The UCMD-Causing COL6A1 () Intron Mutation Leads to the Secretion and Aggregation of Single Mutated Collagen VI α1 Chains

Figure 5

Recombinant mutant α1 chains do not assemble into collagen VI tetramers in WI-26 VA4 COL6A1 KO cells. (a) Control WI26 cells (CTRL), COL6A1 knock out (KO) and KO cells transfected with either the α1 wild-type (KO + His α1 WT) or mutant chain (KO + His α1 pex) were incubated for seven days in the presence of ascorbic acid. Labellings were performed with the α3N antibody to detect collagen VI microfibrils and the His antibody in order to localize the recombinant protein; scale . (b) Supernatants from cells (KO and retransfected) were separated on a 6% SDS-PAGE gel under non-reducing (-βMe) or under reducing (+βMe) conditions and detected with antibodies against the α1 chains or the pseudoexon sequence (Pex). (c) Supernatants of the two cell lines were resolved on a composite agarose/polyacrylamide gel and tetramers were detected with the α3N antibody via immunoblot. The mutant chains cannot form tetramers but assemble into extracellular patches. These do not have the organized network structure seen for the wild-type chains.

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