Gene name - dystrophin
Cytological map position - 92A5--10
Function - signaling
Symbol - Dys
FlyBase ID: FBgn0260003
Genetic map position - 3R
Classification - actin binding, rod domain, spectrin repeats
Cellular location - cytoplasmic
Mutations in the human dystrophin gene cause the Duchenne and Becker muscular dystrophies. The Dystrophin protein provides a structural link between the muscle cytoskeleton and extracellular matrix to maintain muscle integrity. Recently, Dystrophin has also been found to act as a scaffold for several signaling molecules, but the roles of dystrophin-mediated signaling pathways remain unknown. To further an understanding of this aspect of the function of dystrophin, Drosophila mutants that lack the large dystrophin isoforms were generated and their role in synapse function at the neuromuscular junction was analyzed. In expression and rescue studies, lack of the large dystrophin isoforms in the postsynaptic muscle cell were shown to lead to elevated evoked neurotransmitter release from the presynaptic apparatus. Overall synapse size, the size of the readily releasable vesicle pool as assessed with hypertonic shock, and the number of presynaptic neurotransmitter release sites (active zones) are not changed in the mutants. Short-term synaptic facilitation of evoked transmitter release is decreased in the mutants, suggesting that the absence of dystrophin results in increased probability of release. Absence of the large dystrophin isoforms does not lead to changes in muscle cell morphology or alterations in the postsynaptic electrical response to spontaneously released neurotransmitter. Therefore, postsynaptic glutamate receptor function does not appear to be affected. These results indicate that the postsynaptically localized scaffolding protein Dystrophin is required for appropriate control of neuromuscular synaptic homeostasis (van der Plas, 2006).
Duchenne (DMD) and Becker muscular dystrophy are caused by mutations in the dystrophin (dys) gene. Dystrophin and its partially redundant homolog Utrophin are associated with a number of other proteins, members of the Dystrophin-glycoprotein complex (DGC). The DGC links the actin cytoskeleton to the extracellular basal lamina, providing tensile strength to muscle fibers. In addition to its structural role, the DGC acts to scaffold signaling molecules (for review, see Rando, 2001). How the DGC and associated signaling partners interact with other cellular pathways remains unclear (van der Plas, 2006).
Members of the mammalian DGC complex, including Dystrophin and Utrophin, are found in the CNS and the extrasynaptic and postsynaptic regions of muscle cells (for review, see Blake, 2002). Reduction of postsynaptic junctional folds and acetylcholine receptor (AChR) clustering are observed at the neuromuscular junction (NMJ) in the dystrophin/utrophin double knock-out mouse, but electrophysiological analyses revealed a normal postsynaptic response to spontaneous neurotransmitter release (Deconinck, 1997a; Grady, 1997). These and other data suggest that dystrophin and utrophin likely play partially redundant and subtle roles at the mammalian NMJ (Lyons, 1991; Deconinck, 1997b). A single dystrophin ortholog exists in Drosophila (Greener, 2000); its isoforms are predominantly expressed in the muscle and nervous system (Neuman, 2001; Dekkers, 2004). This study investigates the role of the postsynaptically localized dystrophin-like protein 2 (DLP2) Dystrophin isoform at the Drosophila NMJ (van der Plas, 2006).
During maturation and modification of synaptic contacts, homeostatic mechanisms match neurotransmitter release levels to changing postsynaptic requirements, keeping depolarization levels within a narrow range. Studies of myasthenia gravis patient NMJs (in which autoantibodies reduce AChR number) revealed that, in addition to anterograde signaling, the homeostatic machinery encompasses retrograde signals required for the appropriate regulation of presynaptic neurotransmitter release. Similar compensatory neurotransmitter release upregulation was shown in rodents with decreased postsynaptic AChR levels and in Drosophila with reduced DGluRIIA glutamate receptor function (van der Plas, 2006 and references therein).
In Drosophila, several proteins have been implicated in NMJ retrograde signaling, including members of the bone morphogenetic protein (BMP) pathway, the presynaptic type II receptor wit (for wishful thinking), and its muscle-derived ligand gbb (for glass bottom boat). Postsynaptic calcium/calmodulin-dependent kinase (CaMKII) activity has also been reported to modulate retrograde signaling via wit (van der Plas, 2006 and references therein).
This study takes advantage of Drosophila genetics to study the role of dystrophin at the NMJ. dystrophin is shown to be required for appropriate homeostatic control of neurotransmitter release. Absence of postsynaptically localized Dystrophin results in a wit-dependent increase in neurotransmitter release, leading to enhanced muscle depolarization. Furthermore, short-term synaptic facilitation is impaired in the dystrophin mutant, indicating an increase in the probability of release. These results reveal a novel role for dystrophin in the dynamics of neurotransmitter release (van der Plas, 2006).
This study provides evidence that postsynaptic dystrophin is required to maintain appropriate levels of presynaptic neurotransmitter release. The absence of dystrophin from the muscle results in abnormally high QC as determined by three different methods. The NMJ homeostatic machinery is apparently engaged in the absence of dystrophin, but it has an inappropriate endpoint. In contrast to members of the BMP signaling pathway implicated previously in NMJ homeostasis but similarly to CaMKII, Dystrophin does not appear to regulate synaptic growth or overall structure. The requirement for dystrophin is haplo-insufficient, indicating that the Drosophila NMJ is highly sensitive to the levels of Dystrophin, possibly reflecting its interaction with rate-limiting signaling factors (van der Plas, 2006).
This study shows, in a number of experiments, that the lack of the postsynaptically localized DLP2 isoform is responsible for the electrophysiological phenotype observed in the dystrophin mutants: (1) expression analyses and semiquantitative RT-PCR studies indicate that, of the known dystrophin isoforms, only expression of DLP2 is affected by the mutation in dysE6; (2) the mutant phenotype can be rescued by expressing DLP2 postsynaptically but not presynaptically, (3) expression of double-stranded RNA directed against sequences present in the DLP2 isoform results in increases in neurotransmitter release similar to those observed in the dysE6 mutants, only when expressed postsynaptically, (4) RNA in situ analyses and RT-PCR assays show that DLP2 is highly expressed in larval muscle, DLP1 is not detectably expressed in the musculature, and all dystrophin large isoforms are absent from the neuropil in which the cell bodies of motoneurons are located, and (5) double labeling of larval body walls with anti-Dystrophin and anti-HRP, which labels the presynaptic membrane, reveals no overlapping expression domains (van der Plas, 2006).
Morphometric analyses and stainings were performed with several muscle- and motoneuron-specific antibodies to examine the general morphological characteristics of the synapse and musculature in the mutants. These studies indicate that muscle and synapse size are not significantly altered by the lack of Dystrophin. Because it was found that Dystrophin colocalizes extrasynaptically with actin, actin distribution was examined in the mutant muscle but no differences were observed relative to the controls. The morphology of the T-tubular network was also unaltered. That the absence of dystrophin does not grossly affect muscle morphology or glutamate receptor density/properties was reflected by the observation of unchanged electrical input resistance, mEJP amplitudes, and anti-glutamate receptor subunit antibody staining in the mutants (van der Plas, 2006).
Although DLP2 is expressed only postsynaptically and changes in synapse elaboration or muscle morphology were not observed in its absence, there are significant changes in the NMJ electrophysiology of the mutant. EJP amplitudes are increased with little or no change in the mEJPs, resulting in increased QC, i.e., the number of quanta released on stimulation, calculated by the direct, failure, or variance methods. Therefore presynaptic mechanisms were examined whose alteration might account for the increase in QC at the mutant NMJ. The increase was correlated to an increase in the probability of release as indicated by reduced EJP facilitation at the mutant synapse (van der Plas, 2006).
In addition to increased release probability, an increased size of the readily releasable vesicle pool may account for the increased QC at the dystrophin mutant NMJ. However, this seems not to be the case in the dystrophin mutant because the increase in mEJP frequency at the mutant NMJ in response to hypertonic shock, a method used to assess the readily releasable pool, did not differ from that at control NMJs. It should, however, be noted that it is not yet clear that only the readily releasable pool of vesicles is released after hypertonic shock of the Drosophila larval NMJ (van der Plas, 2006).
Increased release probability may result from increased efficacy of the translation of presynaptic Ca2+ influx into transmitter release by the neuroexocytotic machinery, possibly attributable to increased sensitivity of Ca2+ sensors. However, the observation that both the mutant and wild-type NMJs have highly similar slope values for the log[Ca2+] versus log[QC] argues against such an effect. An alternative explanation is that aberrant retrograde signaling in the absence of dystrophin results in altered modulation of presynaptic Ca2+ channel activity and thus leads to increased presynaptic Ca2+ influx (van der Plas, 2006).
Ultrastructural analysis of synaptic boutons showed a slight increase of the area occupied by vesicles in the mutants, which is unlikely to be sufficient to explain the increase in QC. Also, no significant changes were observed in the number and size of active zones, whose increase could explain the increased neurotransmitter release. No increase in T-bars, the presumed docking sites for synaptic vesicles, was observed in the mutant boutons. Although at present the precise function of these morphologically defined structures is unclear, an increase in T-bars has also found correlated with increased neurotransmitter release in other studies (van der Plas, 2006).
How does the absence of dystrophin affect the retrograde control of release? The abnormally high EJP amplitudes observed at the dystrophin mutant NMJ suggests the possibility that the Dystrophin-deficient muscle inappropriately signals to the motoneuron that it is inadequately depolarized. Thus, the absence of dystrophin may result in desensitization of an as yet unknown monitor of muscle function during depolarization. This raises the following questions: what is the homeostatic monitor and how does Dystrophin interact with it? Although a postsynaptic monitor regulating NMJ homeostatic pathways has been proposed, its identity has proven elusive. The ligand-gated glutamate receptor channel, which conducts Ca2+, has been an attractive candidate, and mammalian Dystrophin is known to scaffold a variety of postsynaptic Ca2+ channels (for review, see Carlson, 1998). However, no alterations were observed in either glutamate receptor field, sensitivity to spontaneous neurotransmitter release, or localization of the DGluRIIA or DGluRIIB subunits in the dystrophin mutant, suggesting that glutamate receptors are unlikely to play a key role in the homeostatic pathways affected by the lack of dystrophin (van der Plas, 2006).
Glutamate receptor-independent monitors of muscle depolarization, possibly voltage-gated L-type channels, have been shown sufficient to trigger NMJ homeostatic mechanisms. Alteration of L-type Ca2+ channel function or other changes in Ca2+ handling in the dystrophin-deficient larval muscle, which are well documented in mammals (for review, see Gailly, 2002), might inappropriately trigger or prolong the action of the homeostatic machinery, resulting in aberrantly high neurotransmitter release and muscle hyperdepolarization. CaMKII and Ca2+/calmodulin represent other attractive candidates to directly link Dystrophin to homeostatic pathways; Ca2+/calmodulin has been shown to associate with the DGC (Madhavan, 1992), but its role in dystrophin function is not yet understood. Possibly, dystrophin indirectly affects cAMP-dependent mechanisms (Johnson, 2005) that have been shown to be involved in synaptic plasticity (Zucker, 2002) and short-term dynamics of release (van der Plas, 2006).
This study finds that the presynaptically localized type II BMP receptor wit is required for the increased QC observed in the dystrophin mutant, as also shown for increases in QC induced by the postsynaptic inhibition of CaMKII or glutamate receptor function (Haghighi, 2003). Dystrophin and CaMKII are unlikely, however, to signal through the PMad-dependent BMP signaling pathway, because the expression levels and domains of PMad are unchanged when Dystrophin or CaMKII levels are either decreased or increased. The results address the question as to whether the retrograde BMP signal directly participates in homeostatic signaling ('instructive') or is required for the overall development of the synapse ('permissive'). The findings indicate that it is likely permissive, at least for the homeostatic mechanisms induced by perturbation of Dystrophin or CaMKII levels. BMP signaling may simply be required for the development of the presynaptic apparatus to a point at which it can respond to muscle-derived cues (van der Plas, 2006).
The degree of evolutionary conservation of the role of dystrophin in regulating neurotransmitter release at the NMJ and the potential role that defective NMJ homeostasis may play in the onset or progression of muscular dystrophy in humans is, at present, unclear. Differences between species in the severity of muscle wasting in the absence of dystrophin are observed. No muscle degeneration is observed when DLP2 is absent from the musculature; other isoforms may be required for muscle integrity in Drosophila. The dystrophin-deficient cholinergic Caenorhabditis elegans NMJ also shows elevated levels of neurotransmitter, but this is attributable to decreased clearance of acetylcholine subsequent to delocalization of the SNF-6 acetylcholine transporter (Kim, 2004). Recent work on retinal synapses in the mdx mouse suggests that the large Dystrophin isoforms may also influence neurotransmitter release at other types of synapses (Green, 2004). Whether the CNS-specific Dp186 isoform plays roles at Drosophila interneuronal synapses, similar to those played by DLP2 at the NMJ, remains to be evaluated. Disrupted homeostasis at Dystrophin-deficient interneuronal brain synapses might contribute to the poorly understood mental impairments associated with DMD in humans (van der Plas, 2006).
The Drosophila gene encoding the dystrophin-like protein (DLP) is as complex as the mammalian dystrophin gene. Three 5' promoters and three internal promoters regulate the expression of three full-length and three truncated products, respectively. The existence of this complex gene structure in such evolutionary remote organisms suggests that both types of products have diverse important functions. The promoters of both the DLP gene and the mammalian dystrophin gene are located in very large introns. These introns contribute significantly to the large size of the genes. The possible relevance of the conservation of the large size of introns containing promoters to the regulation of promoter activity is discussed (Neuman, 2005).
Dystrophin is a large rod-shaped protein comprised of four domains: N-terminal actin-binding domain, twenty four spectrin-like triple helix repeats, a cysteine-rich domain, and a unique C-terminal domain. In muscle, dystrophin is a part of a large complex that links the actin cytoskeleton, the sarcolemma, and the extracellular matrix. Other proteins in the complex are collectively called dystrophin-associated proteins (DAPs). Absence of dystrophin or some components of the DAPs complex, results in progressive damage to muscle fibers during cycles of contraction and relaxation. Recent studies suggest that the dystrophin-DAPs complex may also be involved in signaling in muscle and other tissues (Neuman, 2005).
Dystrophin is encoded by one of the largest and most complex mammalian genes. Three 5′ promoters and four internal promoters regulate the 2.2 mega base gene. Thus, the gene expresses, in a tissue and cell-type specific manner, three full-length dystrophin isoforms, and four truncated proteins, each consisting of the cysteine-rich and C-terminal domains of dystrophin, with various extensions into the spectrin-like repeats domain. The 5′ promoters are active in muscle and glial cells (the M promoter), in neurons (the B promoter) and in Purkinji cells in the brain (the P promoter). The smaller proteins, named according to their molecular weights, include: Dp71, Dp116, Dp140, and Dp260 (Neuman, 2005).
Dystrophin is very similar in its domain structure, amino acids sequence (50% identity), and gene structure to another cytoskeletal protein, utrophin. While all vertebrates studied so far have both the dystrophin and utrophin genes, invertebrates have only one related gene, which is the orthologue of the ancestral dystrophin/utrophin gene. It is therefore suggested that the gene duplication event forming the dystrophin and utrophin genes, occurred during early evolution of vertebrates (Neuman, 2005).
The Drosophila homologue of the dystrophin/utrophin ancestral gene, the dystrophin-like protein (DLP) gene. contains three 5′ promoters, which regulate the expression of full-length products, and one internal promoter that regulates the expression of a 186 Kd protein, Dp186. This study describes two additional truncated gene products, showing that the gene is controlled by at least six promoters. Three promoters regulate the expression of full-size dystrophin-like proteins, and three regulate the expression of smaller proteins. According to the location of their first exons (in relation to the coding sequence), one of the proteins is the orthologue of mammalian and sea urchin Dp116. The other two Drosophila truncated products have no known vertebrate orthologues (Neuman, 2005).
Interestingly, the promoters of the DLP gene are situated in the largest introns of the gene. Likewise, the promoters of human and mouse dystrophin genes are situated in very large introns, whose size is conserved in these two species. These findings show that the large introns, which contain promoters, are major contributors to the unusually large size of the dystrophin gene in various organisms(Neuman, 2005).
A Drosophila transcript is described that is initiated in intron 16 of the DLP gene. The encoded protein, Dp186, consists of four spectrin-like repeats, the cysteine-rich domain, and the C-terminal domain of DLP. On the basis of its location in relation to the coding sequence, intron 16 of the DLP gene does not have a homologue in the human dystrophin gene (Neuman, 2005).
Human introns 29 and 44, which contain the promoters and first exons of Dp260 and Dp140, respectively, do not have homologous introns in the dmDLP gene. Human introns 55 and 62, which contain the promoters and first exons of Dp116 and Dp71, respectively, have homologous introns in the fly gene, introns 22 and 29, respectively. Since intron 29 is very small (66 bp) it is unlikely to accommodate a unique first exon and a promoter. Indeed, using the 5′RACE technique, no evidence was found of a transcript initiated in this intron. In contrast, intron 22 is very large (47 kb), and its homologues in sea urchin and mammals harbor internal promoters. Gene prediction analysis was performed on intron 22 of the DLP gene and several putative coding exons were identified. Using RT/PCR, it was found that one of these putative exons was spliced in frame to exon 23 of the gene. The predicted coding sequence begins with an ATG translation start site. The presence of 5′non-coding sequences upstream from the initiator ATG was predicted by computer analysis and confirmed by RT/PCR analysis. In addition, no evidence by RT/PCR analysis was found for splicing of the new exon to upstream known exons of the DLP gene. Using the 5′RACE technique, the transcription start site of the mRNA was found in an additional 5′ non-coding exon, residing in the same intron 22. The computer programs used did not predict the existence of this exon. The 5′RACE analysis also suggests the existence of a second transcription start site in the non-coding sequence of the second exon. The encoded protein consists of a unique large N-terminus, two spectrin-like repeats, hinge region no. 4, and the cysteine-rich and C-terminal domains of dmDLP. According to the location of the unique exons in relation to the protein coding sequences, the encoded protein is the evolutionary homologue of Dp116. However, unlike the unique first exon of Dp116, which encodes a short product-specific N-terminus consisting of 10 amino acids, the unique exons of its Drosophila orthologue encode a large polypeptide of 870 amino acids. The molecular weight of the encoded protein is 205 Kd (Accession No. AV875639). The protein, according to the nomenclature of internal products of the dystrophin gene family, is called Dp205. RT/PCR analysis showed that Dp205 mRNA is expressed in embryos, larvae, and adult flies (Neuman, 2005).
Potential new Drosophila coding sequences have been identified and confirmed to be expressed as mRNAs. By searching the database it was found that one of the annotated sequence (Accession No. BK002672) encodes the specific N-terminus of Dp205, with some additional amino acids at the C-terminus. However, the annotated protein lacks the entire sequence shared by Dp205 and DLP. RT/PCR and 5′RACE analysis indicate that this clone represents the second exon of Dp205. The analysis suggests that the extra 188 bps of the 3′ end of the annotated sequence BK002672 (which are not found in Dp205 mRNA) consist of an intronic sequence (Neuman, 2005).
A cDNA clone that was sequenced as part of a high throughput process to clone Drosophila gene collection encodes a protein consisting of the cysteine-rich and C-terminal domains of DLP and a unique 269 amino acids N-terminus. The calculated molecular weight of the encoded protein is 117Kd. The presence of 5′UTR, and RT/PCR analysis that was performed showed that the cDNA represents a third internal transcript of the DLP gene and not an alternative splicing form of DLP mRNA. The encoded protein is therefore named Dp117. Analysis of the genomic DNA shows that the 5′ unique sequence of the mRNA is transcribed from two exons localized in intron 26 of the DLP gene. The predicted initiator ATG is preceded by 635 bp of non-coding sequence. Interestingly, using RT/PCR analysis an alternative splicing form of the 5′ UTR of Dp117 mRNA was confirmed in which the donor site of exon 1 recognizes an acceptor site within exon 2. The promoter of this transcript resides in intron 26 of the DLP gene. Dp117 consists of a unique N-terminus (269 amino acids), hinge region no. 4 and the cysteine-rich and C-terminal domains of DLP. RT/PCR analysis indicates that Dp117 mRNA is expressed in all the developmental stages of Drosophila (Neuman, 2005).
The three internal products of the DLP gene contain long product-specific N-termini. Blast analysis of the unique amino acid sequences of the N-terminal regions of Dp186, Dp117 and Dp205 did not reveal significant similarity to known proteins. In addition, no significant similarity in amino acid sequence was detected between the specific N-termini of the three truncated gene products, suggesting that these sequences have different functions. A search for functional motifs revealed various potential sites for modifications, such as phosphorylation by several kinases, myristoyalation, as well as sequences that may be involved in protein-protein interactions. The functional significance of these motifs and of the unique N-termini of Dp186, Dp205 and Dp117 await further studies (Neuman, 2005).
Computer sequence analysis and comparison showed that the specific N- termini of Dp205 and Dp117 have been conserved in other Drosophila species. 92.5% and 96% sequence identity was found between the specific N-termini of Dp117 and Dp205 of Drosophila melanogaster and Drosophila yakuba, and 76% and 85% sequence identity between these specific N-termini of Drosophila melanogaster and Drosophila pseudoobscura. The estimated evolutionary distances between D. yakuba and D. melanogaster and between D. pseudoobscura and D. melanogaster are 13 and 55 million years, respectively. The conservation of the specific N-termini of the truncated products for at least 55 million years indicates that they have important functions (Neuman, 2005).
The DLP gene is one of the largest genes of Drosophila (about 130 kb). Introns comprise about 90% of the gene. Several introns are noticeably large and are the major contributors to the size of the gene. The four largest introns of the gene harbor internal promoters. Thus, the introns of the DLP gene, which contain promoters, comprise about 75% of the gene. In D. yakuba and D. pseudoobscura these introns comprise more than 80% of the gene (Neuman, 2005).
Also in the dystrophin genes of human and mouse, the internal promoters are situated in very large introns. Likewise, the 5′ introns that contain the muscle type and purkinji-type dystrophin promoters are very large. Unlike the Drosophila DLP gene, the mammalian dystrophin genes contain several additional large introns, which are not known to contain promoters (Neuman, 2005).
Due to an overall reduction in intron length, the chicken dystrophin gene is smaller than its mammalian orthologue -- its size is about one mega base. Nevertheless, the 5′ introns and the introns harboring the promoters of the internal products remained among the largest introns in the gene (Neuman, 2005).
Like Drosophila, Caenorhabditis elegans has only one gene encoding a dystrophin-like protein. The worm gene is only 31kb long, it does not have large introns, and there are as yet no reports describing multiple 5′ or internal promoters in the gene (Neuman, 2005).
Thus the DLP gene encodes three full-length gene products, DLP 1, 2, and 3 and a truncated protein Dp186 as well as two additional internal products, Dp117 and Dp205. These findings show that the DLP gene is as complex as the mammalian dystrophin gene. Thus, the structure of a large gene encoding several full-length products and several truncated products has been conserved for at least 600 million years, indicating that both type of products have important functions (Neuman, 2005).
Dp205 is the evolutionary orthologue of mammalian Dp116. Also sea urchin has a Dp116 orthologue. These findings, and the identification of a Dp116 homologue encoded by the utrophin gene, Up116, show that Dp116 is an old and conserved internal dystrophin gene product. Dp117 and Dp186 do not have mammalian homologues. Thus, with regards to location of the promoters in relation to the coding sequence, only one internal product identified so far, is found in both vertebrates and invertebrates. Due to the large evolutionary distance and the lack of sequence similarity it could not be determine whether the 5′ promoters of the fly gene are evolutionary related to the 5′ promoters of the mammalian gene (Neuman, 2005).
The variability in the location of the internal promoters in the dystrophin genes of various organisms raises the question of the origin of these promoters. One possibility is that the ancestral dystrophin gene contained all the currently existing promoters of the gene in vertebrates and invertebrates, and different promoters were lost in various evolutionary pathways. Alternatively, new promoters could be differentially formed, differentially inserted into existing introns, or differentially inserted with new introns into the genes. It is also possible that like introns, promoters have been shuttled from and into the gene, perhaps with the introns in which they are located. Currently available data are not sufficient to differentiate between these alternatives (Neuman, 2005).
The products of the dystrophin gene in mammals have very short (few amino acids) N-termini, which are product-specific. It is likely that the existence of multiple promoters (and the specific first exons) is related to a requirement for the cell-type specific expression of the various gene products. Alternatively, a common feature of the three internal products of the DLP gene is the large unique N-termini, consisting of hundreds of amino acids. These N-termini may have specific functions, possibly interactions with other proteins. However, since the three N-termini are not similar to other proteins in the database, biological functions to these parts of the proteins cannot be assigned (Neuman, 2005).
Defects in the dystrophin complex (DC) underlie several human genetic disorders, but dissection of its function is complicated by potential redundancy of the multiple vertebrate isoforms of most DC components. This analysis shows that the fly retains all essential components of the DC, but with substantially less diversity. Seventeen known human components (three dystrophin-related proteins, two dystrobrevins, five sarcoglycans, five syntrophins, one dystroglycan and one sarcospan) appear to be reduced to eight in Drosophila (one, one, three, two, one and none, respectively). The simplicity of this system recommends it as a model for its human counterpart (Greener 2000).
Residing within the genomic scaffold clone AE003726, the Drosophila orthologue of dystrophin (DmDYS) consists of an unusual gene of at least 130 kb in length (the size could not established because of the uncertainty of one intron), with 31 introns ranging from 61 bp up to 48 kb. No genes were detected within the large introns using NIX to apply a range of bioinformatic gene-detecting algorithms, and the reason for the large size of this gene remains as enigmatic as the human dystrophin gene size. The intron/exon structure is virtually unrelated to that of the human gene (one coding exon is an extraordinary 3.5 kb in length). The 2.7 Mb human dystrophin gene encodes a large protein of 3685 amino acids in length, comprising four main sections: an actin-binding N-terminus, a rod-like spectrin repeat region, a cysteine-rich region (containing a WW domain, four EF-hands and a ZZ domain) and a C-terminal region. Using the highly conserved C-terminal end, a similar sequence representative of the sole Drosophila dystrophin-like protein has been isolated. The recent characterisation of the complete coding sequence for DmDYS reveals a shorter protein (3124 residues) that retains all four sections that are distinctive of its vertebrate counterpart. The cysteine-rich and C-terminal regions remain the most highly conserved part of DmDYS, with an identity of 54% between human and fly. These regions of the vertebrate protein contain sequences known to interact with β-dystroglycan, the syntrophins and dystrobrevins (Greener 2000).
An alignment of the N-terminus reveals a lower degree of conservation in the predicted actin-binding region (43% identity, 62% similarity); the C. elegans sequence is correspondingly divergent (22% identity, 43% similarity). Assuming that the N-termini of worm and fly dystrophins bind actin, it is apparent that the mode of interaction is subject to fewer evolutionary constraints than are those of the C-terminal domains. Unlike the human protein, there does not appear to be any recognisably unique basic region within the central rod domain that may represent an alternative means of F-actin-binding (Greener 2000).
Analysis of the rod domain shows a high level of degeneracy, and unlike the C. elegans DYS-1, the fly rod domain is 20% shorter than its human dystrophin counterpart (and 12% shorter than utrophin). The central region of the DmDYS rod domain departs from the canonical dystrophin-like spectrin repeat motif: its characteristic tryptophan residues show that any specific resemblance between human and fly dystrophin is replaced for much of the rod domain by a more generic adherence to a looser repeat motif. Finally, the first and last few spectrin repeats of both fly and worm dystrophins show a high degree of continuous specific similarity to human dystrophin, suggesting a more critical role for these particular repeats (Greener 2000).
The gene which is defective in Duchenne muscular dystrophy (DMD) is the largest known gene containing at least 79 introns, some of which are extremely large. The product of the gene in muscle, dystrophin, is a 427 kDa protein. This gene encodes at least two additional non-muscle full length dystrophin isoforms transcribed from different promoters located in the 5'-end region of the gene, and four smaller proteins transcribed from internal promoters located further downstream, that lack important domains of dystrophin. Several other genes, encoding evolutionarily related proteins, have been identified. To study the evolution of the DMD gene and the significance of its various products, genes encoding dystrophin-like proteins have been sought in sea urchin and in Drosophila. A sea urchin gene encodes a protein which is an evolutionary homologue of Dp116, one of the small products of the mammalian DMD gene, and the partial sequencing of a large product of the same gene is reported. The full-length product shows strong structural similarity and sequence identity to human dystrophin and utrophin. A Drosophila gene closely related to the human dystrophin gene is described. Like the human gene, the Drosophila gene encodes at least three isoforms of full length dystrophin-like proteins (dmDLP1, dmDLP2 and dmDLP3,), regulated by different promoters located at the 5' end of the gene, and a smaller product regulated by an internal promoter (dmDp186). As in mammals, dmDp186 and the dmDLPs share the same C-terminal and cysteine-rich domains which are very similar to the corresponding domains in human dystrophin and utrophin. In addition, dmDp186 contains four of the spectrin-like repeats of the dmDLPs and a unique N-terminal region of 512 amino acids encoded by a single exon. The full length products and the small product have distinct patterns of expression. Thus, the complex structure of the dystrophin gene, encoding several large dystrophin-like isoforms and smaller truncated products with different patterns of expression, existed before the divergence between the protostomes and deuterostomes (Neuman, 2001).
The Drosophila dystrophin gene encodes at least four protein isoforms bearing a number of highly conserved domains (Greener, 2000; Neuman, 2001). The three large isoforms DLP1, DLP2, and DLP3 have an N-terminal actin-binding domain, spectrin repeats, and a C-terminal cysteine-rich domain, which in mammals has been shown to interact with other DGC proteins. A shorter isoform, Dp186, has a unique N-terminal domain appended to the pan-Dystrophin C-terminal domain (van der Plas, 2006).
date revised: 12 May 2006
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