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Is SPARC an Evolutionarily Conserved Collagen Chaperone?

¹ Department of Cell and Systems Biology, University of Toronto, 25 Harbord Street, Toronto, ON, Canada M5S 3G5;
² CIHR Group in Matrix Dynamics, Faculty of Dentistry, University of Toronto, 150 College Street, Toronto, ON, Canada M5S 3E2

View larger version (48K): [in this window] [in a new window]   Figure 1. Immunolocalization of SPARC in mouse periodontal tissues. (A) Immunostaining for SPARC in the first molar and surrounding tissues of a three-day-old mouse. SPARC is present in most cells, including the enamel epithelium, but is more strongly stained in odontoblasts (green arrows) and bone cells (red arrows). (B) In an enlarged section shown by the red rectangle in A, strong staining for SPARC is evident in osteoblasts, osteocytes, and the surrounding periosteal cells, with no staining evident in the bone matrix. (C) Immunostaining for SPARC in the incisor and surrounding tissues of the same mouse. Collagen-expressing periodontal ligament fibroblasts (magenta arrows) and cementoblasts (blue arrows), osteoblasts and osteocytes of the mandibular bone (red arrows), and odontoblasts (green arrows) all stain strongly for SPARC. However, while there was some staining of the matrix of the developing periodontal ligament and cementum, no staining was evident in the mineralized bone and dentin matrices. A weak staining for SPARC was also evident in the ameloblasts (black arrows). The SPARC was identified with the use of affinity-purified rabbit antibodies and specificity ascertained by staining similar sections from SPARC-null mice (not shown).

View larger version (25K): [in this window] [in a new window]   Figure 2. Schematic representation of the modular organization of SPARC and the EC-collagen-binding domain. The N-terminal domain of SPARC in vertebrates can bind up to 8 Ca2+-ions with low-affinity (Kd 10–3 to 10–5 M), whereas 2 EF-hands bind a single Ca2+-ion, each with high affinity (Kd 10–7 M). X-ray crystallographic studies have indicated that the EC-domain of SPARC binds to the triple-helical domains of several types of fibrillar collagen and the basal laminae-associated network-forming collagen type IV. The illustrations are based on schematics by Sasaki et al.(1998) and an NCBI Cn3D v4.1 3D structure viewer (NCBI, http://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?form=6&db=t&Dopt=s&uid=6694). Human SPARC (with signal peptide attached, lower-case letters) is shown as an example and color-coded to highlight the 3 modules.

View larger version (18K): [in this window] [in a new window]   Figure 3. Increased acidity of the low-affinity, high-capacity Ca2+-binding N-terminal domain I during evolution. Domain I of SPARC in invertebrates has an almost equal number of acidic and basic amino acid residues. However, despite the presence of several basic amino acids in vertebrates, this domain can bind with low affinity to several Ca2+ ions (Maurer and Hohenester, 1997). Interestingly, basic amino acid residues are not found in Domain I of vertebrate SPARC, suggesting that SPARC may have been recruited during vertebrate evolution to function in the formation of mineralized tissues. Acidic amino acids (red), basic amino acids (blue), cysteine residues (amber).

View larger version (24K): [in this window] [in a new window]   Figure 4. Comparison of SPARC amino acid sequences in the EC-domain essential for Ca2+-dependent collagen-binding. Bold letters highlighted in red indicate 5 amino acids found by site-directed mutagenesis to be essential for the Ca2+-dependent binding of SPARC to human type I collagen. Note that the essential amino acids are 100% conserved among vertebrates (upper-case underscored letters). Within invertebrates, conservative amino acid substitutions are found at essential sites (upper-case, underscored, and italicized letters). The conservative amino acid changes would be expected to have only minor topographical impact on the collagen-binding domain of SPARC. Fig. adapted from data presented by Sasaki et al.(1998).

View larger version (27K): [in this window] [in a new window]   Figure 5. Model of SPARC acting as a collagen chaperone. Molecular chaperones and folding enzymes ensure that secretory proteins are correctly folded into their native state prior to exiting the endoplasmic reticulum. The unique and complex folding and assembly of procollagen -chains into triple-helical collagen molecules are orchestrated by several endoplasmic reticulum resident molecular chaperones, including the collagen-specific chaperone HSP47. Analysis of the data indicates that the amino acids essential for the Ca2+-binding of SPARC to collagens are evolutionarily conserved in organisms ranging from nematodes to mammals. Studies in Drosophila have indicated that SPARC expression is required for the deposition of collagen IV in basal laminae by hemocytes during embryonic development. We hypothesize that, in invertebrates that do not code for HSP47, intracellular SPARC functions as the principal collagen-specific molecular chaperone, stabilizing triple helices prior to their export from the endoplasmic reticulum. We further hypothesize that SPARC may also promote the assembly and maturation of collagen fibrils by regulating the binding of molecules, such as decorin, which bind directly to collagens and inhibit fibrillogenesis. The latter may, in part, account for the smaller fibrils in SPARC-null mice.