Immunological analyses of alkyl-dihydroxyacetonephosphate synthase in human peroxisomal disorders

Alkyl-dihydroxyacetonephosphate synthase (alkyl-DHAP synthase) is a peroxis omal enzyme involved in the biosynthesis of ether phospholipids. To localiz e the enzyme in human peroxisomal disorders, indirect immunofluorescence an d immunoblot analysis was performed. In Zellweger syndrome and rhizomelic c hondrodysplasia punctata fibroblast cell lines, alkyl-DHAP synthase protein levels on immunoblots were strongly decreased and residual immunofluoresce nce was diffusely localized throughout the cytoplasm. In a particular neona tal adrenoleukodystrophy cell line, characterized by the absence of a funct ional peroxisomal targeting signal 1 receptor, the precursor form of the en zyme was detected in Western blots at levels comparable to that of the matu re enzyme in control fibroblasts, Similarly, fibroblasts from patients with a single deficiency in the activity of either alkyl-DHAP synthase or DHAP- acyltransferase showed normal levels of the mature alkyl-DHAP synthase prot ein on immunoblots. Immunofluorescence experiments revealed a peroxisomal l ocalization of both the precursor and the mature form of the enzyme Collect ively, these results visualize the peroxisomal localization of alkyl-DHAP s ynthase, indicate that the enzyme is unstable outside its target organelle and explain that normal enzyme protein levels found in some peroxisomal dis orders result from protection against cytoplasmic degradation through impor t into peroxisomes. Additionally, alkyl-DHAP synthase could be detected in rat mesangial cells and murine NIH-3R3 fibroblasts by immunofluorescence as well as immunoblot analysis. Immunoelectron microscopy showed that the enz yme is predominantly located on the lumenal side of the peroxisomal membran e in rat and guinea pig liver.