Functional characterization of alpha- mannosidosis associated sequence variants

Abstract

Alpha-mannosidosis is a rare autosomal recessive lysosomal-storage disorder resulting from deficiency of lysosomal alpha-mannosidase activity. The disease shows a wide range of clinical phenotypes caused by intracellular accumulation of mannose-containing oligosaccharides, which ultimately may lead to mental retardation, hearing loss, skeletal changes and immune deficiency. Lysosomal alpha-mannosidase is a hydrolase that cleaves alpha-linked mannose residues from the non-reducing end of N-linked glycans of glycoproteins. So far, 40 alpha-mannosidosis associatied sequences have been reported, but no correlation of clinical and disease associated sequence variations has been detected so far. In previous work, the seven alpha-mannosidosis associated mutations, p.C55F,p.H200L, p.P263L, p.S318L, p.S453F, p.S457E and p.T745R in the LAMAN gene were identified. In order to characterize the functional of these sequence variants at the biochemical and cellular levels to investigate if they are disease-causing mutations. The mutations were introduced into an expression vector containing the wild-type LAMAN cDNA by in vitro mutagenesis, and the resulting proteins were expressed in COS-7 and BHK cells. This study had revealed that the previous indentified mutation p.C55F suggested to cause human alpha-mannosidosis result in no enzyme significant activity in transfected COS-7 and BHK cells. This missense-mutation probably resulted in misfolding and ER-retention. This was consistent with the low enzyme expression level and non lysosomal localization in transfected cells. The six other mutant variants resulted either in no significant residual or low residual activity, but they were intracellularly proteolytically processed and partially sorted to the lysosomes similar to wild-type.