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2 Department of and Anatomy Cell Biology, Columbia University College of Physicians and Surgeons, New York, NY 10032
Address correspondence to Ronald K.H. Liem, Department of Pathology, Columbia University College of Physicians and Surgeons, 630 West 168th St., New York, NY 10032. Tel.: (212) 305-4078. Fax: (212) 305-5498. E-mail: RKL2{at}columbia.edu
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Abstract |
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Key Words: intermediate filaments; actin; microfilaments; microtubules; plakin
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Introduction |
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Microtubule (MT) actin cross-linking factor (MACF) is a divergent member of the plakin family (Leung et al., 1999b). At its NH2 terminus, MACF exhibits high homology to the NH2 terminus of BPAG1-n, including the ABD and the plakin domain. However, MACF does not contain a CC rod domain or an IFBD. Instead, MACF bears a 23 spectrin repeat (SR)-containing rod domain and a novel COOH-terminal MT-binding domain (MTBD). Recently, we characterized the COOH-terminal portion of a protein (MACF2) that is highly homologous to the MTBD of MACF (Sun et al., 2001). In this report, we describe a detailed characterization of the BPAG1 locus and the alternatively spliced isoforms of BPAG1 in mice. We found that MACF2 is structurally similar to MACF, and that it is an alternatively spliced form of the BPAG1 gene; hence, we renamed it BPAG1-a. BPAG1-a appears to be the predominant form of BPAG1 expressed in the nervous system. In striated muscle we discovered another BPAG1 isoform that possesses a putative novel IFBD in the center of the molecule, and we named this isoform BPAG1-b.
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Results and discussion |
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Sequence analysis of BPAG1 isoforms
The composite BPAG1-a cDNA is 17.2 kb (GenBank/EMBL/DDBJ accession no. AF396878) and encodes a 615-kD protein (Fig. S1 A). The domain structure of BPAG1-a is similar to that of MACF (Fig. 1 B). The ABD and plakin domains of BPAG1-a are identical to those reported for BPAG1-n (Yang et al., 1996). However, the rod domain of BPAG1-a is made up of 23 SRs (Fig. 1 B). BPAG1-a contains two EF-hand motifs and an MTBD at its COOH terminus (Sun et al., 2001). The primary sequences of MACF and BPAG1-a share a 52% identity and a 70% homology, suggesting that they originated from the same ancestral gene. Most of the homologies are located at the NH2 and COOH termini, whereas the sequences of the SRs are more divergent.
The composite BPAG1-b cDNA is 23.2 kb (GenBank/EMBL/DDBJ accession no. AF396879) and codes for an 834-kD protein (Fig. S1 B). The transcript of BPAG1-b is identical to that of BPAG1-a except that it has four additional exons in the middle of the molecule, including the
5-kb ORF-containing exon that was found in our genomic clone (Fig. 1 A). Part of the deduced amino acid sequence of the
5-kb ORF exhibits significant homology to the IFBD of BPAG1-e (Fig. 2)
; hence, we designated this domain as IFBD2 and the COOH-terminal domain of BPAG1-e/n as IFBD1. In contrast to IFBD1 which contains two repeats, IFBD2 harbors only one repeat. Aside from the IFBD2, the extra sequences of BPAG1-b do not show significant homology to other proteins. The composite cDNA of mouse BPAG1-e was
9.0 kb (GenBank/EMBL/DDBJ accession no. AF396877) and encoded a 302-kD protein (Fig. S1 C). The protein structure of mouse BPAG1-e is similar to its human orthologue (Sawamura et al., 1991).
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To extend our studies on the distribution of BPAG1 isoforms during development, we used probes corresponding to various domains of BPAG1 for in situ hybridization analysis on mouse embryonic day E14.5 embryos. The hybridization patterns of probes against the CC rod domain and IFBD1 were identical, as both labeled the epidermis and mucosal epithelia along the digestive tract (Fig. 4 A and unpublished data). These probes gave no signal in the nervous system, confirming that the BPAG1-n isoforms were not expressed in detectable quantities. The IFBD2 probe specifically labeled BPAG1-b mRNA in myocardium, skeletal muscle masses, vertebrae cartilage, and epithelia of the tongue (Fig. 4 B). More ubiquitous labeling patterns were observed with probes prepared for the ABD, plakin domain, SR-containing rod domain, and MTBD that would label BPAG1-a and BPAG1-b (Fig. 4 C and unpublished data). Strong signals were found with these probes in nervous tissues, especially at the pituitary primordium, the cranial ganglia, and the dorsal root ganglia (DRG). To determine the major form of BPAG1 expressed in the DRG, we hybridized a series of adjacent transverse sections with probes against various domains. Hybridization signals were obtained only with probes against the ABD, plakin domain, SR-containing rod domain, and MTBD, but not with the CC rod domain, IFBD1, and IFBD2 probes. These results demonstrated that BPAG1-e, BPAG1-n, and BPAG1-b are not expressed in high quantities in the DRG, and that the major form there is BPAG1-a (Fig. 4, DF, and unpublished data).
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Like MACF, BPAG1-a is a mammalian homologue of the Drosophila protein kakapo/short stop. Mutations in the short stop/kakapo gene result in defects in axonal outgrowth, as well as in the local development of dendritic processes (Prokop et al., 1998; Lee et al., 2000). During early development, the growth cones of both sensory and motor neurons fail to continue advancing after formation of the normal trajectory. By analogy, the neuropathology of dt mice could be the result of defects in axonal outgrowth and/or dendritic sprouting in sensory neurons. However, even though BPAG1-a is expressed broadly in the nervous system, only sensory neurons of the DRG degenerated in the dt mice. A possible explanation is that MACF can carry out the same functions as BPAG1-a. As DRG neurons express less MACF, there is less compensation from MACF for the loss of BPAG1-a, thus resulting in degeneration.
BPAG1-b is structurally similar to BPAG1-a, except that it has an additional putative IFBD in the middle of the molecule. Hence, BPAG1-b could potentially associate with microfilaments, IFs, and MTs; however, this remains to be shown. BPAG1-b is more confined to muscles, and its absence may be responsible for the muscle weakness observed in dt mice (Dalpe et al., 1999). The transcripts for the epithelial-specific BPAG1-e can be detected in the epidermis and mucosal epithelia of the digestive tracts (Fig. 4 A). BPAG1-e anchors keratin filaments to hemidesmosomes and the skin of BPAG1-null mice is more fragile in its absence (Guo et al., 1995).
We did not detect mRNAs of the proposed BPAG1-n isoforms in the central nervous system by Northern blot analyses, RPA, and in situ hybridization. However, when we performed 35 cycles of RT-PCR, we detected trace amounts of BPAG1 transcripts that have the plakin domain connected to the CC rod domain, presumably the BPAG1-n isoforms. Previously, independent studies have reported that BPAG1-n is widely expressed in the nervous system. However, the probes and antibodies used in these studies recognize not only BPAG1-n, but also BPAG1-a and BPAG1-b (Bernier et al., 1995b; Dowling et al., 1997; Dalpe et al., 1998; Yang et al., 1999). In conclusion, we propose that BPAG1-a is the primary form of BPAG1 expressed in the nervous system, and that its deficiency could account for the neurological phenotype observed in dt mice.
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Materials and methods |
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Northern blot analysis
The boundaries of the probes used were: ABD probe (nt 1915 of BPAG1-a); plakin probe (nt 25493184 of BPAG1-a); CC rod domain probe (nt 48705383 of BPAG1-e); IFBD1 probe (nt 64917601 of BPAG1-e); IFBD2 probe (nt 59376655 of BPAG1-b); SR-containing rod domain probe (nt 1198712660 of BPAG1-a); and MTBD probe (nt 1524116143 of BPAG1-a). 30 µg of total RNA was resolved by electrophoresis in 0.8% formaldehyde agarose gel. To enhance the transfer of large-size RNAs, gels were treated with 0.05 M sodium hydroxide for 20 min before blotting onto nylon membranes. Hybridization was carried out at 68°C in hybridization buffer (6x SSC, 2x Denhardt's reagent, and 0.1% SDS) overnight. The membranes were washed once with SSC and 0.1% SDS at room temperature, and three times with 0.2x SSC and 0.1% SDS at 68°C.
RPA
Riboprobes were synthesized and labeled with biotinylated UTP using MAXIscript in vitro transcription kit (Ambion). The IFBD1 antisense probe included nt 76757852 of BPAG1-e, whereas the IFBD2 and SR-containing rod domain antisense probes encompassed nt 63526583 and nt 1840118688 of BPAG1-b, respectively. The template for the actin riboprobe was supplied by the manufacturer. RPAs were performed according to the manufacturer's protocol (RPA III; Ambion).
RT-PCR analysis
RT-PCR was performed using Titanium OneStep RT-PCR kit (CLONTECH Laboratories, Inc.). The forward primer recognized the plakin domain, 5'-ATTCAAGAGTTCATGGACCTACGGACAC-3'. The isoform-specific reverse primers were: BPAG1-a, 5'-TAATTAGGCGGTTTTCAGTCTGGGTGAG-3'; BPAG1-b, 5'-CAATAAGGCCTCTTAAAACTGCCTGAAA-3'; and BPAG1-e/n, 5'-TTTCCTGCAGCTGGCTCCGGAAGTTGCG-3'. The actin primer set was provided by the manufacturer. 0.2 µg of the total RNAs from various tissues was used for each reaction. The RT-PCR cycling parameters included an RT incubation step at 50°C for 60 min, followed by an RT step at 94°C for 5 min, 25 cycles at 94°C for 30 s and at 68°C for 2 min, and a final extension step of 68°C for 5 min. 3 µl of the reaction products was subjected to electrophoresis. For the skin samples, 20 µl of the reaction products was used. The PCR products were sequenced to confirm their identities.
In situ hybridization
Both sagittal and transverse cryostat sections were prepared from E14.5 mouse embryos. Templates used for the generation of riboprobes were constructs obtained by cloning the Northern blot probes described above into pGEM7 vectors (Promega). Both sense and antisense [35S]UTPlabeled cRNA transcripts were synthesized in vitro using Riboprobe Gemini Systems (Promega) and purified using Sephadex G-50 columns (Roche). In situ hybridization was conducted as described previously (Zheng et al., 1998). Hybridization with sense control probes yielded low background staining in all cases.
Online supplemental material
The primary deduced amino acid sequences of the three major isoforms of mouse BPAG1 described in this paper, BAG1-a, BAG1-b, and BAG1-e, are available online as Fig. S1 at http://www.jcb.org/cgi/content/full/200012098.
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Footnotes |
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* Abbreviations used in this paper: ABD, actin-binding domain; BPAG1, bullous pemphigoid antigen 1; CC, coiled coil; DRG, dorsal root ganglia; dt, dystonia musculorum; IF, intermediate filament; IFBD, IF-binding domain; MACF, MT actin cross-linking factor; MT, microtubule; MTBD, MT-binding domain; RPA, ribonuclease protection assay; RT, reverse transcriptase; SR, spectrin repeat.
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Acknowledgments |
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This work was supported by grant NS15182 from the National Institutes of Health, and a grant from the Muscular Dystrophy Association. C.L. Leung was supported in part by training grant AG00189 from the National Institute on Aging.
Submitted: 22 December 2000
Revised: 6 July 2001
Accepted: 13 July 2001
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