Biochemical analysis of GNPTAB missense mutations associated with ML II

Abstract

Mucolipidosis type II and type III (ML II and III) are rare autosomal recessive disorders of lysosomal hydrolase trafficking respectively caused by completely absent or reduced activity of the enzyme GlcNAc-phosphotransferase, which catalyzes the initial step in the synthesis of mannose-6-phosphate recognition marke. This heterohexameric enzyme composed of three subunits (alpha2 beta2 gamma2), is a product of two distinct genes GNPTAB and GNPTG. Mutations in GNPTAB result ML II and ML III, while mutations in GNPTG are only associated with ML III. To date more than 100 different GNPTAB mutations have been described, causing either ML II alpha\beta or ML III alpha\beta. Although splicing and frameshift mutations are usually associated with more severe phenotypes and missense mutations with milder ones, this typical pattern is not observed for all ML II patients. Here we report the impact of two GNPTAB missense mutations upon the protein: while W81L occurred in the portion of the gene that encodes the apha-subunit, R986C affected a genomic region encoding the beta subunit. To address this issue, the entire coding region of the wild-type GNPTAB was cloned into the pcDNAHisMax TOPO vector and the c.440delC (A147AfsX5), c.2956T>C (R986C) and c.242G>T (W81L) were introduced on this vector using the QuikChange Site-directed Mutagenesis kit. The presence of additional mutations, resulting from possible enzymatic misincorporation, was excluded by sequencing all constructs. COS7 cells were transfected with control and mutant plasmids using Lipofectamine 2000 reagent. Protein expression levels and subcellular location were determined through Western Blot and Immunofluorescence, respectively. Results and Conclusions: We analyzed the protein expression levels of three GNPTAB mutations: c.242G>T (W81L), c.2956T>C (R986C) andc.440delC (A147AfsX5). The frameshift c.440delC (A147AfsX5), predicting to introduce a premature stop codon, was used as negative control. and, as expected, no GNPTAB protein product was detectable. Instead, the analysis of both missense mutations, c.2956T>C (R986C) and c.242G>T (W81L), revealed a decrease in GNPTAB protein expression, compared to the control wild type. This concurs with a previous computational assessment by the Polyphen and SIFT algorithms, predicting that the 2 mutations were likely to be potentially damaging. In addition, computational analysis (http://www.ensembl.org/) revealed that both missense mutations occurred at evolutionarily conserved amino acid residues. The results of all these approaches correlate with the severe ML II phenotype of the patients