Lysine methylation of mitochondrial ATP synthase subunit c stored in tissues of dogs with hereditary ceroid lipofuscinosis

Certain forms of ceroid lipofuscinosis, a hereditary degenerative disease, are characterized by accumulation of large amounts of subunit c of mitochondrial ATP synthase in lysosomal storage bodies of numerous tissues. The subunit c protein appears to constitute a major fraction of the total storage body protein. In previous studies it was demonstrated that hydrolysates of total storage body protein from affected humans and sheep contain significant amounts of epsilon-N-trimethyllysine (TML). This finding suggested that one or both of the two lysine residues of subunit c might be methylated in the stored form of the protein. The normal subunit c protein from mitochondria does not appear to be methylated. Using a putative canine model for the juvenile form of ceroid lipofuscinosis, analyses were conducted to determine whether lysosomal storage of subunit c was accompanied by lysine methylation of this protein. In affected dogs, as in humans and sheep with hereditary ceroid lipofuscinosis, the storage bodies were found to contain large amounts of subunit c protein, as indicated by polyacrylamide gel electrophoresis and partial amino acid sequence analysis. The subunit c protein partially purified from isolated storage bodies was found to contain lysine and TML in an almost equimolar ratio. Normal subunit c contains 2 lysine residues, one at position 7 and the other at position 43. Removal of the first 7 residues of the partially purified protein through sequential Edman degradation resulted in a dramatic increase in the TML to lysine ratio in the residual protein. This suggests that lysine residue 43 is methylated. Confirmation that residue 43 of the stored protein is TML was obtained by amino acid sequence analysis after cleavage of the protein with trypsin. This finding strongly suggests that specific methylation of lysine residue 43 of mitochondrial ATP synthase plays a central role in the lysosomal storage of this protein.