Fibromodulin Binds Collagen Type I via Glu-353 and Lys-355 in Leucine-rich Repeat 11

Abstract

Fibromodulin belongs to the small leucine-rich repeat proteoglycan family, interacts with collagen type I, and controls collagen fibrillogenesis and assembly. Here, we show that a major fibromodulin-binding site for collagen type I is located in leucine-rich repeat 11 in the C terminus of the leucine-rich repeat domain. We identified Glu-353 and Lys-355 in repeat 11 as essential for binding, and the synthetic peptide RLDGNEIKR, including Glu-353 and Lys-355, inhibits the binding of fibromodulin to collagen in vitro. Fibromodulin and lumican compete for the same binding region on collagen, and fibromodulin can inhibit the binding of lumican to collagen type I. However, the peptide RLDGNEIKR does not inhibit the binding of lumican to collagen, suggesting separate but closely situated fibromodulin-and lumican-binding sites in collagen. The collagenbinding Glu-353 and Lys-355 residues in fibromodulin are exposed on the exterior of the ␤-sheet-loop structure of the leucine-rich repeat, which resembles the location of interacting residues in other leucine-rich repeat proteins, e.g. decorin.Fibromodulin is an extracellular matrix proteoglycan expressed in dense regular connective tissues, including ligaments, cartilage, and tendons (1). Fibromodulin belongs to the small leucine-rich repeat proteoglycan family (SLRPs) 2 (2, 3), which also includes e.g. biglycan (4), decorin (5), and lumican (6). These SLRPs are involved in the regulation of collagen fibril formation and matrix assembly, as demonstrated in SLRP-deficient mice (7). Decorin-deficient mice have fragile skin (8), fibromodulin deficiency leads to weak tendons and ligaments, causing osteoarthritis (9, 10), and lumican-deficient mice have opaque corneas (11). All these genotypes mediate an abnormal development of collagen matrices.Fibromodulin and lumican are differentially expressed in developing tendons and possibly act in a coherent manner to regulate collagen matrix assembly (12), as they bind to the same region of collagen, different from the decorin-binding site (13). Fibromodulin binds preferentially in the gap region of assembled collagen fibrils (14) and affects the growth of collagen type I fibrils in vitro, as it binds to collagen monomers and delays their accretion to the growing fibrils (15). Collagen fibrils in tendon of fibromodulin-deficient mice are irregularly shaped with a higher proportion of thin fibrils, in contrast to fibrils formed in wild type mice (9). In addition, fibromodulin also binds collagen type XII (16) and transforming growth factor-␤ (17).It has also been proposed that fibromodulin and other collagen-binding SLRPs have a function in protecting collagen from collagenase degradation in connective tissues (18). Genomic analysis suggests a role for fibromodulin in metastasis (19), and the proteoglycan is also up-regulated in chronic lymphoid leukemia (20). Fibromodulin is one of just 14 genes progressively changed in expression with age and in progeria (21,22). Fibromodulin is also implicated in pathways of inflammatory response ...

Homologous Sequence in Lumican and Fibromodulin Leucine-rich Repeat 5-7 Competes for Collagen Binding

“…Because the closest homologue of lumican is fibromodulin and both bind to the same region of collagen, we also tested whether any fibromodulin fragment used in our previous study (24) could inhibit the lumican-collagen interaction. Fmod5-7, containing LRRs 5-7, impaired the binding of lumican to collagen (K i ϳ 250 nM), whereas other fragments were ineffective.…”

“…fibromodulin and decorin (24,27). In general, this implies that LRR domains contain interaction sites near the distal parts of the conserved ␤-sheet structures and include charged amino acids.…”

“…Prior to use, protein was dialyzed against PBS. Similar procedure was used for making fibromodulin, as described earlier (24). The proteins migrated faster on a non-reduced SDS-PAGE than on a reduced gel, indicating the presence of disulfide bonds.…”

“…The protein concentration was determined with Coomassie Blue protein assay reagent (Pierce). A similar procedure was used for making fibromodulin fragments Fmod5-7 and Fmod11-12 (24). Proteins usually co-purify with some free GST protein (around 28 kDa), but free GST does not bind to collagen (data not shown).…”

“…For mutation E251Q in the Fmod5-7 fragment, we used similar procedure, as described previously (24).…”