Amyloid Aggregation of Streptococcus mutans Cnm Influences Its Collagen-Binding Activity

Streptococcus mutans is a keystone pathogen that promotes caries by acidifying the dental biofilm milieu. The collagen- and laminin-binding glycoprotein Cnm is a virulence factor of S. mutans . Expression of Cnm by S. mutans is hypothesized to contribute to niche expansion, allowing colonization of multiple sites in the body, including collagen-rich surfaces such as dentin and heart valves.

Cell Adhesion-Dependent Biphasic Axon Outgrowth Elucidated by Femtosecond Laser Impulse

Axon outgrowth is promoted by the mechanical coupling between the dynamic actin cytoskeleton and adhesive substrates via clutch and adhesion molecules in the axonal growth cone. In this study, we utilized a femtosecond laser-induced impulse to break the coupling between an axonal growth cone and an adhesive substrate, enabling us to evaluate the strength of the binding between proteins in the growth cone and a laminin substrate, and also determine the contribution of adhesion strength to axon outgrowth. We found that the adhesion strength of axonal L1 cell adhesion molecule (L1CAM)-laminin binding increased with the density of the laminin substrate. In addition, fluorescent speckle microscopy revealed that the retrograde flow of actin filaments in the axonal growth cone was dependent on the laminin density such that the flow speed reduced with increasing L1CAM-laminin binding. However, axon outgrowth did not increase monotonically with increased L1CAM-laminin binding but rather exhibited biphasic behavior, in which the outgrowth was suppressed by excessive L1CAM-laminin binding. Our quantitative evaluations of the adhesion strength suggest that the biphasic outgrowth is regulated by the balance between traction force and adhesion strength as a result of changes in the number of L1CAM-laminin interactions. These results imply that adhesion modulation is key to the regulation of axon guidance.

Sarcospan increases laminin binding capacity of α-dystroglycan to ameliorate DMD independent of Galgt2

In Duchenne muscular dystrophy (DMD), mutations in dystrophin result in a loss of the dystrophin-glycoprotein complex at the myofiber membrane, which functions to connect the extracellular matrix with the intracellular actin cytoskeleton. The dystroglycan subcomplex interacts with dystrophin and spans the sarcolemma where its extensive carbohydrates (matriglycan and CT2 glycan) directly interact with the extracellular matrix. In the current manuscript, we show that sarcospan overexpression enhances the laminin-binding capacity of dystroglycan in DMD muscle by increasing matriglycan glycosylation of α-dystroglycan. Furthermore, we find that this modification is not affected by loss of Galgt2, a glycotransferase which catalyzes the CT2 glycan. Our findings reveal that the matriglycan carbohydrates, and not the CT2 glycan, are necessary for sarcospan-mediated amelioration of DMD. Overexpression of Galgt2 in the DMD mdx murine model prevents muscle pathology by increasing CT2 modified α-dystroglycan. Galgt2 also increases expression of utrophin, which compensates for the loss of dystrophin in DMD muscle. We found that combined loss of Galgt2 and dystrophin reduced utrophin expression; however, it did not interfere with sarcospan rescue of disease. These data reveal a partial dependence of sarcospan on Galgt2 for utrophin upregulation. In addition, sarcospan alters the cross-talk between the adhesion complexes by decreasing the association of integrin β1D with dystroglycan complexes. In conclusion, sarcospan functions to re-wire the cell to matrix connections by strengthening the cellular adhesion and signaling which, in turn, increases the resilience of the myofiber membrane.

High degree of conservation of the enzymes synthesizing the laminin-binding glycoepitope of α-dystroglycan

The dystroglycan (DG) complex plays a pivotal role for the stabilization of muscles in Metazoa. It is formed by two subunits, extracellular α-DG and transmembrane β-DG, originating from a unique precursor via a complex post-translational maturation process. The α-DG subunit is extensively glycosylated in sequential steps by several specific enzymes and employs such glycan scaffold to tightly bind basement membrane molecules. Mutations of several of these enzymes cause an alteration of the carbohydrate structure of α-DG, resulting in severe neuromuscular disorders collectively named dystroglycanopathies. Given the fundamental role played by DG in muscle stability, it is biochemically and clinically relevant to investigate these post-translational modifying enzymes from an evolutionary perspective. A first phylogenetic history of the thirteen enzymes involved in the fabrication of the so-called ‘M3 core’ laminin-binding epitope has been traced by an overall sequence comparison approach, and interesting details on the primordial enzyme set have emerged, as well as substantial conservation in Metazoa. The optimization along with the evolution of a well-conserved enzymatic set responsible for the glycosylation of α-DG indicate the importance of the glycosylation shell in modulating the connection between sarcolemma and surrounding basement membranes to increase skeletal muscle stability, and eventually support movement and locomotion.

Laminin-binding Integrins Regulate Angiogenesis by Distinct and Overlapping Mechanisms in Organotypic Cell Culture Models

Endothelial cells engage extracellular matrix and basement membrane components through integrin-mediated adhesion to promote angiogenesis. Our previous studies demonstrated that endothelial expression of laminin-411 and laminin-511 as well as α6 integrins is required for endothelial sprouting and tube formation in organotypic angiogenesis assays. These studies demonstrated that α6 integrins promote migration and regulate the expression of ANGPT2 and CXCR4 and that α6-dependent regulation of CXCR4 contributes to endothelial morphogenesis in our assays. However, these studies did not identify specific roles for the α6β1, α6β4, or α3β1 laminin-binding integrins. Here, we employ RNAi technology to parse the contributions of these integrins. We demonstrate that α6β4 promotes migration, sprouting, and tube formation, and also positively regulates the expression of ANGPT2, but does not promote CXCR4 expression, suggesting that α6β1 functions in this regulation. Additionally, we show that α3β1 regulates endothelial sprouting and tube formation, but is not required for migration in our assays or for the expression of ANGPT2 or CXCR4. Integrin α3β1 promotes the expression of NRP1 and ID1 RNAs, both of which are known to promote angiogenesis. Taken together, our results indicate that laminin-binding integrins play distinct roles during endothelial morphogenesis and do not compensate for one another in organotypic culture.

Cell Surface Glycan Engineering Reveals that Matriglycan Alone can Recapitulate Dystroglycan Binding and Function

Alpha-Dystroglycan (alpha-DG) is uniquely modified on O-mannose sites by a repeating disaccharide (-Xylalpha1,3-GlcAbeta1,3-)n termed matriglycan, which is a receptor for laminin-G domain-containing proteins and employed by old-world arenaviruses for infection. Using chemoenzymatically synthesized matriglycans printed as a microarray, we demonstrated length-dependent binding to Laminin, Lassa virus GP1, and the clinically-important antibody IIH6. Utilizing an enzymatic engineering approach, an N-linked glycoprotein was converted into a IIH6-positive Laminin-binding glycoprotein. Engineering of the surface of cells deficient for either alpha-DG or O-mannosylation with matriglycans of sufficient length recovered infection with a Lassa-pseudovirus. Finally free matriglycan in a dose and length dependent manner inhibited viral infection of wildtype cells. These results indicate that matriglycan alone is necessary and sufficient for IIH6 staining, Laminin and LASV GP1 binding, and Lassa-pseudovirus infection and support a model in which it is a tunable receptor for which increasing chain length enhances ligand-binding capacity.

Transcriptomic analysis of human brain microvascular endothelial cells exposed to laminin binding protein (adhesion lipoprotein) and Streptococcus pneumoniae

Streptococcus pneumoniae invades the CNS and triggers a strong cellular response. To date, signaling events that occur in the human brain microvascular endothelial cells (hBMECs), in response to pneumococci or its surface adhesins are not mapped comprehensively. We evaluated the response of hBMECs to the adhesion lipoprotein (a laminin binding protein—Lbp) or live pneumococci. Lbp is a surface adhesin recently identified as a potential ligand, which binds to the hBMECs. Transcriptomic analysis was performed by RNA-seq of three independent biological replicates and validated with qRT-PCR using 11 genes. In total 350 differentially expressed genes (DEGs) were identified after infection with S. pneumoniae, whereas 443 DEGs when challenged with Lbp. Total 231 DEGs were common in both treatments. Integrative functional analysis revealed participation of DEGs in cytokine, chemokine, TNF signaling pathways and phagosome formation. Moreover, Lbp induced cell senescence and breakdown, and remodeling of ECM. This is the first report which maps complete picture of cell signaling events in the hBMECs triggered against S. pneumoniae and Lbp. The data obtained here could contribute in a better understanding of the invasion of pneumococci across BBB and underscores role of Lbp adhesin in evoking the gene expression in neurovascular unit.

NAD+ enhances ribitol and ribose rescue of α-dystroglycan functional glycosylation in human FKRP-mutant myotubes

Mutations in the fukutin-related protein (FKRP) cause Walker-Warburg Syndrome (WWS), a severe form of congenital muscular dystrophy. Here we established a WWS human induced pluripotent stem cell-derived myogenic model that recapitulates hallmarks of WWS pathology. We used this model to investigate the therapeutic effect of metabolites of the pentose phosphate pathway in human WWS. We show that functional recovery of WWS myotubes is promoted not only by ribitol but also its precursor ribose. Moreover, we found that the combination of each of these metabolites with NAD+ results in a synergistic effect, as demonstrated by rescue of α-dystroglycan glycosylation and laminin binding capacity. Mechanistically, we find that FKRP residual enzymatic capacity, characteristic of many recessive FKRP mutations, is required for rescue as supported by functional and structural mutational analysis. These findings provide the rationale for testing ribose/ribitol in combination with NAD+ to treat WWS and other diseases associated with FKRP mutations.