Commercial Dysferlin antibodies are available from the following companies:
- Novocastra Laboratories Ltd. (catalog numbers: NCL-Hamlet, NCL-Hamlet-2)
- Santa Cruz Biotechnology, Inc. (catalog numbers: C-19, E-20)
- Abcam (catalog number: ab15108)
Click on the facts below to learn more.
Commercial Dysferlin antibodies are available from the following companies:
The following Dysferlin antibodies have been published:
The following antibodies are directed towards the C-terminus of Dysferlin:
The following antibodies are directed towards the N-terminus of Dysferlin:
The following antibodies are directed towards the internal region of Dysferlin:
The following antibodies react with human Dysferlin:
The following antibodies react with mouse Dysferlin:
The NCL-Hamlet antibody from Novocastra Laboratories Ltd. reacts with rat, rabbit, hamster, pig and dog Dysferlin besides the mouse and human protein.
Based on their synthetic peptide, the following Dysferlin antibodies are not predicted to cross-react with Myoferlin by the authors:
The following Dysferlin antibodies are polyclonal:
The following Dysferlin antibodies are monoclonal:
The following antibodies have been successfully used for Dysferlin immunoblotting:
The following antibodies have been successfully used for Dysferlin immunoprecipitation:
The following antibodies have been successfully used for Dysferlin IF or IHC:
The epitopes for the various Dysferlin antibodies map to the following regions of the human protein:
The epitopes for the various Myoferlin antibodies map as follows:
The species reactivity of Myoferlin antibodies is as follows:
Based on the sequence of their synthetic peptide, the following antibodies are not predicted to react with Dysferlin by the authors:
The following Myoferlin antibodies are polyclonal:
The Myoferlin antibody published in Jaiswal JK et al., 2007. Traffic 8:77-88 is monoclonal.
The following Myoferlin antibodies work well for immunoblotting:
The following Myoferlin antibodies work well for IF:
The following Myoferlin antibodies have been published, and one is available commercially:
Full-length human dysferlin cDNA construct has been described in: Hernandez-Deviez et al., Human Molecular Genetics 15(1):129-142
Full-length human dysferlin cDNA is also commercially available from OriGene (Catalog numbers: RC219485, SC303318, SC315658, www.origene.com)
Full-length mouse dysferlin cDNA construct has been described in: Vafiadaki et al., Neuroreport 12(3): 625-629.
Yes, N-terminal GFP-tagged and C-terminal Myc-tagged human dysferlin has been described in: Hernandez-Deviez et al., Human Molecular Genetics 15(1):129-142.
Yes, the human dysferlin promoter has been cloned, and is described in: Foxton et al., European Journal of Human Genetics 12:127-131.
Full-length human myoferlin cDNA constructs have been described in the following references:
Yes, 3’ HA-tagged human myoferlin has been described in: Bernatchez et al., JBC 282(42):30745-30753
Yes, full-length human myoferlin constructs are commercially available from:
Dysferlin is predominantly localized at the sarcolemma of skeletal muscle cells and can be clearly visualized at the cell periphery by immunostaining of normal muscle. This surface localization is absent or markedly reduced in patients suffering from MM/LGMD2B (Anderson et al., Human Molecular Genetics 8 (5): 855-861; Glover and Brown, Traffic 8: 785-794; Piccolo et al., Ann Neurol 48: 902-912). Dysferlin also shows cytoplasmic presence, and associates with the T-tubule network (Ampong et al., Acta Myologica 24: 134-144; Klinge et al., FASEB J 21: 1768-1776). In fact, Dysferlin localization in C2C12 myotubes is largely intracellular, and gives a reticular staining pattern. It is widely believed that cytoplasmic vesicles also harbor Dysferlin, though a direct localization to the vesicular compartment has not been demonstrated yet (Piccolo et al., Ann Neurol 48: 902-912; Bansal et al., Nature 423: 168-172).
The earliest presence of Dysferlin protein in human fetal muscle tissue can be detected at embryonic weeks 5-6, a stage when limbs begin to show regional differentiation (Anderson et al., Human Molecular Genetics 8 (5): 855-861). Dysferlin shows differential expression and subcellular distribution during early myogenesis. Data from primary human muscle cultures indicates that the level of dysferlin RNA progressively increases as myoblasts differentiate into myotubes (deLuna et al., J of Neuropath Exp Neuro 63 (10): 1104-1113). Consistent with this transcriptional pattern, C2C12 cultures show that Dysferlin protein expression begins at later stages of myoblast fusion. Also, in contrast to mature muscle fibres, Dysferlin shows a cytoplasmic distribution in myotubes (see question 3 below; Klinge et al., FASEB J 21: 1768-1776).
The highest expression of Dysferlin protein is found in skeletal and cardiac muscle, and kidneys. Dysferlin expression has also been reported in satellite cells (SCs) and certain immune cells like monocytes and macrophages. In addition, a shorter transcript of 4kb is detectable in the brain tissue and faint, lower molecular weight bands can be detected in the brain stem, spinal cord and sciatic nerve (Glover and Brown, Traffic 8: 785-794; deLuna et al., J of Neuropath Exp Neuro 63 (10): 1104-1113; Anderson et al., Human Molecular Genetics 8 (5): 855-861). Recently Dysferlin expression was also detected in human placental microvillus syncytiotrophoblasts (Vandre et al., Biol Reprod 77: 533-542). Dysferlin positive staining is also observed in case of stomach, lung, liver and spleen tissue from rats (Anderson et al., Human Molecular Genetics 8 (5): 855-861).
The human Dysferlin gene is located on chromosome 2, position 2p13.3-p13.1
The coding sequence of Dysferlin is comprised of 55 exons. Two splice variants of Dysferlin have been described, “original” and “variant-1” that are 6907bp and 6543bp in length respectively. Please see below for more information on Dysferlin splice variants.
There are two splice variants of human Dysferlin that differ from each other with respect to their first exon (exon 1). The two isoforms are designated as DYSF (original) and DYSF_v1 (variant 1), and their GenBank accession numbers are NM_003494 and DQ267935 respectively. Corresponding splice-isoforms are also found in mice, and their accession numbers are NM_001077694 (mouse transcript variant 2; similar to “original” human Dysferlin) and NM_021469 (mouse transcript variant 1; similar to “variant-1” human dysferlin). These splice isoforms are available through the Jain Foundation at no cost. Please contact the foundation to request the plasmids.
For additional information on Dysferlin splice variants please click here.
Annexins are Ca++ dependant phospholipids-binding proteins. Annexins form a multi-membered superfamily whose constituents are generally cytosolic, and are implicated in functions such as membrane organization, exocytosis, endocytosis, ion fluxes, signal transduction etc.
Moss and Morgan, Genome Biology 5:219
Gerke and Moss, Physiol Rev 82: 331-371
Annexin A1 (Annexin 1 or Lipocortin) has been implicated in the regulation of phagocytosis, cell signaling, proliferation, inflammation and control of anterior pituitary hormone release. A requirement for Annexin A1 in Ca++ induced plasma membrane repair has also been described.
McNeil et al., JBC 281 (46): 35202-35207
Annexin A2 (Annexin II or Calpactin) plays a role in functions such as vesicle trafficking, osteoclast formation and bone resorption, and anticoagulant reactions.
Annexin A6 binds to skeletal muscle at the triad junctions, goes to the sarcolemma following injury in zebra fish and increases opening time of ryanodine receptors in smooth muscle (this is not known for skeletal muscle). Annexin A6 morphant zebrafish exhibit a muscle pathology similar to dysferlin deficiency.
Dysferlin was shown to interact with Annexins A1 and A2 in a Ca++ and membrane-injury dependant fashion. The interaction was demonstrated by coimmunoprecipitation.
Yes, mouse knockout models have been generated for both Annexin A1 and A2.
Annexin A1 knockout mice exhibit resistance to anti-inflammatory effects of glucocorticoids and an exacerbated response to certain stimuli-induced inflammation. Macrophages from these mice have aberrant phagocytosis.
Hannon et al., FASEB 17: 253-255
Annexin A2 knockout mice are developmentally normal but show microvascular fibrin accumulation, impaired clearance of injury-induced arterial thrombi and impairment of postnatal angiogenesis.
Ling et al., Journal of Clinical Investigation 113 (1): 38-48
Annexin A6 knockout mice appear phenotypically normal. Annexin A6 overexpressing mice leads to cardiomyopathy within the first 4 weeks of life.
No human disorders have been attributed to Annexin A1 or A2 deficiency as of yet.
Microarray analysis of eight dysferlinopathy patients (7-Miyoshi, 1-LGMD phenotype) showed upregulation of Annexin A2.
Campanaro et al., Human Molecular Genetics 11: 3283-3298
A positive correlation has been also been observed between disease severity and levels of Annexin A1 and A2 from dysferlinopathic patients (as well as those suffering from other muscular disorders).
Cagliani et al., Human Mutation 26 (3): 283
Localization of both the Annexins at the sarcolemma is disrupted in dysferlinopathic muscle sections.
Lennon et al., JBC 278 (50): 50466-50473
Affixin (beta-parvin) is a focal adhesion protein that is ubiquitously expressed, with highest expression in heart and skeletal muscles.
Yamaji et al., JCB 153 (6): 1251-1264
Affixin was shown to interact with dysferlin in mouse and human skeletal muscles by coimmunoprecipitation. The C-terminal intracellular region of Dysferlin was identified as the Affixin binding domain and the CH1 domain of Affixin was identified as Dysferlin-interacting region.
Matsuda et al., J Neuropathol Exp Neurol 64 (4): 334- 340
Affixin knockout mice have not yet been described.
No human disorders have been attributed to Affixin deficiency as of yet.
Deficiency of Dysferlin in MM and LGMD2B causes secondary reduction of Affixin at the sarcolemma.
Carboxy-terminal domains of both AHNAK proteins (AHNAK1 and 2) interact directly with C2A-Dysferlin. The N-terminal portion of Myoferlin also interacts with the C-terminus of both AHNAK proteins.
Huang et al., FASEB J 21 (3): 732-742
AHNAK knock-out mice have no developmental irregularities and do not exhibit any abnormal phenotype. Upon further investigation, it was uncovered that another related protein exists, AHNAK2, which probably compensates for the loss of AHNAK/Desmoyokin.
Komuro et al., PNAS 101 (12): 4053-4058
Kouno et al., J Invest Dermatol. 123 (4): 700-707
No human disorders have been attributed to AHNAK deficiency as of yet.
Secondary reduction of AHNAK was reported in muscle tissue from LGMD2B patients, though it persisted in blood-vessels.
Huang et al., FASEB J 21 (3): 732-742
Also called Desmoyokin, AHNAK is a very large (700kd) protein that is ubiquitously expressed in many cell types. In epithelial cells, it is localized to the plasma membrane, whereas in non-epithelial cells, it is expressed in the nucleus and cytoplasm. It shuttles between nucleus and cytoplasm depending on the extracellular Ca++ concentration. It has been shown to interact with Annexin 2/S100A10 complex and regulate Actin cytoskeleton organization and cell membrane architecture. No in vivo functions are known for AHNAK.
Amagai, Journal of Investigative Dermatology 123 (4): xiv-xv. A Mystery of AHNAK/Desmoyokin Still Goes On
Dysferlin and Calpain-3 were shown to interact by coimmunoprecipitation in human muscle homogenates. Full-length as well as degradation products of Calpain-3 were shown to interact.
Huang et al., European Journal of Human Genetics 13 (6): 721-730
Yes, Calpain-3 knockout mice have been generated. They have small foci of muscle necrosis, and though myogenic cells fuse normally, they lack well-organized sarcomeres.
Kramerova et al., Human Molecular Genetics 13 (13): 1373-1388
Primary mutations in Calpain-3 cause LGMD2A.
Secondary reduction in Calpain-3 levels were reported in eight out of sixteen cases of LGMD2B.
Anderson, Neuromuscular Disorders 10 (8): 553-559
However, microarray studies in Dysferlin-deficiencies did not show any changes in Calpain-3 transcript levels.
Patients: Campanaro et al., Human Molecular Genetics 11 (26): 3283-3298
Mice: Hagena et al., Neuromuscular Disorders 15 (12): 863-877
Calpains are intracellular Ca++ modulated non-lysosomal cys-proteases. Calpain-3 is primarily expressed in skeletal muscle.
Caveolae are small membrane invaginations on the surface of cells that participate in membrane-trafficking, sorting, transport and signal transduction. Caveolins play a role in the formation of caveolar membranes. Caveolin-3 or M-Caveolin is a muscle-specific form that is localized to the sarcolemma.
Caveolin-3 and Dysferlin were shown to interact weakly in normal biopsied muscle by coimmunoprecipitation. Seven sites in Dysferlin correspond to micro-domains believed to bind the caveolin-3 scaffolding region.
Matsuda et al., Human Molecular Genetics 10 (17): 1761-1766
Caveolins are required for Dysferlin trafficking of Caveolin-1 or Caveolin-3 mutants which cause accumulation of Dysferlin in the Golgi complex.
Hernandez-Deviez et al., Human Molecular Genetics 15 (1): 129-142
Caveolin-3 and Dysferlin show only limited co-localization at the sarcolemma in mature muscle fibers and Dysferlin is not particularly enriched in caveolae. It was suggested that the weak association between the two proteins may occur during dysferlin trafficking but not at the membrane.
Hernandez-Deviez et al., Human Molecular Genetics 15 (1): 129-142
Caveolin-3 knockout mice show mild myopathic changes in their skeletal muscle fibers, which are also characterized by aberrant trafficking of the dystrophin-glycoprotein complex to lipid raft microdomains and abnormal organization of the T-tubule system.
Galbiati et al., JBC 276 (24): 21425-21433
Caveolin-3 knockout also results in progressive cardiomyopathy in the heart, and in addition is associated with increased adiposity and whole body insulin resistance. Woodman et al., JBC 277 (41): 38988-38997
Capozza et al., Am J Physiol Cell Physiol 288: C1317-C1331
Mutations in Caveolin-3 are associated with numerous muscle pathologies - autosomal dominant LGMD1C, distal myopathy, hyperCKemia and rippling muscle disease (RMD).
Dysferlin has been reported to be abnormally localized in LGMD1C. Membrane staining of Dysferlin is patchy and variable in intensity. In one LGMD1C patient, speckled cytoplasmic staining was observed. Though Caveolin-3 deficiency secondarily reduces Dysferlin, the opposite is not true all the time. It was proposed that this may be because Caveolin-3 is more tightly bound to the membrane and does not change when Dysferlin is absent.
Matsuda et al., Human Molecular Genetics 10 (17): 1761-1766
Walter et al., J Neurology 250 (12): 1431-1438
Campanaro et al., Human Molecular Genetics 11 (26): 3283-3298
The “original” human Dysferlin splice variant contains 2080 amino acids, whereas “variant-1” is 2081 amino acids long.
A structure for the Dysferlin protein has not yet been elucidated. However, several domains have been identified based on sequence homology to other proteins. Please click here to see a color-coded domain-distribution of Dysferlin.
C2 domains are Ca2+-binding motifs that exhibit a wide range of functions such as phospholipid-binding, signaling and membrane-trafficking. The function of DysF (dysferlin domain) is not known. The protein contains two copies of DysF, one of which is nested within the other, thereby giving rise to a DysFN and DysFC at the N- and C-termini of DysF respectively. The function of the conserved ferlin-family domains is unknown. The transmembrane-domain facilitates membrane insertion of Dysferlin.
Dysferlin is predominantly localized at the sarcolemma of skeletal muscle cells and can be clearly visualized at the cell periphery by immunostaining of normal muscle. Dysferlin also shows cytoplasmic presence, and associates with the T-tubule network. Data suggests that cytoplasmic vesicles also harbor Dysferlin, though a direct localization to the vesicular compartment has not been demonstrated yet.
Two naturally occurring mouse strains routinely used in a number of laboratory studies unrelated to dysferlinopathy, the SJL/J mouse and the A/J mouse strains, are dysferlin deficient. The dysferlin mutation of the SJL mouse has been transferred to the C57 Black 10 strain (this cross is called C57BL/10.SJL-Dysf), so that the C57BL/10 mouse can be used as a control in experiments. Similarly the mutation of the A/J mouse has been transferred to the C57 Black 6 strain (this cross is called B6.A-DysfPrmdGene/J), so that the C57BL/6 mouse can be used as a control in experiments. C57BL/6J-Chr6A/J/NaJ mice are C57BL/6 with the full A/J chromosome 6 substituted into the genome, which also makes this strain dysferlin deficient.
B6.A-DysfPRMDGene/J mice and B6.CB17-Prkdcscid mice were crossed by the laboratory of Dr. Yvan Torrente at the University of Milan to generate a B6.A-PrkdcscidDysfprmd mouse that lacks both dysferlin and the adaptive immune response.
Two research groups have also created Dysferlin knockout mice by introducing nonsense mutations into the dysferlin gene. These are the 129-Dysftm1Kcam/J and B6.129-Dysftm1Kcam/J mice developed in the laboratory of Dr. Kevin Campbell at the University of Iowa, and the Dysf-/- mouse developed in the laboratory of Dr. Robert Brown at the Massachusetts General Hospital. While the 129.Dysftm1Kcam/J and B6.129-Dysftm1Kcam/J mouse strains are commercially available, the Dysf-/- mouse strain has not been maintained and is not available for further research applications.
In addition to these dysferlin deficient lines, the laboratory of Dr. Kevin Campbell has made a line of mice expressing human dysferlin publically available: the B6.Cg-Tg(Ckm-DYSF)3Cam/J mouse strain.
For more information, see our section on mouse models.
The SJL/J mice are homozygous for a missense mutation in dysferlin. The mutation is in the 3’ splice junction of Exon 45, which causes a deletion of 171 base pairs corresponding to amino acids 1628-1685 of the Dysferlin protein. This deletion removes a part of the fifth C2 domain (C2E) of the protein. SJL mice have partial Dysferlin expression; expression has been reported to be about 15% of the WT level. In addition, Dysferlin is mislocalized, and is spread throughout the muscle cells rather than being concentrated at the sarcolemma. Dysferlin expression by the C57BL10.SJL-Dysf strain is likely to be similar to that seen in SJL mice.
The A/J mouse has an ETn retrotransposon (5-6kb) inserted in Intron 4 of the dysferlin gene. The mice are homozygous for this insertion. The mice do not express any dysferlin. Similarly, dysferlin expression in the B6.A-DysfPRMDGene/J and C57BL/6J-Chr6A/J/NaJ mice strains is likely to be absent.
In the 129.Dysftm1Kcam/J and B6.129-Dysftm1Kcam/J mice, a 12-kb region of the genome, containing the last three exons (Exons 53-55, aa1983-2080) of dysferlin, is deleted. This deletion removes the transmembrane domain of the protein. 129.Dysftm1Kcam/J mice are of a mixed 129SvJ and C57BL/6 background and B6.129-Dysftm1Kcam/J mice are on a C57BL/6 background. Both strains are homozygous for this deletion and do not express any dysferlin.
In all Dysferlin-deficient strains except A/J, the age of onset is similar, with muscle weakness appearing at about 2 months of age. In A/J, the onset and progression is somewhat slower, with weakness appearing at about 3 months of age. In general, the mice strains show initial weakness primarily in the proximal and abdominal muscles, although the C57BL/10.SJL-Dysf strain has been reported to have variable patterns of weakness between animals.
For a detailed description of phenotypes, and a comparison of the different models, please click here.
Since the mutations found in A/J and SJL/J mice are naturally occurring, choosing an appropriate control strain is challenging. The SWR/J strain has been used as a dysferlin sufficient control for the SJL/J strain and the A/WySnJ or A/HeJ strains used as dysferlin sufficient controls for the A/J strain. It is important to recognize that the SWR, A/HeJ and A/WySnJ strains are only genetically similar to, and NOT true congenic controls for, either SJL or A/J animals. It is also important to note that at some foreign repositories in Europe and Australia, A/J mice do not have the mutated dysferlin gene, so researchers in those countries have imported the dysferlin deficient A/J strain (calling it A/J DYSFPRMD) and use the A/J mice from their local mouse repositories as dysferlin sufficient controls.
C57BL/10 mice are the appropriate control for the C57BL/10.SJL-Dysf mouse strain, and C57BL/6 mice are the appropriate control for B6.A-Dysfprmd/GeneJ mice, which contain the SJL and A/J mutations, respectively. The Jackson Laboratories recommend using wild type mice from their colony of B6.129-Dysftm1Kcam/J mice as controls for that targeted dysferlin deficient strain, but it is also important to note that this line has been backcrossed to C57BL/6 for seven generations, which should serve as an appropriate control strain.
The following mice can be obtained from the Jackson Laboratories:
C57BL/6J-Chr6A/J/NaJ mice: http://jaxmice.jax.org/strain/004384.html
The Jain Foundation also maintains private colonies of B6.A-Dysfprmd/GeneJ and B6.A-DysfPRMDGene/J mice at the Jackson Laboratories in order to provide the dysferlinopathy research community with a live repository of breeding pairs and aged mice. For more information about their availability, please contact the Jain Foundation.
None have been identified yet. Animal models of muscular dystrophy in larger animals, for instance the model for Duchenne Muscular Dystrophy in Golden Retriever dogs, are sometimes much more similar in phenotype to the same disease in humans. This is often advantageous for testing proposed therapies before human trials.
The first Ferlin to be discovered was the protein Fer-1, in the nematode C. elegans. This protein is required for reproduction in C. elegans (it is involved in vesicle fusion during spermatid maturation), and therefore its name is an abbreviation for “fertilization factor 1”. The nomenclature for the human Ferlins, Fer1Lx, means “Fer-1 like number x”.
There are six known human Ferlins. In the order of their discovery, they are: Dysferlin (Fer1L1), Otoferlin (Fer1L2), Myoferlin (Fer1L3), Fer1L4, Fer1L5, and Fer1L6. It appears from a search of the reference sequences of the human genome, that there are no other undiscovered human Ferlins.
A deficiency of the Dysferlin protein causes the autosomal recessive disorders LGMD2B, and Miyoshi Myopathy. A lack of Otoferlin, which is normally expressed in the inner ear and brain, causes a recessive form of deafness, DFNB9. No human diseases have been associated with Myoferlin yet, although a mouse model lacking Myoferlin shows a myopathy and has very small muscles. The other three Ferlins, Fer1L4, Fer1L5, and Fer1L6, have not been associated with any diseases in humans or animals.
The Ferlins can be grouped into two sub-families with three proteins each, which more closely resemble each other than they do with members of the other sub-family. One group contains Dysferlin, Myoferlin, and Fer1L5; the other contains Otoferlin, Fer1L4, and Fer1L6. An easy way to remember the grouping is that all the odd numbers (Fer1L1, Fer1L3, Fer1L5), and all the even numbers (Fer1L2, Fer1L4, Fer1L6) group together. So, Myoferlin and Fer1L5 are the two human Ferlins which most closely resemble Dysferlin.
Although the genomes of most other mammals have not yet been analyzed as closely as the human genome, all of the identified Ferlins have also been identified in many other mammals: mice, rats, dogs, chimpanzees, cows, etc. The sequences are very similar to the human sequence. For instance, the human and mouse sequences for Dysferlin have 94% amino acid identity, and 97% amino acid similarity.
There is a ferlin gene in Drosophila called misfire, which like fer-1, is also involved in reproduction. In the purple sea urchin (Strongylocentrotus purpuratus), there are three identified Ferlins. Also, some proteins in yeast contain functional domains that are unique to Ferlins.
It appears that in general Ferlins are involved in vesicle fusion events. When cell membranes fuse together there is an energy barrier to the fusion process, and various factors, including Ferlins and other proteins such as Synaptotagmins, are implicated in facilitating the fusion process. For example, membrane fusion is involved in the repair of muscle sarcolemma , and critically requires Dysferlin. Also, Otoferlin has been shown to interact with SNAREs and functions in synaptic exocytosis in cochlear hair cells
Dysferlin is now realized to be a member of a family of proteins, called Ferlins, which are found in humans as well as many other organisms.