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 bead-to-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.
Highlights
Tissues such as lung, arteries, and elastic ligaments
(H)NT was not detected by this method, suggesting that the binding site on this fragment was irreversibly labile to treatment with SDS detergent (Fig. 2B). These findings suggest that microfibril-associated glycoprotein (MAGP)-2 has distinct binding activities to Fib-1 (H)NT and Fib-1 N(H)
MAGP-1 was found to bind to the same fibrillin fragments as MAGP-2 (Fig. 3)
Summary
The microfibrils occur as elastin-free bundles in the extracellular matrix of a diverse range of tissues including ocular zonule, skeletal muscle, skin, and kidney [3,4,5,6] As their name suggests, the major structural components of these microfibrils are the fibrillins that are large and rod-like 350-kDa glycoproteins (6 – 8). It has been proposed that MAGP-1 may stabilize the head-to-tail interaction of fibrillin monomers within the beads [37] and/or act as an elastin-binding protein on the surface of the microfibril [41]. In the present study we show that MAGP-2 is covalently and periodically located along the fibrillin-containing microfibrils of the developing nuchal ligament, strongly suggesting that the protein is an integral component of most, if not all, of the microfibrils in this elastic fiber-rich tissue. The findings suggest that MAGP-2 may be involved in the stabilization of folded fibrillin monomers within the microfibril by forming interdomain and/or inter-monomer connections
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