Multiple functions of the von Willebrand Factor A domain in matrilins: secretion, assembly, and proteolysis
© Zhang et al; licensee BioMed Central Ltd. 2008
Received: 13 November 2007
Accepted: 02 June 2008
Published: 02 June 2008
The von Willebrand Factor A (vWF A) domain is one of the most widely distributed structural modules in cell-matrix adhesive molecules such as intergrins and extracellular matrix proteins. Mutations in the vWF A domain of matrilin-3 cause multiple epiphyseal dysplasia (MED), however the pathological mechanism remains to be determined. Previously we showed that the vWF A domain in matrilin-1 mediates formation of a filamentous matrix network through metal-ion dependent adhesion sites in the domain. Here we show two new functions of the vWF A domain in cartilage-specific matrilins (1 and 3). First, vWF A domain regulates oligomerization of matrilins. Insertion of a vWF A domain into matrilin-3 converts the formation of a mixture of matrilin-3 tetramer, trimer, and dimer into a tetramer only, while deletion of a vWF A domain from matrilin-1 converts the formation of the native matrilin-1 trimer into a mixture of trimer and dimer. Second, the vWF A domain protects matrilin-1 from proteolysis. We identified a latent proteolytic site next to the vWF A2 domain in matrilin-1, which is sensitive to the inhibitors of matrix proteases. Deletion of the abutting vWF A domain results in degradation of matrilin-1, presumably by exposing the adjacent proteolytic site. In addition, we also confirmed the vWF A domain is vital for the secretion of matrilin-3. Secretion of the mutant matrilin-3 harbouring a point mutation within the vWF A domain, as occurred in MED patients, is markedly reduced and delayed, resulting from intracellular retention of the mutant matrilin-3. Taken together, our data suggest that different mutations/deletions of the vWF A domain in matrilins may lead to distinct pathological mechanisms due to the multiple functions of the vWF A domain.
In cartilage, extracellular matrix (ECM) molecules mediate cell-matrix and matrix-matrix interactions, thereby providing tissue integrity. Matrilins (matn) are a novel ECM protein family which consists at least of four members . All the members of matrilin family contain von Willebrand Factor A domains (vWF A domain), EGF-like domains, and a heptad repeat coiled-coil domain at the carboxyl terminus, which is a nucleation site for the oligomerization of the molecule [2, 3]. Among the four members, matrilin-1 and matrilin-3 are expressed specifically in cartilage. Matrlin-1 forms a homotrimer and matrilin-3 forms a mixture of homotetramer, -trimer, and -dimer [4, 5], in addition to the hetero-oligomers matn-1 and -3 form together [4, 6]. It is not known how matn-1 forms a trimer only while matn-3 forms a mixture of tetramer, trimer and dimer. The major structural difference between matn-1 and -3 is that matn-1 contains two vWF A domains while matn-3 contains only one; the second vWF A domain flanking the coiled coil domain is missing from matn-3. In addition, matn-3 contains four EGF repeats, while matn-1 contains only one EGF-like domain. Previously we have shown that the number of the EGF repeats does not affect the assembly of matrilins . In this study, we investigate whether the presence or absence of the vWF A domain adjacent to the coiled-coil is involved in modulating oligomeric formation of matrilins.
The vWF A domain is one of the most widely distributed domains involved in cell adhesion and the formation of multiprotein complexes. These vWF A domain containing molecules include both subunits of the intergrin receptor (α and β), sixteen collagens, and non-collagenous ECM proteins such as matrilins. The property of the vWF A domain in cell adhesion and protein-protein interaction is mediated, in many cases, by the metal-ion dependent adhesion site (MIDAS) located within the domain . We have shown previously that the deletion of the vWF A domain or mutations of the MIDAS motif in MATN-1 abolish its ability to form pericellular filamentous network . This indicates that one of the functions of the vWF A domain of matrilins is to act as an adhesion site for its matrix ligands including collagens and proteoglycans [10, 11]. However, this function may not be the only function of the vWF A domain. This is indicated by the recent identification of the mutations of MATN-3 in multiple epiphyseal dysplasia (MED) patients .
MED is an osteochondrodysplasia primarily characterized by delayed and irregular ossification of the epiphyses and early-onset osteoarthritis . Two different recessive mutations in the exon encoding the vWF A domain of MATN-3 cause the EDM5 form of MED . These point mutations result in single amino acid changes of V194D or R121W. Subsequent genetic analysis indicates that the R121W mutation is recurrent in multiple families with common or different ancestries . Interestingly, although these residues are conserved in all matrilin family members across species, they are not part of the MIDAS motif . This suggests that these residues in the vWF A domain may play other important roles in addition to protein-protein interactions.
To determine these unknown roles of the vWF A domain in matrilins, we performed a series of deletions and mutations of the vWF A domain in cartilage-specific MATN-1 and -3. We found several novel functions of the vWF A domain of matrilins including regulation of protein secretion, oligomeric assembly, and proteolysis by matrix proteases.
Materials and methods
Cloning and Construction of Matrilin-3 cDNAs
Primers used in this study
Primer Sequences (5'-->3')
Results shown in Figures
TAA TAC GAC TCA CTA TAG GG
T7, amplifying inserts from pCDNA3.1
AAG GAC GAT GAT GAC AAA GCT GCA AAT ACA TGT GCA CT
Adding a flag tag
TGT CAT CAT CGT CCT TAT AGT CCC CCC AGA CTC CAC AGC T
GAG GAG AGG GTT AGG GAT AGG CTT A
Amplifying inserts from pCDNA3.1
ACT GCA AGC TGA GCA AGT CTT CTT G
Adding a vWFA domain into minimatn3 (combining with the PCR product of primers 1 and 4)
ATCTGC GTT AGA GCC ACA ACA AGC AGT
ACT GCA AGC TGA GCA AGT CTT CTT G
Replacing minimatn3 coiled-coil domain with that of matn1 (combining with the PCR product of primers 1 and 4)
ATC TGC GTT AGA GCC ACA ACA AGC AGT
AAA GAA CAA CCT GGG TGG CAG TCA TGA
Introducing R116W mutation in the vWFA domain
TCA TGA CTG CCA CCC AGG TGG TTC TTT
GAT GAC AAA GCA CCT CCT CAG CCC AGA
Adding a flag tag
ATC TTC CTC ACT GCA GGT CTT CCC ATC ATT
ACC TGC AGT GAG GAA GAT CCA TGC GAA TGT
Creating Δmatn1 by deleting vWFA2
ACC TGC AGT TGC GAA TGT AAA TCT ATA GT
ACA TTC GCA ACT GCA GGT CTT CCC ATC AT
Creating Δmatn1_del by deleting 4 amino acids from Δmatn1
ACT TGC TCA GCT TGC AGT GGT GGG TCA
TGT GGC TCT AAC GCA GAT TTT CAT TTG
Amplifying mantn1 coiled-coil domain
ACT TGC TCA GCT GTC AGT GGT GGG TCA
TCT GGC TCT AAC GCA GAT TTT CAT TTG
Amplifying mantn1 vWFA2 domain
Transfection of Matrilin cDNAs
cDNA constructs of matrilin-3 and -1 were transfected into COS-7 cells (Monkey Kidney Fibroblast Cells) or MCT cells (Immortalized Mouse Chondrocytes)  using LIPOFECTAMINE (Life technology, Rockville, MD) according to manufacturer instruction. Briefly, COS-7 cells or MCT chondrocytes were trypsinized and counted. Each 60 mm plate were seeded with 6 × 105 cells, with were allowed to attach overnight and reach 70% confluence in DMEM supplied with 10% FBS (Life technology). The following day, the cells were rinsed with DMEM and subjected to a DNA/LIPOFECTAMINE(Life technology) mix for 5–24 hours. Five μg cDNA were used for single transfection and 4 μg/each cDNA were used for co-transfection, respectively. The DNA/LIPOFECTAMINE mixture was aspirated and replaced with 3 ml DMEM supplied with 1% FBS. The media from transfected cell culture were collected at different time points (1, 2, 3, and 4 days) after transfection. Cells were lysed on ice for 10 minutes in a lysis buffer as previously described . Cell lysates were centrifuged at 4°C for 10 minutes. Supernatant of the cell lysate as well as the conditioned medium were analyzed using western blot. Some transfected cells were treated with matrix protease inhibitors including EDTA and actinonin at indicated concentrations for 48 hours before the conditioned medium was collected for analysis.
SDS-Polyacrylamide Gel Electrophoresis and Western Blot
Western blot analysis was performed with collected conditioned medium or cell lysates from transfected cell culture. For non-reducing condition, collected samples were mixed with standard 2× SDS gel-loading buffer. For reducing conditions, the loading buffer contains 5% b-mercaptoethanol and 0.05 M DTT. Samples were boiled for 10 minutes before loaded onto 10% SDS-PAGE gels, or 4–20% gradient gels as indicated. After electrophoresis, proteins were transferred onto Immobilon-PVDF membrane (Millipore Corp., Bedford, MA) in 25 mM Tris, 192 mM glycine, and 15% methanol. The membranes were blocked in 2% bovine serum albumin fraction V (Sigma Co., St. Louis, MO) in PBS for 30 minutes and then probed with antibodies. The primary antibodies used were a monoclonal antibody against the V5 tag (diluted 1:5000) (Invitrogen), and a monoclonal antibody against FLAG (diluted 1:1000) (Affinity BioReagents). Horseradish peroxidase conjugated goat anti-mouse or goat anti-rabbit IgG (H+L) (Bio-Rad Laboratories, Melville, NY), diluted 1:3,000, was used as a secondary antibody. Visualization of immunoreactive proteins was achieved using the ECL Western blotting detection reagents (Amersham Corp., Heights, IL) and exposing the membrane to Kodak X-Omat AR film. Molecular weights of the immunoreactive proteins were determined against two different sets of protein marker ladders.
COS-1 or MCT cells were cultured in DMEM + 10% FBS in 12-well plates overnight. Matrilin-3 or MED-mutant matrilin-3 cDNA was transfected into the cells using Lipofectamin 2000 (Invitrogen). Three days after transfection, cells were starved for 2 hours in 0.5 ml cysteine and methionine free medium (Sigma), pulse-labeled in 100 μCi/ml medium of S-35 methionine (Amersham) for 1 hour, and chased in normal medium. After harvest of culture supernatants, monolayer cells were lysed in 1% NP-40, 50 mM Tris, pH 7.4. Immunoprecipitation was carried out by incubating culture supernatant or cell lysate with 1.5 μl anti-V5 antibody (Invitrogen) at 4°C for 2 hours, followed by coupling to protein A/G plus agarose (Santa Cruz) overnight at 4°C. After precipitation, the samples were eluted by boiling after washing 3 times with 0.5% Triton 100 in TBS. The eluted proteins were separated by electrophoresis in a 4–15% SDS-PAGE gel, followed by transferring to a PVDF membrane and exposed to X-ray films.
After cells from Cos-1 and MCT cell lines were seeded onto 8-well chamber slides, 1 μg wild-type matrilin-3 or MED-mutant matrilin-3 cDNA was transfected into each well using Lipofectamin 2000 (Invitrogen). Three days after transfection, monolayer cells were fixed with 70% ethanol, 50 mM glycine for 1 hour. Immunofluorescence staining was performed by incubation of anti-V5 primary antibody (Invitrogen) at 1:200 for 2 hours, followed by incubation with donkey anti-mouse rhodamine secondary antibody (Jackson Laboratory) at 1:200 dilutions in the presence of Hoechst Stain Solution (Sigma). Slides were mounted with coverslips in Gel/Mount (Biomed).
MED mutation in the vWFA domain of MATN3
Insertion of vWFA2 domain into MATN3
Deletion of vWFA2 domain from MATN1
Proteolysis of matn1
Exchange of the coiled-coil domain between MATN1 and MATN3
Our study suggests that the matrilin vWF A domain, a widely distributed structural module in integrins and ECM proteins, plays a role in regulating protein secretion, assembly, and proteolysis, in addition to its well-documented role in cell-matrix adhesion . These newly discovered functions of the vWF A domain of matrilins are discussed as follows.
We show that a single point mutation in the vWF A domain of mouse MATN3 (R116W), equivalent to the MED mutation (R121W) in human MATN3, leads to a deficiency of matrilin secretion in vitro which is consistent with previous reports. In addition to the decrease of the amount of the mutant protein secreted into the medium (Fig. 2B), the secretion time course is markedly delayed for 24 hours (Fig. 2C, D). In the meantime, excessive amount of the mutant protein is accumulated intracellularly (Fig. 2B, E). These observations indicate that intracellular retention of the mutant protein is responsible for the deficiency of protein secretion in quantity and speed. Consistent with this hypothesis, we observed a great increase of intracellular vesicles that contain mutant matrilin-3 (Fig. 3). The vWF A domain is composed of about 200 amino acid residues arranged into multiple α-β units, which results in a three dimensional structure of a central β sheet core flanked by α helices . Because R121 is located in one of the β strands, despite the molecular mechanism is still under investigation, it strongly suggests that abnormal protein folding contributes to the secretion deficiency of the mutant protein.
Although matrilin-3 is the only matrilin family member that has been associated with chondrodysplasia so far, more and more point mutations within the vWF A domain of matrilin-3 have been reported to cause MED. They include mutations A219D, I192N, T120M, and E134K . Interestingly, all of these MED-causing mutations are located in the β strands in the center of the vWF A domain, which are important for the folding of the protein structure . It suggests that the secretion deficiency due to intracellular retention of the mutant protein, as demonstrated by this study, is a common mechanism of matrilin-3 associated MED. Such mechanism is similar to that of a point mutation of cartilage oligomeric matrix protein (COMP), which also leads to MED or related pseudoachondroplasia. It has been demonstrated previously that the mutant COMP is retained in the rough endoplasmic reticulum . This retention in turn results in excessive accumulation of the proteins that are associated with COMP such as collagen type IX, whose mutation also leads to similar clinical manifestation. Our observation that cells expressing mutant matrilin-3 exhibit expanded cytoplasm with multiple vacuoles, which is similar to the phenotype of mutant COMP expressing cells [18, 20], suggests that mutated matrilin-3 or COMP may lead to common cellular phenotype. In light of the recent discovery that COMP interacts with matrilin-1, -3, and -4, our finding here lends support to the hypothesis that mutations in any of these interacting proteins including matrilin, COMP, or collagen IX, result in a secretion defect, which manifests in common chondrodysplasia pathological phenotypes. It should also be noted that a portion of the mutant protein is secreted into the medium. However, we do not know whether the mutant protein is defective in its adhesion to matrix ligands or subject to extracellular proteolysis. These possibilities remain to be determined in future studies.
The oligomeric assembly of matrilins is complex. This complexity is two fold. First, in contrast to some ECM protein families such as collagens that always form a trimeric structure, different matrilin member forms different set of oligomers. While the major oligomeric forms of matrilin-1, -2 and -4 are trimers, matrilin-3 is a tetramer [4, 22]. Second, in addition to the major oligomeric form, each matrilin has minor oligomeric forms. For example, matrilin-2 has a tetramer and a dimer in addition to a trimer, and matrilin-3 has a trimer and a dimer in addition to a tetramer. So far, two theories have been proposed to explain the cause of heterogeneity of matrilin oligomers. One is proteolytic processing, which proposes that the heterogeneity of the matrilin derives from the proteolytic cleavage of a single matrilin oligomer . Indeed, studies using the peptide of the coiled-coil domain demonstrate that each matrilin peptide forms a single homo-oligomer, with matrilin-1, -2, and -4 being a trimer and matrilin-3 being a tetramer [23, 24]. Furthermore, Klatt et al. demonstrated that proteolytic cleavage of a matrilin-4 trimer generates a dimer and a monomer . However, the proteolytic processing theory cannot explain all the heterogeneity of matrilin oligomers. For example, it cannot explain how a matrilin-2 trimer gives rise to a tetramer through proteolytic cleavage.
We proposed an alternative theory that heterogeneity of oligomeric forms of matrilins may arise from imperfect oligomerization , in addition to protein processing. The imperfect oligomerization hypothesis was based on the fact that the amino acid sequence of the oligomeric nucleation site coiled-coil domain, although strongly favours one oligomeric form, has ambiguity for alternate forms . This ambiguity is modulated by the vWF A domain next to the coiled-coil domain. Our study here put this hypothesis to test. First, replacing the coiled-coil domain of matrilin-3 with that of matrilin-1 changes the matrilin-3 oligomeric forms from a combination of a tetramer, a trimer, and a dimer into a combination of a trimer and a dimer, reminiscent of those of matrilin-1 (Fig. 7). Thus, the coiled-coil domain primarily determines the oligomeric forms of matrilins. Second, the vWF A domain next to the coiled-coil further modulates the diversity of matrilin oligomeric forms. Deletion of the vWFA2 domain from matrilin-1 converts the formation of a predominant trimer into a mixture of trimer and dimer (Fig. 5), while insertion of the vWFA2 domain into matrilin-3 converts the formation of a mixture of tetramer, trimer, and dimer into a tetramer only (Fig. 4). The vWFA domain may achieve this modulatory role in two ways, by affecting either matrilin processing or assembly. The identification of a latent matrilin-1 cleavage site (EEDP) at the junction of the vWFA2 domain and the coiled-coil domain seems to suggest that different oilgomeric forms of matrilins arise from processing at this site. However, deletion of this cleavage site, which clearly eliminates protein processing, does not reduce the number of different matrilin oilgomeric forms (Fig. 6B). Thus, the diversity of the matrilin oligomeric forms cannot be attributed to protein processing alone. The vWFA2 domain, therefore, must play a role in regulating matrilin oligomeric formation as well.
The junction region of matrilins contains potential proteolytic cleavage sites.
Amino Acid Sequence
KLKKGICEALEDSDGRQDSPAGELPKTVQQPT ESEPVTINIQDLLSCSNFAVQHRYLFEE DNLL RSTQKLSHSTKPSGSPLEE
KLKEGICEALEDSGGRQDSAAWDLPQQAHQP TEPEPVTIKIKDLLSCSNFAVQHRFLFEE DN LSRSTQKLFHSTKSSGNPLEE
One of the major functions of the junction region containing these cleavage sites is to process matrilins and generate proteolytic fragments. The cleavage in the junction region of matrilins separates the vWF A domain that binds matrix ligands from the coiled-coil domain that oligomerizes matrilins. Such proteolytic cleavage may destabilize or destroy matrilin filamentous network in extracellular matrix. Our study raises a possibility that mutation/deletion of the vWF A domain may change its conformation to expose the mutant matrilin for accelerated proteolytic degradation.
Different mutations/deletions of the vWF A domain in matrilins may lead to distinct pathological mechanisms due to the multiple functions of the vWF A domain. This may explain how different mutations within matrilin-3 lead to a variety of cartilage diseases.
polyacrylamide gel electrophoresis
reverse-transcription polymerase chain reaction
multiple epiphyseal dysplasia
cartilage oligomeric matrix protein
We thank Benoit deCrombrugghe for providing MCT chondrocyte cell line. This study is supported by grants AG14399, AG17021 from NIH to QC and a Human Growth Foundation grant to YZ. QC and YZ are also supported by the Arthritis Foundation.
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