Abstract
BackgroundEpitope tags and fluorescent fusion proteins have become indispensable molecular tools for studies in the fields of biochemistry and cell biology. The knowledge collected on the subdomain organization of the two subunits of the adhesion complex dystroglycan (DG) enabled us to insert the 10 amino acids myc-tag at different locations along the α-subunit, in order to better visualize and investigate the DG complex in eukaryotic cells.ResultsWe have generated two forms of DG polypeptides via the insertion of the myc-tag 1) within a flexible loop (between a.a. 170 and 171) that separates two autonomous subdomains, and 2) within the C-terminal domain in position 500. Their analysis showed that double-tagging (the β-subunit is linked to GFP) does not significantly interfere with the correct processing of the DG precursor (pre-DG) and confirmed that the α-DG N-terminal domain is processed in the cell before α-DG reaches its plasma membrane localization. In addition, myc insertion in position 500, right before the second Ig-like domain of α-DG, proved to be an efficient tool for the detection and pulling-down of glycosylated α-DG molecules targeted at the membrane.ConclusionsFurther characterization of these and other myc-permissive site(s) will represent a valid support for the study of the maturation process of pre-DG and could result in the creation of a new class of intrinsic doubly-fluorescent DG molecules that would allow the monitoring of the two DG subunits, or of pre-DG, in cells without the need of antibodies.
Highlights
Epitope tags and fluorescent fusion proteins have become indispensable molecular tools for studies in the fields of biochemistry and cell biology
We show how the α subunit can be tagged with the myc epitope inserted at specific sites within its N-terminal domain in order to better analyze intracellular and membrane-targeted α-DG and its possible processing within cells
We generated different DG constructs characterized by a myc-tagged α-subunit and the C-terminus of the βsubunit fused to the intrinsic fluorescent protein green fluorescence protein (GFP)
Summary
Epitope tags and fluorescent fusion proteins have become indispensable molecular tools for studies in the fields of biochemistry and cell biology. Dystroglycan (DG) is a widely expressed transmembrane protein that connects the extracellular matrix to the cytoskeleton. It is composed of two subunits, α and β, encoded by a single gene and expressed as a unique precursor (pre-DG) that is cleaved into two proteins by an early post-translational processing [1]. In skeletal muscle DG forms, together with sarcoglycans, sarcospan, syntrophins, and dystrobrevins, the dystrophin-glycoprotein complex (DGC). This complex links the extracellular matrix with the actin cytoskeleton and provides stability to the muscle fiber sarcolemma against contractile forces [1]. ΑDG can act as a direct receptor for Arenaviruses and, in complex with laminin, as a receptor for Mycobacterium leprae [5,6]
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