Monitoring Editor: Paul T. Matsudaira MAGP-1 and fibrillin-1, two protein components of extracellular microfibrils, were shown by immunoprecipitation studies to interact with the chondroitin sulfate proteoglycan decorin in the medium of cultured fetal bovine chondrocytes. Decorin interacted with each protein individually and with both proteins together to form a ternary complex. Expression of truncated fibrillin-1 proteins in Chinese hamster ovary cells localized proteoglycan binding to an amino-terminal region near the proline-rich domain. A spatially analogous fibrillin-2 truncated protein did not coprecipitate the same sulfated molecule, suggesting that chondroitin sulfate proteoglycan binding in this region is specific for fibrillin-1. An interaction between fibrillin and MAGP-1 was also observed under culture conditions that abrogated decorin secretion, suggesting that the two microfibrillar proteins can associate in the absence of the proteoglycan. Sulfation of matrix proteins is important for elastic fiber assembly because inhibition of sulfation was shown to prevent microfibrillar protein incorporation into the extracellular matrix of cultured cells.

Alignment of tropoelastin molecules during the process of elastogenesis is thought to require fibrillin-containing microfibrils. In this study, we have demonstrated that amino-terminal domains of two microfibrillar proteins, fibrillin-1 and fibrillin-2, interact with tropoelastin in solid phase binding assays. The tropoelastin-binding site was localized to a region beginning at the glycine-rich and proline-rich regions of fibrillin-2 and fibrillin-1, respectively, and continuing through the second 8-cysteine domain. Characterization of the binding requirements using the fibrillin-2 construct found that a folded, secondary structure was necessary for binding. Furthermore, binding between tropoelastin and fibrillin was mediated by ionic interactions involving the lysine side chains of tropoelastin. The importance of the lysine side chains was corroborated by the finding that the fibrillin-2 construct did not bind to mature elastin, whose lysine side chains have been modified to form cross-links. Interestingly, there was no interaction between the fibrillin constructs and tropoelastin in solution phase, suggesting that binding of tropoelastin to a solid substrate exposes a cryptic binding site. These results suggest that fibrillin plays an important role in elastic fiber assembly by binding tropoelastin and perhaps facilitating side chain alignment for efficient cross-linking.

Aggregation of fibrillin molecules via disulphide bonds is postulated to be an early step in microfibril assembly. By expressing fragments of fibrillin 1 and fibrillin 2 in a mammalian expression system, we found that the N-terminal region of each protein directs the formation of homodimers and that disulphide bonds stabilize this interaction. A large fragment of fibrillin 1 containing much of the region downstream from the N-terminus remained as a monomer when expressed in the same cell system, indicating that this region of the protein lacks dimerization domains. This finding also confirms that the overexpression of fibrillin fragments does not in itself lead to spurious dimer formation. Pulse-chase analysis demonstrated that dimer formation occurred intracellularly, suggesting that the process of fibrillin aggregation is initiated early after biosynthesis of the molecules. These findings also implicate the N-terminal region of fibrillin 1 and fibrillin 2 in directing the formation of a dimer intermediate that aggregates to form the functional microfibril.

Microfibrils are found in the extracellular matrices of most tissues where they serve several functional roles, including the binding and sequestration of growth factors, supplying informational signals through receptor signaling, and providing the basic structural elements for elastic fiber assembly. Many different proteins have been localized to microfibrils, although it is not clear whether all are structural proteins or proteins that simply associate with other components of microfibrils (1, 2).The fibrillins provide the basic functional framework of microfibrils. This multigene family consists of three related proteins (fibrillin-1, -2, and -3) that have a similar domain structure but differ in expression pattern and biological function (3). The fibrillins are secreted from the cell as dimers that then pack together in ways that are not completely understood to form the microfibril. Extracted microfibrils are typically visualized as a linear string of beads with multiple arms extending into the interbead region. The beads show a ϳ50-nm spacing, and several models of fibrillin assembly have been proposed to account for packing a 150-nm fibrillin monomer into a structure with 50-nm periodicity (4, 5).A second protein associated with microfibrils is a small molecular mass glycoprotein called microfibril-associated glycoprotein. MAGP-1 1 contains 183 amino acids that form two distinctive domains; the N-terminal half of the molecule is highly acidic, enriched in proline, and contains a clustering of glutamine residues, whereas the C-terminal portion contains all 13 cysteine residues and contains the matrix-binding domain that mediates the association of MAGP-1 with microfibrillar components (6). The protein undergoes post-translational transglutamination, sulfation of specific tyrosine residues, and O-linked glycosylation (7). In mouse development, MAGP-1 mRNA is widely expressed, mainly in mesenchymal and connective tissue cells where it is easily detected as early as day 8.5 of development (8). MAGP-1 is associated with all microfibrils with only two known exceptions: those directly adjacent to the plasma membrane of aortic endothelial cells and those at the junction of the zonule and the lens capsule of the eye (9 -11).MAGP-2 is a second member of the MAGP family that has close sequence similarity to MAGP-1 in a central region of 60 amino acids, where the spacing of 7 cysteine residues is identical in both proteins. The MAGP-2 molecule is otherwise rich in serine and threonine residues and lacks the proline-, glutamine-, and tyrosine-rich sequences characteristic of the Nterminal domain of MAGP-1 (12-15). MAGP-2 exhibits a more restricted pattern of tissue localization and developmental expression than MAGP-1 (15), suggesting that MAGP-1 and MAGP-2 are structurally divergent proteins sharing one functional motif (encoded by two conserved exons) rather than being functionally closely related molecules. Both MAGPs have been shown to directly bind to purified fibrillin, and for MAGP-2, a prominent binding site w...

Microfibril-associated glycoprotein-1 (MAGP-1) is a small molecular weight protein associated with extracellular matrix microfibrils. Biochemical studies have shown that MAGP-1 undergoes several posttranslational modifications that may influence its associations with other microfibrillar components.To identify the sites in the molecule where posttranslational modifications occur, we expressed MAGP-1 constructs containing various point mutations as well as front and back half truncations in CHO cells. Characterization of transiently expressed protein showed that MAGP-1 undergoes O-linked glycosylation and tyrosine sulfation at sites in its amino-terminal half. This region of the protein also served as a major amine acceptor site for transglutaminase and mediated self-assembly into high molecular weight multimers through a glutamine-rich sequence. Fine mapping of the modification sites through mutational analysis demonstrated that Gln20 is a major amine acceptor site for the transglutaminase reaction and confirmed that a canonical tyrosine sulfation consensus sequence is the site of MAGP-1 sulfation. Our results also show that O-glycosylation occurs at more than one site in the molecule.