Abstract
Testicans are modular proteoglycans of the extracellular matrix of various tissues where they contribute to matrix integrity and exert cellular effects like neurite outgrowth and cell migration. Using testican-2 as a representative member of the family, we tackle the complete lack of general structural information and structure–function relationship. First, we show using isothermal titration calorimetry and modeling that extracellular calcium-binding domain (EC) has only one active calcium-binding site, while the other potential site is inactive, and that testican-2 is within extracellular matrix always in the calcium-loaded form. Next, we demonstrate using various prediction methods that N- and C-terminal regions plus interdomain connections are flexible. We support this by small-angle X-ray-scattering analysis of C-terminally truncated testican-2, which indicates that the triplet follistatin-EC-thyroglobulin domain forms a moderately compact core while the unique N-terminal is disordered. Finally, using cell exclusion zone assay, we show that it is this domain triplet that is responsible for promoting cell migration and not the N- and C-terminal regions.
Highlights
Testicans/SPOCKs are proteoglycans of the vertebrate extracellular matrix (ECM)
Sequence alignment and motif identification using ScanProsite [27] revealed that all three testicans have two potential calcium-binding sites, corresponding to two EF-hand motifs within the extracellular calcium-binding domain (EC) domain (Figure 2a)
We believe that our findings are at least partially applicable to other two members of the testican family, in terms of their overall structural organization
Summary
Together with other proteoglycans and glycoproteins they form a complex network which provides hydration, compression resistance, and binding sites for soluble and cell surface molecules, thereby extending the functionality of the fibrous components of the ECM (collagens, fibronectins and elastans) [1,2] All these interconnected components are critically involved in intercellular communication and migration, which are both hallmarks of normal physiological processes like morphogenesis and differentiation, as well as pathological conditions, for example, cancer cell invasion [3]. A member of the testican family, testican-1, has been shown to be involved in the proliferation, migration and invasion of cancer cells via the PI3K/Akt and Wnt/β-catenin pathway, and represents a potential therapeutical target [4,5] Despite their involvement in these and other important processes outlined below, the data on their structure and structure–function relationship are virtually non-existent. Promotes cell migration in both testican-1 and -2
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