Selective intracellular retention of extracellular matrix proteins and chaperones associated with pseudoachondroplasia
Introduction
Pseudoachondroplasia (PSACH), an autosomal dominant skeletal dysplasia characterized by short-limbed dwarfism and early onset osteoarthritis, is caused by mutations in the gene encoding COMP (reviewed in Horton and Hecht, 2000). COMP, a pentameric glycoprotein and member of the thrombospondin family of proteins, is found in the extracellular matrix of developing and adult cartilage, and is present in ligament, tendon and synovium (Delot et al., 1998, Di Cesare et al., 1999, Hedbom et al., 1992, Smith et al., 1997). The COMP monomer is composed of several modular domains each of which are thought to fold independently of one other. The amino terminal α-helical coiled-coil domain is important in pentamerization of the COMP molecule (Efimov et al., 1994, Malashkevich et al., 1996), followed by four epidermal growth factor (EGF)-like domains and the thrombospondin (TSP) type 3 domain. The TSP type 3 domain has recently been shown to bind calcium (Chen et al., 2000, Maddox et al., 2000) and is where the majority of the mutations associated with PSACH are found to occur (Ballo et al., 1997, Briggs et al., 1995, Briggs et al., 1998, Hecht et al., 1995, Loughlin et al., 1998). Finally, each COMP monomer contains a C-terminal globular domain which has been shown to bind to collagen I and II with high affinity, an interaction dependent on the presence of divalent cations (Adams and Lawler, 1993, Rosenberg et al., 1998). This suggests a role for COMP in the organization of collagen fibrils contributing to tissue structure and function.
The hallmark of PSACH is the presence of enlarged, distinctive lamellar rER vesicles within chondrocytes (Maynard et al., 1972, Stanescu et al., 1982). These have previously been shown to contain COMP and type IX collagen (Delot et al., 1998, Maddox et al., 1997), two components normally expressed and secreted by chondrocytes. Type II collagen was shown to be absent from the ER vesicles in PSACH, indicating a selective process by which specific matrix proteins are either retained or secreted. Large oligomeric glycoproteins such as thrombospondin I and thyroglobulin have been shown to specifically associate with rER proteins during their intracellular processing and secretion (Kuznetsov et al., 1997). These rER proteins most likely function as molecular chaperones in the protein folding process, and they may serve as retention anchors within the quality control system in the secretory pathway for immature or misfolded proteins (Ellgaard et al., 1999, Hammond and Helenius, 1995). Presumably, such factors play a role in the cellular response to a mutant COMP molecule, as in the case of PSACH. Whether the abnormal rER accumulations in PSACH are due to selective interactions with mutant COMP molecules or are the result of a secretory pathway impaired by abnormal COMP is not known. In an effort to better understand not only the pathology of PSACH, but also the selective secretory processes for ECM molecules, we have identified distinct ECM and molecular chaperone proteins retained within the rER inclusions of chondrocytes from a patient with PSACH. These studies suggest a shared secretory pathway whereby intracellular accumulation of mutant COMP molecules results in the selective retention of certain ECM molecules, a process that is mediated by specific molecular chaperones.
Section snippets
Patient samples
The patient was an 11-year-old female diagnosed with PSACH. She exhibited the typical features of PSACH: short-limb short stature, joint laxity and abnormal radiology. A sample of iliac crest cartilage was removed following informed consent protocol and used as the source of material for subsequent analyses. Tissue samples of iliac crest were also obtained from a 10-year old control for immunofluorescence. The chondrocytes were isolated following collagenase digestion: patient cartilage sample
Mutation analysis
Tissue from the PSACH patient was extracted and analyzed for mutations in the COMP gene. Direct sequencing of a PCR fragment from exon 14 in the patient showed a heterozygous A/G peak at position 1544 of the coding sequence (GenBank accession no. L32137). This base substitution is predicted to result in a change from an aspartic acid to a glycine residue at position 515 in the amino acid sequence (Fig. 1). Blood samples from the unaffected parents were analyzed and the DNA was found to carry
Discussion
In this study we report a novel D515G mutation in the calcium-binding TSP type 3 domain of COMP in a patient diagnosed with PSACH. It is in this region of the protein where the majority of reported PSACH mutations have been found to occur, demonstrating the importance of calcium binding to the structure and function of COMP. Although the function of COMP remains unclear, the mutations associated with PSACH presumably interfere with the protein's ability to bind calcium thereby affecting the
Acknowledgements
This work was supported by grants from Shriners Hospital for Children (to H.P.B., D.R.K., L.Y.S. and W.A.H.) and by Grant AR45582 from the Department of Health and Human Services (to H.P.B).
References (52)
- et al.
The thrombospondin family
Curr. Biol.
(1993) - et al.
Calnexin: a membrane-bound chaperone of the endoplasmic reticulum
Trends Biochem. Sci.
(1994) - et al.
Diverse mutations in the gene for cartilage oligomeric matrix protein in the pseudoachondroplasia-multiple epiphyseal dysplasia disease spectrum
Am. J. Hum. Genet.
(1998) - et al.
Cartilage oligomeric matrix protein is a calcium binding protein and a mutation in its type 3 repeats causes conformational changes
J. Biol. Chem.
(2000) - et al.
Physiological and pathological secretion of cartilage oligomeric matrix protein by cells in culture
J. Biol. Chem.
(1998) - et al.
The thrombospondin-like chains of cartilage oligomeric matrix protein are assembled by a five-stranded alpha-helical bundle between residues 20 and 83
FEBS Lett.
(1994) - et al.
Cartilage matrix proteins. An acidic oligomeric protein (COMP) detected only in cartilage
J. Biol. Chem.
(1992) - et al.
Binding of fibromodulin and decorin to separate sites on fibrillar collagens
J. Biol. Chem.
(1993) - et al.
Procollagen binds to both prolyl 4-hydroxylase/protein disulfide isomerase and HSP47 within the endoplasmic reticulum in the absence of ascorbate
FEBS Lett.
(2000) - et al.
Multiple molecular chaperones complex with misfolded large oligomeric glycoproteins in the endoplasmic reticulum
J. Biol. Chem.
(1997)