untranslated region of ZASP as a probe revealed that skeletal muscle is the major site of expression of this gene ( and ). It is also expressed in heart, but to a lesser degree. A major band was detected at 1.9 kb in human heart and skeletal muscle, and at ~1.6 kb in mouse heart and skeletal muscle ( C). Smaller transcripts could also be seen in pancreas and placenta at ~1 kb, the signal in pancreas being quite strong. When Northern blot analysis of different human tissues was done using as a probe at the 5′ end of ZASP ( D), three transcripts of 1.9 kb, 4 kb, and 5.4 kb were detected both in heart and skeletal muscle. A weak signal could also be detected in brain, at ~6 kb.
Figure 2 Northern blot analysis of human (A, B, and D) and mouse (C) tissues demonstrating patterns of expression of ZASP mRNAs. Blots containing Poly(A)+ RNA from a variety of human and mouse tissues were probed with 3′ untranslated region of ZASP (A–C) (more ...)
Western blot analysis was done to determine the electrophoretic mobility and tissue distribution of ZASP. From the results using human tissue ( A), it can be seen that ZASP is present to a lesser extent in heart than in skeletal muscle tissue. ZASP has the same pattern of distribution in mouse tissue as in human, that is, it is predominantly found in skeletal muscle and, to a lesser extent, in heart (data not shown). When the ZASP antibody is used at high dilutions (1/20,000) it detects two bands ( A). A prominent lower band corresponds to a molecular weight of ~32 kD and an upper band of ~78 kD. However, on using twofold higher amounts of total human protein (20 μg per lane) and 100-fold more concentrated antibody (1/200 dilution), extra bands can be detected in both heart and skeletal muscle ( B) with apparent molecular weights of 22, 27, and 67 kD. Also, there are two extra proteins that can be detected only in heart (43 and 83 kD). Several human tissues (brain, heart, kidney, lung, skeletal muscle, liver, placenta, ovary, testis, and spleen) were screened by Western blotting using a high concentration of ZASP antibody (1/200 dilution) as the probe. To detect bands in tissues other than heart and skeletal muscle, six times more protein had to be used (60 μg), as well as high concentration of antibody. Under these conditions, bands could be detected in brain and placenta ( B). Four bands corresponding to proteins of ~198, 175, 38, and 20 kD could be detected in brain, and one band corresponding to a protein of 43 kD in placenta.
Figure 3 (A) Western blot analysis of heart and skeletal muscle tissue with antibodies to myosin, ZASP, and preimmune sera. Equal amounts of proteins were run in each lane (10 μg) on a 15% SDS-polyacrylamide gel and then blotted onto Immobilon-P membrane. (more ...)
The bands seen at low dilutions (1/200) with the polyclonal ZASP antibody do not appear to be cross-reactions to bacterial proteins, as these bands were not removed by preadsorption of the antiserum with an acetone powder of the bacteria used for the recombinant protein production (data not shown). It is possible that the extra bands seen at low dilutions could be due to cross-reactions with alternative forms of the ZASP protein, or with other PDZ-containing proteins present in muscle, e.g., ALP (39 kD) and the syntrophins (58–60 kD). It is interesting to note that the complex pattern of proteins seen in Western blotting with low dilutions of polyclonal antiserum ( B) was also seen with ZASP mAb (data not shown).
Since ZASP does not contain any cysteine residues, the higher forms, seen when using more protein and a higher concentration of antisera, are unlikely to be due to homodimer formation. Also, the proteins were run on SDS-PAGE under denaturing conditions in the presence of high amounts of DTT. The adsorption of ZASP antisera with either the 32 or 78 kD protein had only the effect of reducing the affinity of the preadsorbed antisera for both proteins, not one in particular (data not shown), with the ratio of the proteins remaining the same, thus suggesting that the anti-ZASP antibody recognizes an epitope, which is also present in the 78-kD protein.
To have an estimate of the level of ZASP present in muscle tissue, we employed a method (Valle et al. 1997
) based on the intensity of the signal obtained from Western blot analysis to determine the relative amount of a specific protein in tissue extracts. This procedure was used to estimate the amount of human α-actin ( A) and ZASP ( B) present in 2.5 and 10 μg of total heart and skeletal muscle protein, respectively. There is less ZASP present in heart than in skeletal muscle tissue: 4.5 ng as opposed to 18 ng in 10 μg of total proteins, which is 0.05 and 0.18%, respectively. From densitometric analysis of the bands, it can be calculated that the actin signal from 2.5 μg of total muscle proteins is approximately equivalent to 500 ng of recombinant α-actin. Therefore, using this method the percentage of actin present in heart and skeletal muscle would be ~20%, which is in agreement with the percentage (19%) previously found in adult rabbit muscle (Pollard 1981
). This method only gives an estimate of the amount of protein present in muscle tissues, based on two main assumptions: that proteins of the same size, blotted under the same conditions have the same rate of blotting; and that the antibody used has the same affinity for the native and recombinant proteins. However, within these limits it gives a reasonable approximation of the percentage of an unknown recombinant protein present in muscle tissue.
The pattern of expression of ZASP and its ability to coimmunoprecipitate other muscle proteins was studied using immunoprecipitation of total human muscle proteins obtained by in vitro translation of muscle mRNA. In and , for both adult and fetal proteins, immunoprecipitation using preimmune sera is shown in lane 1, anti-ZASP antibody in lane 2, and antimyosin antibody in lane 3. Both the 32 and 78 kD proteins could be immunoprecipitated from in vitro translated proteins of adult and fetal skeletal muscle using the ZASP antibody ( B, lane 2). Also, from in vitro translated fetal skeletal muscle, three other proteins of ~43, 50, and 72 kD could be immunoprecipitated ( B, lane 2). The 32-kD ZASP protein was immunoprecipitated, along with proteins of 14.3, 40, and 50 kD, from total adult heart proteins using anti-ZASP antibody ( C, lane 2), whereas in fetal heart ( C, lane 2) only proteins of 26, 40, and 50 kD could be detected. Therefore, it would appear that the 32-kD protein is not detectable in fetal heart. It is clear that there is more ZASP present in skeletal muscle than in heart, this data being confirmed by both Western blot analysis and these immunoprecipitation experiments. The amount of ZASP immunoprecipitated from in vitro translated fetal skeletal muscle was much less than that from adult, which would indicate that ZASP is present at very low levels in fetal tissue. This variation was not due to poor translation of fetal mRNAs, as can be seen by the total protein obtained from in vitro translation ( A), for both heart and skeletal muscle.
Myosin mAb was used in the immunoprecipitation experiments as a positive control. It immunoprecipitated a protein of ~220 kD from the in vitro translated adult and fetal skeletal muscle proteins, as well as from adult and fetal heart proteins. Both myosin and ZASP antibodies immunoprecipitated a protein of 14.3 kD from adult heart proteins.
Localization of ZASP Protein in Human Muscle Cells
Immunofluorescence experiments were undertaken in primary human myoblasts ( A) and myotubes ( B), as well as skeletal ( C) and heart muscle tissues, with the scope of detecting the intracellular localization of the ZASP protein. A fluorescence signal can be detected in some, but not all, of the primary undifferentiated muscle cells incubated with ZASP antibodies. The fluorescence is usually restricted to the pseudopodia and to the area of the cytoplasm around the nuclei ( A), where it can be seen as strongly fluorescing dots. The fluorescence intensity in individual cells may be similar in differentiated and undifferentiated cells, but the strong fluorescence seen in the undifferentiated cells is restricted to a small percentage of the total cells (5–10%), whereas in differentiated cells, the percentage is much higher (90%). In differentiated cells incubated with antibodies to ZASP, a fluorescent signal can be detected in nearly all of the cells and it is especially strong throughout the myotubes ( B). In these cells, cross-striations can be seen that are reminiscent to those seen in tissue sections incubated with ZASP antibodies ( C). However, there are also cells that show a pattern of strongly fluorescing dots similar to those seen in undifferentiated cells, and these may in fact be cells in the early stages of differentiation. A weak fluorescent signal can be detected in undifferentiated and differentiated cells incubated with preimmune serum ( and ), as well as undifferentiated cells incubated with myosin ( A). However, in differentiated cells incubated with myosin ( B), strong fluorescence can be detected in the cytoplasm near the nuclei and as cross-striations throughout the myotubes.
Figure 6 Indirect immunofluorescence of undifferentiated human myoblasts (A) and differentiated human myotubes (B). Preimmune sera and ZASP pAb were used at a dilution of 1/50; myosin mAb (MF 20) was used at 1/100 dilution. FITC-conjugated anti-mouse immunoglobulin (more ...)
In tissue sections of human heart (not shown) and skeletal muscle ( C), an alternate banding pattern could be detected by indirect immunofluorescence experiments using antibodies to ZASP (red) and actin (green). From double fluorescence experiments, the ZASP and actin signals seem to be coincident, as seen in C, which would suggest that the ZASP protein is present in the I-band.
Immunoelectron microscopy of heart and skeletal muscle tissue sections demonstrated that ZASP is located within the Z-band, as can be seen in and , the latter showing a higher magnification of the same section. Therefore, ZASP would appear to be present throughout the Z-band.
Figure 7 Localization of ZASP in human skeletal muscle by immunoelectron microscopy. Immunoelectron microscopy with ZASP pAb as the primary antibody and anti-mouse IgG whole molecule conjugated with 5-nm gold particles (Sigma Chemical Co.; G7527) as the (more ...)
Characterization of Alternative Forms of ZASP
The full-length cDNA sequence of ZASP was used to search for similar sequences in the Genbank/EMBL/DDBJ databases. Two regions of ZASP (322 and 203 bp) were found to be identical to KIAA0613, as shown in . As mentioned previously, KIAA0613 is a sequence obtained from systematic sequencing of a brain library (Ishikawa et al. 1998
). Interestingly, the 3′ end region of KIAA0613 matches perfectly with a cluster of ESTs from the 3′ end skeletal muscle catalogue mentioned above. This cluster is referred to as HSPD1333 and contains four ESTs, which are found with a frequency of 0.012% (about five times less than ZASP). Therefore, we decided to investigate further the following two points: is the KIAA0613 actually expressed in muscle, as the 3′ end tag would indicate? And, are ZASP and KIAA0613 two alternatively spliced forms encoded by the same gene?
Figure 8 Schematic representation of the ZASP transcript, two alternative muscle variants, and brain transcript KIAA0613. Boxes (not always in scale) represent the coding region and the numbers inside each box indicate the length in bases. The 3′ and 5′ (more ...)
To verify whether the entire KIAA0613 is actually expressed in muscle, we screened by PCR our full-length cDNA library of skeletal muscle, using two primers designed respectively on the 5′ and 3′ end of the KIAA0613 sequence. As a result, we obtained two variant bands that were sequenced, neither of which corresponded to the KIAA0613 sequence (). An identical result was obtained from a heart library. Therefore, we do not have any evidence that KIAA0613 is expressed in skeletal muscle or in heart. However, the two variant transcripts that were identified give further support and complexity to the idea of alternative splicing. In the schematic view presented in , it can be seen that the four transcripts are composed of different combinations of ten fragments. The perfect identity of these fragments in the four transcripts, and the way that they are assorted, is compatible with the hypothesis of alternative splicing.
To address more specifically whether these transcripts could have originated by alternative splicing from the same gene, we amplified human genomic DNA using a forward oligo designed on box 365 (see ), and a reverse oligo designed on the 203-bp box. As a result, a band >10,000 bases was obtained (data not shown). This band was used as a template for a PCR reaction, directed by primers specific for box 197 of ZASP, giving an amplified fragment identical to that of a control performed on genomic DNA. This result confirms the hypothesis of alternative splicing and indicates that the putative exon corresponding to box 197 of ZASP is located after the exon with box 365 of KIAA0613.
The 3′ end region of KIAA0613 was analyzed by the radiation hybrid technique and found to map at 10q22.3-10q23.2, the same position of the 3′ end region of ZASP.
The PDZ Domain of ZASP Interacts with α-Actinin-2
To identify muscle proteins, which could bind to the PDZ domain at the NH2-terminal of ZASP, three cDNA libraries were screened by the yeast two-hybrid system.
The segment consisting of the first 321 coding bases of ZASP was subcloned into the pHybridLex/Zeo vector as a bait and transformed into L40 yeast strain. Then, 2,500,000 transformants were screened from various muscle libraries: 280,000 clones from the pGAD10 human skeletal muscle library (pGAD10S), 600,000 clones from the pGAD10 human heart library (pGAD10H), and 1,750,000 clones from the pDisplayTarget human heart library (pDTH).
Growing clones were picked from the different libraries: 17 clones from the pGAD10S, 9 clones from the pGAD10H, and 87 clones from pDTH, and their interactions confirmed with the β-galactosidase filter assay. The inserts associated with the activation domains of the positive clones were directly amplified from yeast cells by PCR and 30 were sequenced. The inserts of 23 clones were identified as fragments of the α-actinin-2 gene (Beggs et al. 1992
), whereas the other seven clones matched mitochondrial genes and transcription factors, typical false positives of the yeast two-hybrid system.
All the clones containing α-actinin-2 cDNA, although they start from different positions, extend to the end of the coding region, as shown in . The region of α-actinin-2 binding to the PDZ domain of ZASP can be inferred from the clones containing the shortest cDNA inserts that have only the final 155 amino acids of the COOH-terminal region of the α-actinin-2 protein.
Figure 9 Schematic representation of the positive α-actinin-2 clones found using the yeast two-hybrid system. The domains of α-actinin-2 are shown at the top, whereas the remaining part of the figure shows the coding regions of the α-actinin-2, (more ...)