title: Insertion of a myc-tag within α-dystroglycan domains improves its biochemical and microscopic detection.
abstract: BACKGROUND
  Epitope 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.
  
  
  RESULTS
  We 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.
  
  
  CONCLUSIONS
  Further 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.
authors: Istituto di Chimica del Riconoscimento Molecolare (CNR) c/o Istituto di Biochimica e Biochimica Clinica, Università Cattolica del Sacro Cuore, Largo F, Vito 1, Rome, Italy. andrea.brancaccio@icrm.cnr.it
authors: Simona
authors: S
authors: Morlacchi
authors: Francesca
authors: F
authors: Sciandra
authors: Maria Giulia
authors: MG
authors: Bigotti
authors: Manuela
authors: M
authors: Bozzi
authors: Wolfgang
authors: W
authors: Hübner
authors: Antonio
authors: A
authors: Galtieri
authors: Bruno
authors: B
authors: Giardina
authors: Andrea
authors: A
authors: Brancaccio
mesh_terms: D002460:Cell Line; D049030:Dystroglycans / Q000737:chemistry* / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D018613:Microscopy, Confocal; D008958:Models, Molecular; D016271:Proto-Oncogene Proteins c-myc / Q000737:chemistry*
chemical_list: D016271:Proto-Oncogene Proteins c-myc; D049030:Dystroglycans