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Analysis of ?-Dystroglycan/LG Domain Binding Modes: Investigating Protein Motifs That Regulate the Affinity of Isolated LG Domains.

ABSTRACT: Dystroglycan (DG) is an adhesion complex that links the cytoskeleton to the surrounding extracellular matrix in skeletal muscle and a wide variety of other tissues. It is composed of a highly glycosylated extracellular ?-DG associated noncovalently with a transmembrane ?-DG whose cytodomain interacts with dystrophin and its isoforms. Alpha-dystroglycan (?-DG) binds tightly and in a calcium-dependent fashion to multiple extracellular proteins and proteoglycans, each of which harbors at least one, or, more frequently, tandem arrays of laminin-globular (LG) domains. Considerable biochemical and structural work has accumulated on the ?-DG-binding LG domains, highlighting a significant heterogeneity in ligand-binding properties of domains from different proteins as well as between single and multiple LG domains within the same protein. Here we review biochemical, structural, and functional information on the LG domains reported to bind ?-dystroglycan. In addition, we have incorporated bioinformatics and modeling to explore whether specific motifs responsible for ?-dystroglycan recognition can be identified within isolated LG domains. In particular, we analyzed the LG domains of slits and agrin as well as those of paradigmatic ?-DG non-binders such as laminin-?3. While some stretches of basic residues may be important, no universally conserved motifs could be identified. However, the data confirm that the coordinated calcium atom within the LG domain is needed to establish an interaction with the sugars of ?-DG, although it appears that this alone is insufficient to mediate significant ?-DG binding. We develop a scenario involving different binding modes of a single LG domain unit, or tandemly repeated units, with ?-DG. A variability of binding modes might be important to generate a range of affinities to allow physiological regulation of this interaction, reflecting its crucial biological importance.

SUBMITTER: Dempsey CE

PROVIDER: S-EPMC6450144 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Analysis of α-Dystroglycan/LG Domain Binding Modes: Investigating Protein Motifs That Regulate the Affinity of Isolated LG Domains.

Dempsey Christopher E CE Bigotti Maria Giulia MG Adams Josephine C JC Brancaccio Andrea A

Frontiers in molecular biosciences 20190329

Dystroglycan (DG) is an adhesion complex that links the cytoskeleton to the surrounding extracellular matrix in skeletal muscle and a wide variety of other tissues. It is composed of a highly glycosylated extracellular α-DG associated noncovalently with a transmembrane β-DG whose cytodomain interacts with dystrophin and its isoforms. Alpha-dystroglycan (α-DG) binds tightly and in a calcium-dependent fashion to multiple extracellular proteins and proteoglycans, each of which harbors at least one, ...[more]

PMID: 30984766

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Project description:BackgroundThe envelope glycoprotein of lymphocytic choriomeningitis virus (LCMV) can efficiently pseudotype lentiviral vectors. Some strains of LCMV exploit high affinity interactions with α-dystroglycan (α-DG) to bind to cell surfaces and subsequently fuse in low pH endosomes. LCMV strains with low α-DG affinity utilize an unknown receptor and display unique tissue tropisms. We pseudotyped non-primate feline immunodeficiency virus (FIV) vectors using LCMV derived glycoproteins with high or low affinity to α-DG and evaluated their properties in vitro and in vivo.MethodsWe pseudotyped FIV with the LCMV WE54 strain envelope glycoprotein and also engineered a point mutation in the WE54 envelope glycoprotein (L260F) to diminish α-DG affinity and direct binding to alternate receptors. We hypothesized that this change would alter in vivo tissue tropism and enhance gene transfer to neonatal animals.ResultsIn mice, hepatic α- and β-DG expression was greatest at the late gestational and neonatal time points. When displayed on the surface of the FIV lentivirus the WE54 L260F mutant glycoprotein bound weakly to immobilized α-DG. Additionally, LCMV WE54 pseudotyped FIV vector transduction was neutralized by pre-incubation with soluble α-DG, while the mutant glycoprotein pseudotyped vector was not. In vivo gene transfer in adult mice with either envelope yielded low transduction efficiencies in hepatocytes following intravenous delivery. In marked contrast, neonatal gene transfer with the LCMV envelopes, and notably with the FIV-L260F vector, conferred abundant liver and lower level cardiomyocyte transduction as detected by luciferase assays, bioluminescent imaging, and β-galactosidase staining.ConclusionsThese results suggest that a developmentally regulated receptor for LCMV is expressed abundantly in neonatal mice. LCMV pseudotyped vectors may have applications for neonatal gene transfer.AbbreviationsArmstrong 53b (Arm53b); baculovirus Autographa californica GP64 (GP64); charge-coupled device (CCD); dystroglycan (DG); feline immunodeficiency virus (FIV); glycoprotein precursor (GP-C); firefly luciferase (Luc); lymphocytic choriomeningitis virus (LCMV); nuclear targeted β-galactosidase (ntLacZ); optical density (OD); PBS/0.1% (w/v) Tween-20 (PBST); relative light units (RLU); Rous sarcoma virus (RSV); transducing units per milliliter (TU/ml); vesicular stomatitis virus (VSV-G); wheat germ agglutinin (WGA); 50% reduction in binding (C50).

Dataset Information

Analysis of ?-Dystroglycan/LG Domain Binding Modes: Investigating Protein Motifs That Regulate the Affinity of Isolated LG Domains.

Publications

Analysis of α-Dystroglycan/LG Domain Binding Modes: Investigating Protein Motifs That Regulate the Affinity of Isolated LG Domains.

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