Microfibril‐associated glycoprotein‐1 binding to tropoelastin

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Abstract
Microfibrils and elastin are major constituents of elastic fibers, the assembly of which is dictated by multimolecular interactions. Microfibril‐associated glycoprotein‐1 (MAGP‐1) is a microfibrillar component that interacts with the soluble elastin precursor, tropoelastin. We describe here the adaptation of a solid‐phase binding assay that defines the effect of divalent cations on the interactions between MAGP‐1 and tropoelastin. Using this assay, a strong calcium‐dependent interaction was demonstrated, with a dissociation constant of 2.8 ± 0.3 nm, which fits a single‐site binding model. Manganese and magnesium bestowed a weaker association, and copper did not facilitate the protein interactions. Three constructs spanning tropoelastin were used to quantify their relative contributions to calcium‐dependent MAGP‐1 binding. Binding to a construct spanning a region from the N‐terminus to domain 18 followed a single‐site binding model with a dissociation constant of 12.0 ± 2.2 nm, which contrasted with the complex binding behavior observed for fragments spanning domains 17–27 and domain 27 to the C‐terminus. To further elucidate binding sites around the kallikrein cleavage site of domains 25/26, MAGP‐1 was presented with constructs containing C‐terminal deletions within the region. Construct M1659, which spans a region from the N‐terminus of tropoelastin to domain 26, inclusive, bound MAGP‐1 with a dissociation constant of 9.7 ± 2.0 nm, which decreased to 4.9 ± 1.0 nm following the removal of domain 26 (M155n), thus displaying only half the total capacity to bind MAGP‐1. These results demonstrate that MAGP‐1 is capable of cumulative binding to distinct regions on tropoelastin, with different apparent dissociation constants and different amounts of bound protein.