Collagen VI microfibril formation is abolished by an {alpha}2(VI) von Willebrand factor type A domain mutation in a patient with Ullrich congenital muscular dystrophy

Abstract

Collagen VI is an extracellular protein that most often contains the three genetically distinct polypeptide chains, α1(VI), α2(VI), and α3(VI), although three recently identified chains, α4(VI), α5(VI), and α6(VI), may replace α3(VI) in some situations. Each chain has a triple helix flanked by N- and C-terminal globular domains that share homology with the von Willebrand factor type A (VWA) domains. During biosynthesis, the three chains come together to form triple helical monomers, which then assemble into dimers and tetramers. Tetramers are secreted from the cell and align end-to-end to form microfibrils. The precise molecular mechanisms responsible for assembly are unclear. Mutations in the three collagen VI genes can disrupt collagen VI biosynthesis and matrix organization and are the cause of the inherited disorders Bethlem myopathy and Ullrich congenital muscular dystrophy. We have identified a Ullrich congenital muscular dystrophy patient with compound heterozygous mutations in α2(VI). The first mutation causes skipping of exon 24, and the mRNA is degraded by nonsense-mediated decay. The second mutation is a two-amino acid deletion in the C1 VWA domain. Recombinant C1 domains containing the deletion are insoluble and retained intracellularly, indicating that the mutation has detrimental effects on domain folding and structure. Despite this, mutant α2(VI) chains retain the ability to associate into monomers, dimers, and tetramers. However, we show that secreted mutant tetramers containing structurally abnormal C1 VWA domains are unable to associate further into microfibrils, directly demonstrating the critical importance of a correctly folded α2(VI) C1 domain in microfibril formation.

FIGURE 1.

Muscle biopsy immunostaining for collagen VI. Frozen sections of control and patient (UCMD21) muscle were stained with antibodies to collagen VI (red) and perlecan (green). In the control muscle biopsy, collagen VI and perlecan co-localize in the basement membrane surrounding muscle fibers (yellow). In UCMD21, collagen VI no longer co-localizes with perlecan but is seen predominantly in the interstitial space between muscle fibers.

FIGURE 2.

UCMD21 has heterozygous recessive α2(VI) mutations. A, genomic DNA PCR and sequencing. The patient, UCMD21, has compound heterozygous mutations. The first, inherited from her unaffected father, is a c.1771–1G → T splice site mutation that leads to skipping of exon 24 during pre-mRNA splicing (data not shown). The second change, inherited from her unaffected mother, is a c.1855_1860del6 mutation causing an in-frame p.V619_I620del2 mutation in the N-terminal region of the α2(VI) C1 VWA domain. B, schematic representation of the α2(VI) protein changes. The N1, C1, and C2 VWA domains are labeled, and the triple helical domain is shown as a black line. Exon 24 skipping causes a reading frameshift, starting from p.T590 at the C-terminal end of the triple helix, and introduces a premature stop codon 148 amino acids downstream. The abnormal protein sequence at the C-terminal end of the α2(VI) chain is indicated in red. The p.V619_I620del2 mutation is near the N-terminal end of the C1 VWA domain.

FIGURE 3.

mRNA containing the exon 24 skip and a premature stop codon is degraded by nonsense-mediated mRNA decay. A, Northern blot analysis. Total RNA from control (lane C) and UCMD21 (lane U21) fibroblasts was separated on a denaturing agarose gel, transferred to nitrocellulose, and probed with [α-³²P]dCTP-labeled α1(VI), α2(VI), and α3(VI) cDNA probes. In the control, α1(VI) and α2(VI) mRNAs are present in similar amounts; however, in UCMD21 α2(VI) mRNA is reduced relative to α1(VI) mRNA, indicating mRNA decay. B, RT-PCR analysis of a 271-bp fragment of α2(VI) that includes both mutations. In UCMD21 fibroblasts, amplification of the α2(VI) mRNA carrying the V619_I620 deletion generates a 265-bp product that is indistinguishable from the 271-bp wild type product in control fibroblasts. Amplification of the mRNA carrying the exon 24 skip generates a faint 225-bp product in UCMD21. The reduced amount of this RT-PCR product indicates that the mRNA, which contains a premature stop codon, is subject to mRNA degradation. The band migrating above the 265-bp product in the UCMD21 sample is a heteroduplex (het) of the 265- and 225-bp products.

FIGURE 4.

Collagen VI biosynthetic analysis in UCMD21 fibroblasts. Control (lane C) and UCMD21 (lane U21) fibroblasts were labeled overnight with [³⁵S]methionine, and collagen VI was immunoprecipitated from the cell and medium with an α3(VI) N1 domain antibody. A, immunoprecipitated collagen VI was analyzed under reducing conditions on 3–8% Tris-acetate gels to resolve the constituent α1(VI), α2(VI), and α3(VI) chains. Mutant UCMD21 α2(VI) p.V619_I620del2 chains associate with α1(VI) and α3(VI) chains to form collagen VI assemblies that are secreted into the medium. B, nonreduced immunoprecipitated collagen VI samples were analyzed on 2.4% acrylamide, 0.5% agarose gels to visualize collagen VI monomers, dimers, and tetramers. Collagen VI dimers and tetramers and unassociated α3(VI) chains are indicated as are fibronectin dimers (FN²). UCMD21 mutant α2(VI) chains are incorporated into stable tetramers that are secreted from the cell. Some unassembled α3(VI) chains are present in the UCMD21 cell fraction, suggesting that they are present in excess. This is consistent with data demonstrating that fewer α2(VI) chains are synthesized because the mRNA from one allele is degraded.

FIGURE 5.

Pulse-chase analysis of collagen VI assembly and secretion. Control and UCMD21 fibroblasts were labeled with [³⁵S]methionine for 30 min and chased for up to 8 h. Collagen VI was immunoprecipitated with an α2(VI) N1 domain antibody. A, collagen VI immunoprecipitated from the cell layer was analyzed under reducing conditions on a 3–8% gradient acrylamide gel. In the control, α3(VI) chains were co-immunoprecipitated from 0.5 h, indicating that they had assembled with α2(VI) chains. In UCMD21, assembly of the mutant α2(VI) chains with α3(VI) was delayed with significant co-immunoprecipitation occurring only after 1 h of chase. B, collagen VI immunoprecipitated from the medium was resolved under reducing conditions on a 3–8% gradient acrylamide gel. The secretion of collagen VI from control cells can be seen after 1 h of chase, reaching a maximum between 2 and 4 h of chase. In contrast, secretion was much slower from UCMD21 cells, with most of the protein appearing in the medium between 4 and 8 h of chase. C–F, in a separate pulse-chase experiment, collagen VI immunoprecipitated from the cell and medium of control and UCMD21 fibroblasts and was analyzed on nonreducing composite 2.4% polyacrylamide, 0.5% agarose gels. C, nonreduced collagen VI immunoprecipitated from the cell of control fibroblasts. Dimers first appear in the cell at 0.5 h, and tetramers first appear at 1 h. D, nonreduced collagen VI immunoprecipitated from the cell layer of UCMD21 fibroblasts. Dimers appear in the cell from 1 h of chase, and tetramers appear from 2 h onward. E, nonreduced collagen VI immunoprecipitated from the medium of control fibroblasts. Tetramers first appear in the medium of control fibroblasts from 1 h, and the majority of radiolabeled protein is present in the medium from 2 h onward. F, nonreduced, collagen VI immunoprecipitated from the medium of UCMD21 fibroblasts. Tetramers appear in the medium after 2 h of chase, and the majority of radiolabeled protein is present in the medium from 8 h. Collectively these data indicate that the α2(VI) C1 domain deletion in UCMD21 delays the assembly of collagen VI in fibroblasts. Fn₂, fibronectin dimer.

FIGURE 6.

Immunostaining of the collagen VI matrix deposited by fibroblasts. Control and UCMD21 fibroblasts were grown for 2 days post-confluence in the presence of sodium ascorbate and stained with a collagen VI antibody. The cell nuclei were stained with DAPI. Control fibroblasts deposited an extensive collagen VI network; however, collagen VI was not detected in the matrix of UCMD21 fibroblasts.

FIGURE 7.

Collagen VI microfibril formation is compromised in UCMD21. A, gel filtration chromatography of control and UCMD21 fibroblast conditioned medium. Control and UCMD21 fibroblasts were grown to confluence and incubated overnight with serum-free DMEM containing sodium ascorbate. Harvested medium was passed over a Sephacryl S1000 SF column, and proteins were eluted in 0.5-ml fractions. Collagen VI was detected by dot-blot using a collagen VI primary antibody and a fluorescent secondary antibody. Collagen VI from control medium eluted as a broad peak with most of the protein in fractions 70–81. UCMD21 collagen VI eluted later than control collagen VI; the majority was in fractions 75–81. This suggested that mutant tetramers form smaller extracellular assemblies than their wild type counterparts. B, Blue Native-PAGE analysis of secreted collagen VI. To further investigate extracellular microfibril assembly, control and patient fibroblasts were labeled overnight with [³⁵S]methionine, and collagen VI was immunoprecipitated from the medium with an α3(VI) N1 domain antibody and resolved by Blue Native-PAGE. Control medium contains a small number of individual tetramers (40) as well as assemblies containing two tetramers (tet₂); however, the majority of collagen VI exists as microfibrils containing three or more tetramers (≥tet₃). In UCMD21 medium, mutant tetramers are only present as single or double assemblies and do not associate into microfibrils containing three or more tetramers. Fibronectin dimers (FN₂), which bind nonspecifically to the protein A-Sepharose, are also indicated. C, negative staining electron micrographs of collagen VI microfibrils in medium from control and UCMD21 fibroblasts. D, quantitative analysis of collagen VI tetramer-tetramer association. Collagen VI secreted into the medium of control (white bars) and UCMD21 (black bars) fibroblasts was visualized by negative staining electron microscopy, and the ability of the tetramers to associate end-to-end was quantitated. The occurrence of microfibrils containing one to ten tetramers is shown as a percentage of the total number of microfibrils. Tetramer-tetramer association was severely compromised in UCMD21 compared with control.

FIGURE 8.

Recombinant expression of the α2(VI) C1 domain. HEK293-EBNA cells were transfected with episomal expression vectors containing an N-terminal StrepII tag and the sequence for either the wild type α2(VI) domain or the α2(VI) domain harboring the V619_I620 deletion. The insoluble cell lysate (I), soluble cell lysate (S), and medium (M) from an untransfected control and from cells expressing either the wild type (WT) or mutant domains were analyzed by nonreducing SDS-PAGE and immunoblots using a monoclonal antibody to the StrepII tag. When compared with the medium, the loading of soluble and insoluble cell fractions was four times higher as a proportion of the total extract. Wild type α2(VI) C1 domains (∼30 kDa) are soluble and secreted into the medium as autonomously folded units. The mutant α2(VI) C1 domains are predominantly present in the insoluble cell fraction where they form multimers of molecular masses consistent with two (60 kDa), three (90 kDa), four (120 kDa), five (150 kDa), or greater than five disulfide-bonded C1 domains. Furthermore, mutant domains are not present in the medium of transfected cells, indicating that the mutation has detrimental effects on the structure and folding of the C1 domain. The StrepII antibody did not recognize a 30-kDa protein in the untransfected cell and medium, confirming the specificity of the antibody for the recombinant protein; however, the antibody regularly, but not always, recognized two unknown proteins of ∼80 and ∼200 kDa in the medium of control and transfected cells.