Abstract
Secreted modular calcium-binding proteins 1 and 2 (SMOC-1 and SMOC-1) are extracellular calcium- binding proteins belonging to the BM-40 family of proteins. In this work we have identified a highly basic region in the extracellular calcium-binding (EC) domain of the SMOC-1 similar to other known glycosaminoglycan-binding motifs. Size-exclusion chromatography shows that full length SMOC-1 as well as its C-terminal EC domain alone bind heparin and heparan sulfate, but not the related chondroitin sulfate or dermatan sulfate glycosaminoglycans. Intrinsic tryptophan fluorescence measurements were used to quantify the binding of heparin to full length SMOC-1 and the EC domain alone. The calculated equilibrium dissociation constants were in the lower micromolar range. The binding site consists of two antiparallel alpha helices and mutagenesis experiments have shown that heparin-binding residues in both helices must be replaced in order to abolish heparin binding. Furthermore, we show that the SMOC-1 EC domain, like the SMOC-2 EC domain, supports the adhesion of epithelial HaCaT cells. Heparin-binding impaired mutants failed to support S1EC-mediated cell adhesion and together with the observation that S1EC in complex with soluble heparin attenuated cell adhesion we conclude that a functional and accessible S1EC heparin-binding site mediates adhesion of epithelial cells to SMOC-1.
Highlights
Both secreted modular calcium-binding proteins 1 and 2 (SMOC-1 and SMOC-2) are involved in direct or indirect modulation of growth factor signaling pathways and play diverse roles in physiological processes involving extensive tissue remodeling
The overall comparison of the model with the crystal structure of the basement membranes (BMs)-40 extracellular calcium-binding (EC) domain shows that apart from the minor insertion between helices E and F, the major structural difference between both is the absence of helix C in the SMOC EC domain (Figure 1E), which is conserved in both SMOC paralogs
Charged residues are shown as sticks. (D) Surface potential of the S1EC domain calculated with APBS Software. (E) Superposition of the S1EC model and the BM-40 EC domain
Summary
Both secreted modular calcium-binding proteins 1 and 2 (SMOC-1 and SMOC-2) are involved in direct or indirect modulation of growth factor signaling pathways and play diverse roles in physiological processes involving extensive tissue remodeling. It has been shown that SMOC-1 acts as a regulator of osteoblast differentiation [1] and is involved in inhibition of transforming growth factor-b signaling through production of nitric oxide [2]. The SMOCs are members of the BM-40 family, which comprises proteins that contain a follistatin-like domain (FS) and an extracellular calcium-binding (EC) domain with two EF hands. In most members of the family the FS and EC domains occur in tandem, in the SMOCs, they are separated by two thyroglobulin type-1 domains and a domain, unique to SMOC proteins [8,9]
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