Characterization And Localization Of A Novel Membrane Skeletal Protein

The mechanism of skeletal myoblast differentiation has been of great interest to several laboratories for years. Cell-substratum adhesion and cell-cell recognition are two early steps in the development of fusion competence which is brought about by a family of cell surface proteins known as integrins. It is generally accepted that extracellular matrix-receptor complexes convey regulatory signals to the nucleus through interactions with cytoskeletal or membrane skeletal proteins. Therefore, it is important to study those membrane skeletal proteins which interact with cell surface proteins.;The approach was to raise monoclonal antibodies against L6 myoblasts and screen for those antibodies which were against the plasma membrane. A large molecular weight membrane skeletal protein was identified which has never been described in the literature. This protein is referred to as endossin , ( endo meaning inside and ossium meaning skeleton in Latin) based on its localization in the membrane skeleton.;Initial characterization with anti-endossin antibodies revealed that two large polypeptides of approximately 700 kDa and 500 kDa ({dollar}\alpha{dollar} and {dollar}\beta{dollar} endossin) in size, cross-react with this antibody. Both endossin polypeptides do not require any precursors for their synthesis. {dollar}\alpha{dollar}-Endossin has a long half-life of approximately 24 hours in myoblast. These polypeptides are very susceptible to proteolysis in vitro and break down into a ladder of fragments, a characteristic which some of the other membrane skeletal proteins share.;Studies of endossin during myoblast differentiation were done. {dollar}\beta{dollar}-Endossin is phosphorylated at serine residues in myoblast but dephosphorylated in myotubes. In addition, the amount of endossin relative to total protein concentration decreases two-fold during or prior to the onset of differentiation. The significance of the latter two observations in the mechanism of differentiation is not known.;Endossin is present in various cell types, most notable is the red blood cells. Immunofluorescence studies confirmed its localization in the membrane endossin. Upon examination of frozen muscle tissue slices, it was revealed that endossin lines the T-tubules and/or sarcoplasmic reticulum. There appears to be similar amounts of endossin lining the cytoplasmic side of the plasma membrane of slow twitch muscle fibres compared to fast twitch fibres. However, there is a much higher amount of endossin in the T-tubules and/or sarcoplasmic reticulum of fast twitch muscle fibres when compared to slow twitch fibres.;Membrane skeletal proteins such as fodrin and dystrophin are bound directly or indirectly to cell surface glycoproteins. Endossin appears to be bound to cell surface glycoproteins as it co-caps along with ConA-binding proteins.;The results from the initial characterization and localization of endossin indicate that it is a novel membrane skeletal protein