Dysferlin is a Plasma Membrane Protein and is Expressed Early in Human Development

Abstract

Recently, a single gene, DYSF, has been identified which is mutated in patients with limb-girdle muscular dystrophy type 2B (LGMD2B) and with Miyoshi myopathy (MM). This is of interest because these diseases have been considered as two distinct clinical conditions since different muscle groups are the initial targets. Dysferlin, the protein product of the gene, is a novel molecule without homology to any known mammalian protein. We have now raised a monoclonal antibody to dysferlin and report on the expression of this new protein: immunolabelling with the antibody (designated NCL-hamlet) demonstrated a polypeptide of approximately 230 kDa on western blots of skeletal muscle, with localization to the muscle fibre membrane by microscopy at both the light and electron microscopic level. A specific loss of dysferlin labelling was observed in patients with mutations in the LGMD2B/MM gene. Furthermore, patients with two different frameshifting mutations demonstrated very low levels of immunoreactive protein in a manner reminiscent of the dystrophin expressed in many Duchenne patients. Analysis of human fetal tissue showed that dysferlin was expressed at the earliest stages of development examined, at Carnegie stage 15 or 16 (embryonic age 5-6 weeks). Dysferlin is present, therefore, at a time when the limbs start to show regional differentiation. Lack of dysferlin at this critical time may contribute to the pattern of muscle involvement that develops later, with the onset of a muscular dystrophy primarily affecting proximal or distal muscles.

Protein defects in neuromuscular diseases

“…Although a partial deficiency has been reported in LGMD2B patients (46), dysferlin deficiency seems to be specific to LGMD2B in our patients, and has not been seen as a secondary effect in other forms of muscular dystrophy (44).…”

“…Dysferlin, coded by a gene on 2p12-14, is a ubiquitously expressed 230-kDa molecule localized in the periphery of muscle fibers, linked to the sarcolemmal membrane (46). Dysferlin can be detected also in blood, skin and in chorionic villus biopsies (44,47).…”

Genetic Manipulation of Dysferlin Expression in Skeletal Muscle

“…Dysferlin has been shown to reside at the plasma membrane in skeletal muscle, as well as some association with cytoplasmic vesicles. 4,16 Immunohistochemistry for dysferlin in sections of quadriceps muscle showed the protein to be predominantly localized at and near the sarcolemma of Dysf-TG mice, similar to wild-type ( Figure 1C). Routine H&E staining of the quadriceps of Dysf-TG mice at 11 months of age showed no histopathology associated with mild dysferlin overexpression ( Figure 1D).…”

Characterisation of the dysferlin skeletal muscle promoter

“…1,2 Dysferlin expression is detectable very early during the specification of the skeletal muscle and is strongly expressed in the adult skeletal muscle, where it localises to the sarcolemma, 3 although it is not part of the dystrophin -glycoprotein complex. 4 Dysferlin has been shown to associate with caveolin-3 by coimmunoprecipitation, 5 and is detected intracellularly in a number of limb-girdle muscular dystrophies.…”