Elsevier

Matrix Biology

Volume 20, Issue 7, November 2001, Pages 439-450
Matrix Biology

Selective intracellular retention of extracellular matrix proteins and chaperones associated with pseudoachondroplasia

https://doi.org/10.1016/S0945-053X(01)00148-2Get rights and content

Abstract

Mutations in the cartilage oligomeric matrix protein (COMP) gene result in pseudoachondroplasia (PSACH), which is a chondrodysplasia characterized by early-onset osteoarthritis and short stature. COMP is a secreted pentameric glycoprotein that belongs to the thrombospondin family of proteins. We have identified a novel missense mutation which substitutes a glycine for an aspartic acid residue in the thrombospondin (TSP) type 3 calcium-binding domain of COMP in a patient diagnosed with PSACH. Immunohistochemistry and immunoelectron microscopy both show abnormal retention of COMP within characteristically enlarged rER inclusions of PSACH chondrocytes, as well as retention of fibromodulin, decorin and types IX, XI and XII collagen. Aggrecan and types II and VI collagen were not retained intracellularly within the same cells. In addition to selective extracellular matrix components, the chaperones HSP47, protein disulfide isomerase (PDI) and calnexin were localized at elevated levels within the rER vesicles of PSACH chondrocytes, suggesting that they may play a role in the cellular retention of mutant COMP molecules. Whether the aberrant rER inclusions in PSACH chondrocytes are a direct consequence of chaperone-mediated retention of mutant COMP or are otherwise due to selective intracellular protein interactions, which may in turn lead to aggregation within the rER, is unclear. However, our data demonstrate that retention of mutant COMP molecules results in the selective retention of ECM molecules and molecular chaperones, indicating the existence of distinct secretory pathways or ER-sorting mechanisms for matrix molecules, a process mediated by their association with various molecular chaperones.

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).

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