Andrea Brancaccio

Binding of the G domains of laminin α1 and α2 chains and perlecan to heparin, sulfatides, α‐dystroglycan and several extracellular matrix proteins

The C‐terminal G domain of the mouse laminin α2 chain consists of five lamin‐type G domain (LG) modules (α2LG1 to α2LG5) and was obtained as several recombinant fragments, corresponding to either individual modules or the tandem arrays α2LG1‐3 and α2LG4‐5. These fragments were compared with similar modules from the laminin α1 chain and from the C–terminal region of perlecan (PGV) in several binding studies. Major heparin‐binding sites were located on the two tandem fragments and the individual α2LG1, α2LG3 and α2LG5 modules. The binding epitope on α2LG5 could be localized to a cluster of lysines by site‐directed mutagenesis. In the α1 chain, however, strong heparin binding was found on α1LG4 and not on α1LG5. Binding to sulfatides correlated to heparin binding in most but not all cases. Fragments α2LG1–3 and α2LG4‐5 also bound to fibulin‐1, fibulin‐2 and nidogen‐2 with Kd = 13–150 nM. Both tandem fragments, but not the individual modules, bound strongly to α‐dystroglycan and this interaction was abolished by EDTA but not by high concentrations of heparin and NaCl. The binding of perlecan fragment PGV to α‐dystroglycan was even stronger and was also not sensitive to heparin. This demonstrated similar binding repertoires for the LG modules of three basement membrane proteins involved in cell–matrix interactions and supramolecular assembly.

A new crystal structure, Ca2+ dependence and mutational analysis reveal molecular details of E‐cadherin homoassociation

Electron microscopy of ECADCOMP, a recombinant E‐cadherin ectodomain pentamerized by the assembly domain of cartilage oligomeric matrix protein, has been used to analyze the role of cis‐dimerization and trans‐interaction in the homophilic association of this cell adhesion molecule. The Ca2+ dependency of both interactions was investigated. Low Ca2+ concentrations (50 μM) stabilized the rod‐like structure of E‐cadherin. At medium Ca2+ concentration (500 μM), two adjacent ectodomains in a pentamer formed cis‐dimers. At high Ca2+ concentration (>1 mM), two cis‐dimers from different pentamers formed a trans‐interaction. The X‐ray structure of an N‐terminal domain pair of E‐cadherin revealed two molecules per asymmetric unit in an intertwisted X‐shaped arrangement with closest contacts in the Ca2+‐binding region between domains 1 and 2. Contrary to previous data, Trp2 was docked in the hydrophobic cavity of its own molecule, and was therefore not involved in cis‐dimerization of two molecules. This was supported further by W2A and A80I (a residue involved in the hydrophobic cavity surrounding Trp2) mutations in ECADCOMP which both led to abrogation of the trans‐ but not the cis‐interaction. Structural and biochemical data suggest a link between Ca2+ binding in the millimolar range and Trp2 docking, both events being essential for the trans‐association.

ALTERNATIVE SPLICING OF AGRIN ALTERS ITS BINDING TO HEPARIN, DYSTROGLYCAN,AND THE PUTATIVE AGRIN RECEPTOR

Agrin is a heparan sulfate proteoglycan that induces aggregation of acetylcholine receptors (AChRs) at the neuromuscular synapse. This aggregating activity is modulated by alternative splicing. Here, we compared binding of agrin isoforms to heparin, alpha-dystroglycan, and cultured myotubes. We find that the alternatively spliced 4 amino acids insert (KSRK) is required for heparin binding. The binding affinity of agrin isoforms to alpha-dystroglycan correlates neither with binding to heparin nor with their AChR-aggregating activities. Moreover, the minimal fragment sufficient to induce AChR aggregation does not bind to alpha-dystroglycan. Nevertheless, this fragment still binds to cultured muscle cells. Its binding is completed only by agrin isoforms that are active in AChR aggregation, and therefore this binding site is likely to represent the receptor that initiates AChR clustering.

Electron microscopic evidence for a mucin-like region in chick muscle α-dystroglycan

α‐Dystroglycan has been isolated from chicken cardiac muscle and its molecular weight was estimated to be ≈135 kDa. The avian protein interacts with murine Engelbreth‐Holm‐Swarm (EHS) tumor laminin via interaction with the C‐terminal LG4 and LG5 domains (fragment E3) of the laminin α‐chain. This laminin binding is calcium‐dependent and can be competed by heparin. Electron microscopy investigation on the shape of α‐dystroglycan suggests that the core protein consists of two roughly globular domains connected by a segment which most likely corresponds to a mucin‐like central region also predicted by sequence analysis on mammalian isoforms. This segment may act as a spacer in the dystrophin‐associated glycoproteins complex exposing the N‐terminal domain of α‐dystroglycan to laminin in the extracellular space.

Agrin is a high-affinity binding protein of dystroglycan in non-muscle tissue

Agrin is a basement membrane-associated proteoglycan that induces the formation of postsynaptic specializations at the neuromuscular junction. This activity is modulated by alternative splicing and is thought to be mediated by receptors expressed in muscle fibers. An isoform of agrin that does not induce postsynaptic specializations binds with high affinity to dystroglycan, a component of the dystrophin-glycoprotein complex. Transcripts encoding this agrin isoform are expressed in a variety of non-muscle tissues. Here, we analyzed the tissue distribution of agrin and dystroglycan on the protein level and determined their binding affinities. We found that agrin is most abundant in lung, kidney, and brain. Only a little agrin was detected in skeletal muscle, and no agrin was found in liver. Dystroglycan was highly expressed in all tissues examined except in liver. In a solid-phase radioligand binding assay, agrin bound to dystroglycan from lung, kidney, and skeletal muscle with a dissociation constant between 1.8 and 2.2 nm, while the affinity to brain-derived dystroglycan was 4.6 nm. In adult kidney and lung, agrin co-purified and co-immunoprecipitated with dystroglycan, and both molecules were co-localized in embryonic tissue. These data show that the agrin isoform expressed in non-muscle tissue is a high-affinity binding partner of dystroglycan and they suggest that this interaction, like that between laminin and dystroglycan, may be important for the mechanical integrity of the tissue.

Dystroglycan distribution in adult mouse brain : A light and electron microscopy study

Dystroglycan, originally identified in muscle as a component of the dystrophin-associated glycoprotein complex, is a ubiquitously expressed cell-surface receptor that forms a transmembrane link between the extracellular matrix and the cytoskeleton. It contains two subunits, alpha and beta, formed by proteolytic cleavage of a common precursor. In the brain, different neuronal subtypes and glial cells may express dystroglycan in complex with distinct cytoplasmic proteins such as dystrophin, utrophin and their truncated forms. To examine the distribution of dystroglycan in adult mouse brain, we raised antibodies against the recombinant amino- and carboxyl-terminal domains of alpha-dystroglycan. On western blot, the antibodies recognized specifically alpha-dystroglycan in cerebellar extracts. Using light microscopy, alpha-dystroglycan was found in neurons of the cerebral cortex, hippocampus, olfactory bulb, basal ganglia, thalamus, hypothalamus, brainstem and cerebellum, where dystrophin and its truncated isoforms are also known to be present. Electron microscopy revealed that alpha-dystroglycan immunoreactivity was preferentially associated with the postsynaptic specializations. Dystroglycan immunostaining was also detected in perivascular astrocytes and in those facing the pia mater, where utrophin and dystrophin truncated isoforms are present. The cell body and endfeet of astrocytes around blood vessels and the endothelial cells at the blood-brain barrier also expressed dystroglycan. From these data, we suggest that dystroglycan, by bridging the extracellular matrix and the cytoskeleton, may play an important functional role at specialized intercellular contacts, synapses and the blood-brain barrier, whose structural and functional organization strictly depend on the integrity of the extracellular matrix-cytoskeleton linkage.

Identification of an Agrin Mutation that Causes Congenital Myasthenia and Affects Synapse Function

We report the case of a congenital myasthenic syndrome due to a mutation in AGRN, the gene encoding agrin, an extracellular matrix molecule released by the nerve and critical for formation of the neuromuscular junction. Gene analysis identified a homozygous missense mutation, c.5125G>C, leading to the p.Gly1709Arg variant. The muscle-biopsy specimen showed a major disorganization of the neuromuscular junction, including changes in the nerve-terminal cytoskeleton and fragmentation of the synaptic gutters. Experiments performed in nonmuscle cells or in cultured C2C12 myotubes and using recombinant mini-agrin for the mutated and the wild-type forms showed that the mutated form did not impair the activation of MuSK or change the total number of induced acetylcholine receptor aggregates. A solid-phase assay using the dystrophin glycoprotein complex showed that the mutation did not affect the binding of agrin to alpha-dystroglycan. Injection of wild-type or mutated agrin into rat soleus muscle induced the formation of nonsynaptic acetylcholine receptor clusters, but the mutant protein specifically destabilized the endogenous neuromuscular junctions. Importantly, the changes observed in rat muscle injected with mutant agrin recapitulated the pre- and post-synaptic modifications observed in the patient. These results indicate that the mutation does not interfere with the ability of agrin to induce postsynaptic structures but that it dramatically perturbs the maintenance of the neuromuscular junction.

Dystroglycan Expression Is Frequently Reduced in Human Breast and Colon Cancers and Is Associated with Tumor Progression

Dystroglycan (DG) is an adhesion molecule responsible for crucial interactions between extracellular matrix and cytoplasmic compartment. It is formed by two subunits, alpha-DG (extracellular) and beta-DG (transmembrane), that bind to laminin in the matrix and dystrophin in the cytoskeleton, respectively. In this study we evaluated by Western blot analysis the expression of DG in a series of human cancer cell lines of various histogenetic origin and in a series of human primary colon and breast cancers. Decreased expression of DG was observed in most of the cell lines and in both types of tumors and correlated with higher tumor grade and stage. Analysis of the mRNA levels suggested that expression of DG protein is likely regulated at a posttranscriptional level. Evaluation of alpha-DG expression by immunostaining in a series of archival cases of primary breast carcinomas confirmed that alpha-DG expression is lost in a significant fraction of tumors (66%). Loss of DG staining correlated with higher tumor stage (P = 0.022), positivity for p53 (P = 0.033), and high proliferation index (P = 0.045). A significant correlation was also observed between loss of alpha-DG and overall survival (P = 0.013 by log-rank test) in an univariate analysis. These data indicate that DG expression is frequently lost in human malignancies and suggest that this glycoprotein might play an important role in human tumor development and progression.

Anomalous dystroglycan in carcinoma cell lines

Dystroglycan is a receptor responsible for crucial interactions between extracellular matrix and cytoplasmic space. We provide the first evidence that dystroglycan is truncated. In HC11 normal murine and the 184B5 non‐tumorigenic mammary human cell lines, the expected β‐dystroglycan 43 kDa band was found but human breast T47D, BT549, MCF7, colon HT29, HCT116, SW620, prostate DU145 and cervical HeLa cancer cells expressed an anomalous ≈31 kDa β‐dystroglycan band. α‐Dystroglycan was udetectable in most of the cell lines in which β‐dystroglycan was found as a ≈31 kDa species. An anomalous ≈31 kDa β‐dystroglycan band was also observed in N‐methyl‐N‐nitrosurea‐induced primary rat mammary tumours. Reverse transcriptase polymerase chain reaction experiments confirmed the absence of alternative splicing events and/or expression of eventual dystroglycan isoforms. Using protein extraction procedures at low‐ and high‐ionic strength, we demonstrated that both the 43 kDa and ≈31 kDa β‐dystroglycan bands harbour their transmembrane segment.

The dystroglycan complex: From biology to cancer

Dystroglycan (DG), a non‐integrin adhesion molecule, is a pivotal component of the dystrophin–glycoprotein complex, that is expressed in skeletal muscle and in a wide variety of tissues at the interface between the basement membrane (BM) and the cell membrane. DG has been mainly studied for its role in skeletal muscle cell stability and its alterations in muscular diseases, such as dystrophies. However, accumulating evidence have implicated DG in a variety of other biological functions, such as maturation of post‐synaptic elements in the central and peripheral nervous system, early morphogenesis, and infective pathogens targeting. Moreover, DG has been reported to play a role in regulating cytoskeletal organization, cell polarization, and cell growth in epithelial cells. Recent studies also indicate that abnormalities in the expression of DG frequently occur in human cancers and may play a role in both the process of tumor progression and in the maintenance of the malignant phenotype. This paper reviews the available information on the biology of DG, the abnormalities found in human cancers, and the implications of these findings with respect to our understanding of cancer pathogenesis and to the development of novel strategies for a better management of cancer patients.