MAGP-2 Has Multiple Binding Regions on Fibrillins and Has Covalent Periodic Association with Fibrillin-containing Microfibrils

Abstract

The interactions of microfibril-associated glycoprotein (MAGP)-2 have been investigated with fibrillins and fibrillin-containing microfibrils. Solid phase binding assays were conducted with recombinant fragments covering fibrillin-1 and most of fibrillin-2. MAGP-2, and its structure relative MAGP-1, were found to bind two fragments spanning the N-terminal half of fibrillin-1 and an N-terminal fragment of fibrillin-2. Blocking experiments indicated that MAGP-2 had a binding site(s) close to the N terminus of the fibrillin-1 molecule that was distinct from that for MAGP-1 and an additional, more central binding site(s) that may be shared by the two MAGPs. Immunogold labeling of developing nuchal ligament tissue showed that MAGP-2 had regular covalent and periodic (about 56 nm) association with fibrillin-containing microfibrils of elastic fibers in this tissue. Further analysis of isolated microfibrils indicated that MAGP-2 was attached at two points along the microfibril substructure, "site 1" on the "beads" and "site 2" at the "shoulder" of the interbead region close to where the two "arms" fuse. In contrast, MAGP-1 was located only on the beads. Comparison of the MAGP-2 binding data with known fibrillin epitope maps of the microfibrils showed that site 1 correlated with the N-terminal MAGP-2 binding region, and site 2 correlated with the second, more central, MAGP-2 binding region on the fibrillin-1 molecule. Of particular note, immunolabeling at site 2 was markedly decreased, relative to that at site 1, on extended microfibrils with beadto-bead periods over 90 nm, suggesting that site 2 may move toward the beads when the microfibril is stretched. The study points to MAGP-2 being an integral component of some populations of fibrillin-containing microfibrils. Moreover, the identification of multiple MAGP-binding sequences on fibrillins supports the concept that MAGPs may function as molecular cross-linkers, stabilizing fibrillin monomers in folded conformation within or between the microfibrils, and thus MAGPs may be implicated in the modulation of the elasticity of these structures.

Microfibril Structure Masks Fibrillin-2 in Postnatal Tissues

“…Additional antibody labeling studies using epitopemapped mAbs have been used to support various arrangements of fibrillin-1 within the microfibril (11, 12, 16). Antibodies spe- cific for microfibril-associated glycoprotein-1 and -2 (MAGP-1 and -2) have also been used to demonstrate the presence of microfibril-associated glycoproteins near or on the globular beads in the microfibril (21). Double labeling with antibodies specific for fibrillin-1 and -2 showed that fetal microfibrils are composed of both fibrillins (15).…”

New insights into elastic fiber assembly

“…MAGP-1 has also been shown to bind to fibrillin-2 in yeast two-hybrid, ligand blotting, and solid phase binding assays (Werneck et al, 2004). MAGP-2 binds to fibrillin-1 and -2 in yeast twohybrid studies (Penner et al, 2002) and in solid phase binding assays (Hanssen et al, 2004). The ability of MAGP-1 to bind both tropoelastin and fibrillin suggested that it might play an important role in elastic fiber assembly by serving as a bridging molecule between its two binding partners.…”

Enhanced fibrillin-2 expression is a general feature of wound healing and sclerosis: potential alteration of cell attachment and storage of TGF-β

“…After washing three times with TBS, the membranes were incubated for 2 h with monoclonal antibody 16E12 (1:50) in binding buffer against the soluble ligand. 27 The membranes were incubated for 2 h with horseradish peroxidase-conjugated goat-anti mouse antibodies (1:800 diluted in binding buffer) followed by detection with ECL (Amersham, USA) according to the protocol of the manufacturer. Densitometric scanning was performed using ImageJ (Version 1.41) after background subtraction.…”