Mucolipidosis II-Related Mutations Inhibit the Exit from the Endoplasmic Reticulum and Proteolytic Cleavage of GlcNAc-1-Phosphotransferase Precursor Protein (GNPTAB)

Abstract

Mucolipidosis (ML) II and MLIII alpha/beta are two pediatric lysosomal storage disorders caused by mutations in the GNPTAB gene, which encodes an α/β-subunit precursor protein of GlcNAc-1-phosphotransferase. Considerable variations in the onset and severity of the clinical phenotype in these diseases are observed. We report here on expression studies of two missense mutations c.242G>T (p.Trp81Leu) and c.2956C>T (p.Arg986Cys) and two frameshift mutations c.3503_3504delTC (p.Leu1168GlnfsX5) and c.3145insC (p.Gly1049ArgfsX16) present in severely affected MLII patients, as well as two missense mutations c.1196C>T (p.Ser399Phe) and c.3707A>T (p.Lys1236Met) reported in more mild affected individuals. We generated a novel α-subunit-specific monoclonal antibody, allowing the analysis of the expression, subcellular localization, and proteolytic activation of wild-type and mutant α/β-subunit precursor proteins by Western blotting and immunofluorescence microscopy. In general, we found that both missense and frameshift mutations that are associated with a severe clinical phenotype cause retention of the encoded protein in the endoplasmic reticulum and failure to cleave the α/β-subunit precursor protein are associated with a severe clinical phenotype with the exception of p.Ser399Phe found in MLIII alpha/beta. Our data provide new insights into structural requirements for localization and activity of GlcNAc-1-phosphotransferase that may help to explain the clinical phenotype of MLII patients.

Mislocalization of phosphotransferase as a cause of mucolipidosis III αβ

“…S1A) and an undetectable β subunit by Western blotting (Fig. 2B) (10), indicative of misfolding in the ER. Thus, reduced ER exit is unlikely to be the cause of the low β subunit levels of the K4Q and S15Y mutants.…”

Site-1 protease-activated formation of lysosomal targeting motifs is independent of the lipogenic transcription control

“…Confocal immunofl uorescence microscopy of transfected cells, activity determination of the enzyme ␤ -hexosaminidase, and metabolic [ 35 S]methionine labeling of MEF followed by immunoprecipitation of cathepsin Z were described recently ( 18,22 ). Densitometric analyses were performed with the Image J software (The National Institutes of Health) to quantify band intensities in Western blots and X-ray fi lms .…”

“…A myc-tagged, single-chain antibody fragment against M6P residues (scFv M6P-1) and the monoclonal rat antibody against the ␣ -subunit of the GlcNAc-1-phosphotransferase have been described recently ( 21,22 ). The monoclonal anti-myc antibody was purchased from Cell Signaling.…”

“…To deplete or overload the cholesterol content in cells, 10 µM atorvastatin or 100 µg/ml human LDL was added to the DMEM/LPDS medium for 48 h, respectively. Twenty-four hours after addition of atorvastatin (ATV) or LDL, MEFs and human fi broblasts were transfected with cDNA of the ␣ */ ␤ -subunit construct #3 ( 8,22 ) using JetPEI ® according to the manufacturer´s instructions. The ␣ */ ␤ -subunit miniconstruct lacks amino acids 431-819 and results in a shortened ␣ *-subunit exhibiting identical topology, N-and C-terminal ER exit structures, and structural requirements for effi cient proteolytic cleavage by S1P ( 8,22 ).…”

“…human ␣ */ ␤ -subunit precursor construct ( 8,22 ) of the GlcNAc-1-phosphotransferase. Immunofl uorescence microscopy revealed complete costaining of ␣ *-subunit immunopositive material with the cis -Golgi marker protein GM130 independent of high (LDL) or low (ATV) levels of cholesterol ( Fig.…”