Multiple epiphyseal dysplasia mutations inMATN3 cause misfolding of the A-domain and prevent secretion of mutant matrilin-3

Abstract

Multiple epiphyseal dysplasia (MED) is a relatively common skeletal dysplasia that can present in childhood with a variable phenotype of short stature and pain and stiffness in the large joints, and often progresses to early-onset osteoarthritis in adulthood. Mutations in the matrilin-3 gene (MATN3) have recently been shown to underlie some forms of autosomal dominant MED. To date all MED mutations in matrilin-3 cluster in the single A-domain, suggesting that they may disrupt the structure and/or function of this important domain. To determine the effects of MATN3 mutations on the structure and function of matrilin-3 we expressed both normal and mutant matrilin-3 in mammalian cells. Wild-type (wt) matrilin-3 was efficiently secreted into conditioned medium, whereas mutant matrilin-3 was retained and accumulated within the cell. Furthermore, when the mutant A-domains were examined individually, they existed primarily in an unfolded conformation. Co-immunoprecipitation experiments demonstrated that the mutant A-domains were specifically associated with ERp72, a chaperone protein known to be involved in mediating disulfide bond formation. Light microscopy of cartilage from an MED patient with a MATN3 mutation showed the presence of intracellular material within the chondrocytes, whilst the overall matrix appeared normal. On electron micrographs, the inclusions noted at the light microscopy level appeared to be dilated cisternae of rough endoplasmic reticulum and immunohistochemical analysis confirmed that the retained protein was matrilin-3. In summary, the data presented in this paper suggest that MED caused by MATN3 mutations is the result of an intracellular retention of the mutant protein.

Increased expression of matrilin‐3 not only in osteoarthritic articular cartilage but also in cartilage‐forming tumors, and down‐regulation of SOX9 via epidermal growth factor domain 1–dependent signaling

“…Involvement of several missense MATN3 mutations has been observed in MED (6,7). These mutations cause misfolding of the protein (24), resulting in accumulation and stress in the endoplasmic reticulum and apoptosis of chondrocytes due to alterations in the secretion machinery of the chondrocytes (25). Another missense mutation of the T303 residue, which lies in the EGF-1 domain, was found to be statistically significantly associated with hand osteoarthritis (8) without evidence of altered MATN3 secretion (26).…”

Disruption of Extracellular Matrix Structure May Cause Pseudoachondroplasia Phenotypes in the Absence of Impaired Cartilage Oligomeric Matrix Protein Secretion

“…The mutation H587R demonstrates that in certain cases this pathogenic pathway might even be more important than the ER retention. However, in case of a matrilin-3 mutation that leads to intracellular protein accumulation within the ER, the ultrastructure of the matrix was not affected (43). Studies of cell attachment to wild-type and mutant forms of COMP (Fig.…”

Novel mutations in exon 2 ofMATN3affect residues within the α-helices of the A-domain and can result in the intracellular retention of mutant matrilin-3

“…The resulting PCR products were cloned into the expression vector pCEP-4. We have previously generated expression constructs for p.Val194Asp and p.Glu252Lys, which were used as controls in all experiments (Cotterill, et al, 2005). Purified DNA was transfected into 293-EBNA cells (human embryonic kidney cells; Invitrogen) using LipofectAMINE 2000 reagent (Invitrogen).…”

“…Of the 12 MATN3 mutations reported to date, 10 of these affect residues in the 6 β-strands, indeed mutations have now been reported in 5 of the 6 β-strands that comprise the β-sheet (Cotterill, et al, 2005;Jackson, et al, 2004;Mabuchi, et al, 2004;Maeda, et al, 2005;Mostert, et al, 2003). The two exceptions to this rule are p.Arg70His, located in a linker region and only 7 residues downstream from the amino-terminal cysteine residue of the A-domain (Maeda, et al, 2005), and p.Phe105Ser in the α1 helix of the A-domain (Mabuchi, et al, 2004).…”

“…Many of the mutations in these genes cluster in distinct DOI: 10.1002/humu.9518 regions of the protein and usually affect conserved residues that are important for the structure and/or function of the relevant gene products (Briggs and Chapman, 2002). This is particularly the case with MATN3 and to date all MED-causing mutations affect residues within, or closely associated with, the single A-domain of matrilin-3 (Cotterill, et al, 2005;Jackson, et al, 2004;Mabuchi, et al, 2004;Maeda, et al, 2005;Mostert, et al, 2003).…”