Insertion of a myc-tag within α-dystroglycan domains improves its biochemical and microscopic detection.

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 ß-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.

Enzymatic processing of beta-dystroglycan recombinant ectodomain by MMP-9: identification of the main cleavage site.

Dystroglycan (DG) is a membrane receptor belonging to the complex of glycoproteins associated to dystrophin. DG is formed by two subunits, alpha-DG, a highly glycosylated extracellular matrix protein, and beta-DG, a transmembrane protein. The two DG subunits interact through the C-terminal domain of alpha-DG and the N-terminal extracellular domain of beta-DG in a noncovalent way. Such interaction is crucial to maintain the integrity of the plasma membrane. In some pathological conditions, the interaction between the two DG subunits may be disrupted by the proteolytic activity of gelatinases (i.e. MMP-9 and/or MMP-2) that removes a portion or the whole beta-DG ectodomain producing a 30 kDa truncated form of beta-DG. However, the molecular mechanism underlying this event is still unknown. In this study, we carried out proteolysis of the recombinant extracellular domain of beta-DG, beta-DG(654-750) with human MMP-9, characterizing the catalytic parameters of its cleavage. Furthermore, using a combined approach based on SDS-PAGE, MALDI-TOF and HPLC-ESI-IT mass spectrometry, we were able to identify one main MMP-9 cleavage site that is localized between the amino acids His-715 and Leu-716 of beta-DG, and we analysed the proteolytic fragments of beta-DG(654-750) produced by MMP-9 enzymatic activity.

Mutagenesis at the alpha-beta interface impairs the cleavage of the dystroglycan precursor.

The interaction between a-dystroglycan (alpha-DG) and beta-dystroglycan (beta-DG), the two constituent subunits of the adhesion complex dystroglycan, is crucial in maintaining the integrity of the dystrophin-glycoprotein complex. The importance of the alpha-beta interface can be seen in the skeletal muscle of humans affected by severe conditions, such as Duchenne muscular dystrophy, where the alpha-beta interaction can be secondarily weakened or completely lost, causing sarcolemmal instability and muscular necrosis. The reciprocal binding epitopes of the two subunits reside within the C-terminus of alpha-DG and the ectodomain of beta-DG. As no ultimate structural data are yet available on the alpha-beta interface, site-directed mutagenesis was used to identify which specific amino acids are involved in the interaction. A previous alanine-scanning analysis of the recombinant beta-DG ectodomain allowed the identification of two phenylalanines important for alpha-DG binding, namely F692 and F718. In this article, similar experiments performed on the alpha-DG C-terminal domain pinpointed two residues, G563 and P565, as possible binding counterparts of the two beta-DG phenylalanines. In 293-Ebna cells, the introduction of alanine residues instead of F692, F718, G563 and P565 prevented the cleavage of the DG precursor that liberates alpha- and beta-DG, generating a pre-DG of about 160 kDa. This uncleaved pre-DG tetramutant is properly targeted at the cell membrane, is partially glycosylated and still binds laminin in pull-down assays. These data reinforce the notion that DG processing and its membrane targeting are two independent processes, and shed new light on the molecular mechanism that drives the maturation of the DG precursor.

Functional diversity of dystroglycan.

During the last 15 years, following its identification and first detailed molecular characterization, the dystroglycan (DG) complex has taken centre stage in biology and biomedicine. Functions in different cells and tissues have been identified for this complex, ranging from its typical role in skeletal muscle as a sarcolemmal stabilizer, highlighted by the recently identified "secondary dystroglycanopathies", to a variety of very diverse functions including embryogenesis, cancer progression, virus particle entry and cell signalling. Such functional promiscuity can be in part explained when considering the multiple domain organization of the two DG subunits, the extracellular alpha-DG and the transmembrane beta-DG, that has been largely scrutinized, but only in part unraveled, exploiting a variety of recombinant and transgenic approaches. Herein, while rapidly recapitulating some of the functions that nowadays can be assigned safely to each DG domain, we also try to envisage a sort of worry list featuring and dwelling on some of the most compelling "mysteries" that should be solved to finally understand DG's functional diversity.

First molecular characterization and immunolocalization of keratoepithelin in adult human skeletal muscle.

Keratoepithelin (KE) is an extracellular matrix protein that binds collagens, fibronectin, decorin, biglycan and integrins, interconnecting extracellular matrix components with resident cells in several tissues. KE has a molecular mass of 68 kDa and harbours four FAS1 domains named after those identified in the insect cell adhesion molecule fasciclin I. In humans, KE is preferentially expressed by the corneal epithelial layer and liberated towards the corneal stroma but it was also detected in the lung and in the bladder smooth muscle. No detailed information is available on the distribution of this protein in other human tissues. In this work, we have raised a polyclonal antibody against the recombinantly expressed human fourth FAS1 domain which is able to specifically detect KE in human skeletal muscle tissue extracts. Immunofluorescence experiments indicate that KE is localized around the perimysium and endomysium of each skeletal muscle fiber. The same kind of analysis shows that in muscle sections from patients affected by different forms of muscular dystrophy KE is upregulated and widely distributed in fibrotic tissues. The muscle specific expression of KE was also demonstrated by RT-PCR. In human skeletal muscle, KE may help to build up a bridge between collagen VI and yet unidentified muscle receptor(s), adding to the complexity of the adhesive molecular network established between muscle fibers and the surrounding basement membrane.