Thermodynamic and structural studies of carbohydrate binding by the agrin-G3 domain.
Agrin is a key heparan sulfate proteoglycan involved in the development and maintenance of synaptic junctions between nerves and muscles. Agrin's important functions include clustering acetylcholine receptors on the postsynaptic membranes of muscles and binding to the muscle protein alpha-dystroglycan through its glycan chains. ITC and NMR were used to study the interactions of the C-terminal domain, agrin-G3, with carbohydrates implicated in agrin's functions. Sialic acid caps the glycan chains of alpha-dystroglycan and occurs as a posttranslational modification on the muscle-specific kinase component of the agrin receptor. We found that agrin-G3 binds sialic acid in a Ca2+-dependent manner. ITC data indicate that binding is exothermic and occurs with a 1:1 stoichiometry. NMR chemical shift changes map the sialic acid binding site to the loops that control the domain's acetylcholine receptor clustering activity. By contrast, the glycosaminoglycans heparin and heparan sulfate bind independently of Ca2+. Binding is endothermic, and the binding site spans about 12 saccharide units. The binding site for heparin occupies a similar location but is distinct from that for sialic acid. NMR translational diffusion experiments show that agrin-G3 binds heparin with a 2:1 stoichiometry. Comparisons between the muscle (B0) and neuronal (B8) isoforms of the agrin domain showed very similar Ca2+ and carbohydrate binding properties. Our work identifies agrin-G3 as a functional analogue of the concanavalin A-type lectins, highlights functional similarities between agrin and laminin G domains, and provides mechanistic clues about the roles of carbohydrates in agrin's functions.
Sallum
Christine O
CO
Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 06269-3125, USA.
Kammerer
Richard A
RA
Alexandrescu
Andrei T
AT
D018171:Agrin / Q000737:chemistry*; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry; D002151:Calorimetry; D002241:Carbohydrates / Q000737:chemistry*; D006493:Heparin / Q000737:chemistry; D006497:Heparan Sulfate / Q000737:chemistry; D019158:N-Acetylneuraminic Acid / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular; D017434:Protein Structure, Tertiary; D013816:Thermodynamics
D018171:Agrin; D002118:Calcium; D002241:Carbohydrates; D006493:Heparin; D006497:Heparan Sulfate; D019158:N-Acetylneuraminic Acid