Investigation of VPS33A protein function in mucopolysaccharidosis-plus syndrome

Abstract

Previously, we identified a novel disease from the group of hereditary metabolic diseases with an autosomal-recessive type of inheritance - mucopolysaccharidosis-plus syndrome (MPSPS). Using whole exome sequencing, we revealed pathogenic homozygous mutation p.R498W in the VPS33A gene. The objective of study was to analyze the effect of the mutation in the VPS33A gene on the protein level and its function. The cellular model of the disease was generated using Crispr-Cas9 system. Determination of autophagic activity was carried out by analyzing differences in the amount of the LC3-II between samples with and without addition of lysosome inhibitor bafilomycin A1. The level of endocytosis was determined by analysis of EGFR degradation. In the present study we revealed a reduction in the level of the VPS33A protein in cell lines with heterozygous and homozygous p.R498W mutations up to ~43% and ~25%, respectively. We also found that p.R498W mutation does not affect to the main known functions of the VPS33A - endocytosis and autophagy. The fluorescence intensity in the mutant cells was significantly increased compared to the controls, which indicated a shift of lysosomal pH to the acid side. Impairment of domain 2-2 led to dysfunction of the VPS33A protein. The fundamental significance of the study is due to decipher the pathological mechanisms of disease development to find approaches to adequate therapy.