Kinked Collagen VI Tetramers and Reduced Microfibril Formation as a Result of Bethlem Myopathy and Introduced Triple Helical Glycine Mutations

Abstract

Mutations in the genes that code for collagen VI subunits, COL6A1, COL6A2, and COL6A3, are the cause of the dominantly inherited disorder, Bethlem myopathy. Glycine mutations that interrupt the Gly-X-Y repetitive amino acid sequence that forms the characteristic collagen triple helix have been defined in four families; however, the effects of these mutations on collagen VI biosynthesis, assembly, and structure have not been determined. In this study, we examined the consequences of Bethlem myopathy triple helical glycine mutations in the ␣1(VI) and ␣2(VI) chains, as well as engineered ␣3(VI) triple helical glycine mutations. Although the Bethlem myopathy and introduced mutations that are toward the N terminus of the triple helix did not measurably affect collagen VI intracellular monomer, dimer, or tetramer assembly, or secretion, the introduced mutation toward the C terminus of the helix severely impaired association of the mutant ␣3(VI) chain with ␣1(VI) and ␣2(VI). Association of the three chains was not completely prevented, however; and some non-disulfide bonded tetramers were secreted. Examination of the secreted Bethlem myopathy and engineered mutant collagen VI by negative staining electron microscopy revealed the striking finding that in all the cell lines a significant proportion of the tetramers contained a kink in the supercoiled triple helical region. Collagen VI tetramers from all of the mutant cell lines also showed a reduced ability to form microfibrils. These results provide the first evidence of the biosynthetic consequences of collagen VI triple helical glycine mutations and indicate that Bethlem myopathy results not only from the synthesis of reduced amounts of structurally normal protein but also from the presence of mutant collagen VI in the extracellular matrix.Collagen VI is structurally unique within the collagen family of proteins, forming abundant microfibrils in the extracellular matrix of almost all tissues, including skin, cartilage, cornea, and both skeletal and smooth muscle (for reviews see Refs. 1 and 2). Intracellular assembly of collagen VI is a complex multistep process. Association of the three genetically distinct subunits, ␣1(VI), ␣2(VI), and ␣3(VI), to form a triple helical monomer is followed by antiparallel, staggered assembly into disulfide-bonded dimers, which then align to form tetramers, also stabilized by disulfide bonds. Outside of the cell, tetramers, the secreted form of collagen VI, associate end-to-end to form the characteristic beaded microfibrils.Bethlem myopathy is a relatively mild, dominantly inherited disorder, characterized by early childhood onset of proximal muscle weakness and wasting and, commonly, joint contractures mainly involving the elbows, ankles, and fingers (3, 4). This disorder was shown to be linked to the COL6A1 and COL6A2 genes on 21q22.3 (5) and in a separate family to the COL6A3 gene at 2q37 (6), and mutations in these collagen VI genes have subsequently been identified in 8 Bethlem myopathy families. The mutations include a single bas...

Global secretome analysis identifies novel mediators of bone metastasis

“…Collagen is crucial to the bone matrix, but the most common bone collagen is Collagen type 1, with far less being known about COL6A1, though its mutations are linked to muscularskeletal defects such as Bethlam myopathy and Ulrich Congentical Muscular Distrophy [51][52][53][54]. Interestingly, a recent secretome analysis identified COL6A1 as a strongly upregulated protein in the conditioned media of metastatic, as compared to non-metastatic lung cancer cell lines [55].…”

Congenital muscular dystrophy. Part II: a review of pathogenesis and therapeutic perspectives

“…In the first case, mutations towards the N terminal region of the triple helix may cause kinking of the tetramers in the normally straight supercoiled triple helical region, thus reducing their ability to form microfibrils 127 and exerting a dominant negative effect. Baker et al 124 confirmed that the majority of the Bethlem myopathy mutations occur toward the N-terminal end of the triple helix, that is a critical region of the collagen VI monomer (α1:α2:α3) is critical for efficient dimer, tetramer, and microfibril assembly.…”

“…For example, a heterozygous nonsense mutation causing an alpha-1 premature stop codon and a decrease of available alpha-1 chains consequently reduces production and secretion of collagen VI 126 . otherwise, a heterozygous missense mutation interrupting the repetitive amino acid sequence (glycine mutation) that forms the characteristic collagen triple helix either in the alpha-1or alpha-2 chains does not affect collagen VI intracellular monomer, dimer, and tetramer assembly or secretion, but the mutant tetramers are anomalous (kinked) and the extracellular microfibrils are functionally deficient 127 . Patients reported with a heterozygous in-frame deletion in the triple-helical domain resulting in exon skipping in the Col6A1 gene have either a typical clinical picture with finger contractures 125 or a course more severe than that commonly reported 128 .…”

“…lampe and Bushby 69 summarized the most frequent pathogenic mechanisms in Bethlem myopathy: single amino acid substitutions disrupting the Gly-Xaa-yaa motif of the triple helical domain in Col6A1, Col6A2, or Col6A3 77,121,127,130 ,and splice site mutations 75,121,125,128 which cause skipping of Col6A1 exon 14 during pre-mrNA splicing and consequently in-frame deletion of 18 amino acids from the triple helical domain of the a1 chain. In the first case, mutations towards the N terminal region of the triple helix may cause kinking of the tetramers in the normally straight supercoiled triple helical region, thus reducing their ability to form microfibrils 127 and exerting a dominant negative effect.…”

“…The concept of a spectrum of collagen VI-related disorders with marked clinical and genetic heterogeneity has emerged from the recent advances on the molecular mechanism of both diseases 69 . The complex genotype/phenotype correlations that have been broadly analysed in these two conditions clearly indicate that in both collagen VI-related disorders the main pathomechanism is due to the disruption of collagen VI anchorage to the basal lamina of the muscular fibers 74,75,[120][121][122][123][124][125][126][127][128][129][130][131][132][133][134][135][136][137][138] .…”