Abstract
We report the identification of three new collagen VI genes at a single locus on human chromosome 3q22.1. The three new genes are COL6A4, COL6A5, and COL6A6 that encode the alpha4(VI), alpha5(VI), and alpha6(VI) chains. In humans, the COL6A4 gene has been disrupted by a chromosome break. Each of the three new collagen chains contains a 336-amino acid triple helix flanked by seven N-terminal von Willebrand factor A-like domains and two (alpha4 and alpha6 chains) or three (alpha5 chain) C-terminal von Willebrand factor A-like domains. In humans, mRNA expression of COL6A5 is restricted to a few tissues, including lung, testis, and colon. In contrast, the COL6A6 gene is expressed in a wide range of fetal and adult tissues, including lung, kidney, liver, spleen, thymus, heart, and skeletal muscle. Antibodies to the alpha6(VI) chain stained the extracellular matrix of human skeletal and cardiac muscle, lung, and the territorial matrix of articular cartilage. In cell transfection and immunoprecipitation experiments, mouse alpha4(VI)N6-C2 chain co-assembled with endogenous alpha1(VI) and alpha2(VI) chains to form trimeric collagen VI molecules that were secreted from the cell. In contrast, alpha5(VI)N5-C1 and alpha6(VI)N6-C2 chains did not assemble with alpha1(VI) and alpha2(VI) chains and accumulated intracellularly. We conclude that the alpha4(VI)N6-C2 chain contains all the elements necessary for trimerization with alpha1(VI) and alpha2(VI). In summary, the discovery of three additional collagen VI chains doubles the collagen VI family and adds a layer of complexity to collagen VI assembly and function in the extracellular matrix.
Highlights
Collagen VI is an extracellular component that is present in virtually all connective tissues, where it forms abundant and structurally unique microfibrils in close association with basement membranes
Three genetically distinct collagen VI chains, ␣1(VI), ␣2(VI), and ␣3(VI), encoded by the COL6A1, COL6A2, and COL6A3 genes were first described more than 20 years ago (6 – 8)
Mutations in any of the three collagen VI chains lead to two types of congenital myopathies, the relatively mild Bethlem myopathy (BM) and the more severe Ullrich congenital muscular dystrophy (UCMD)
Summary
Cloning of Col6a4, COL6A5, and COL6A6 cDNA Sequences— Partial and full-length human COL6A5 and COL6A6 and mouse Col6a4 cDNAs were amplified by PCR from cDNA templates and cloned into the pCCR1 vector using the CopyControl PCR cloning kit (Epicenter Biotechnologies). CDNAs for ␣4(VI)N6-C2, ␣5(VI)N5-C1, and ␣6(VI)N7-C2 in pCCR1 were amplified using the primers modified to include NotI restriction sites (see supplemental Table 1). Following dehydrogenase forward and reverse primers were used in con- three washes in antibody buffer, anti-rabbit IgG-horseradish trol amplifications. SalI, the PCR products were cloned into the pTHIO-his(A) bac- blocked with 1% bovine serum albumin in PBS and incubated terial expression vector in-frame with the bacterial thioredoxin with dilutions of ␣6(VI)-C1 antiserum in PBS for 1 h. The sections were incubated with either goat anti-rabbit or antimouse secondary antibodies. The cell and media fractions were collected in the presence of protease inhibitors, and the His6-tagged ␣6(VI) chains were immunoprecipitated from the cell and media fractions with an anti-His antibody (0.5 g/ml; Roche Applied Science) or collagen VI polyclonal antisera (Fitzgerald Industries, catalog number 70-XR95) and protein A-Sepharose overnight. The gels were fixed, subjected to fluorography, dried, and exposed to x-ray film
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