Missense mutation in theN-acetylglucosamine-1-phosphotransferase gene (GNPTA) in a patient with mucolipidosis II induces changes in the size and cellular distribution of GNPTG

Abstract

Mucolipidosis type II (ML II; I-cell disease) and mucolipidosis III (ML III; pseudoHurler polydystrophy) are autosomal recessively inherited disorders caused by a defective Nacetylglucosamine 1-phosphotransferase (phosphotransferase). The formation of mannose 6-phosphate markers in soluble lysosomal enzymes is impeded leading to their increased excretion into the serum, to cellular deficiency of multiple hydrolases, and lysosomal storage of non-digested material. Phosphotransferase deficiency is caused by mutations in GNPTA and GNPTG encoding phosphotransferase subunits. Here we report on an adolescent with progressive joint contractions and other signs of mucolipidosis II who survived to the age of 14 years. Impaired trafficking of lysosomal enzymes cathepsin D and β-hexosaminidase in metabolically labeled fibroblasts was documented. Mutations in the GNPTG gene and alterations in the GNPTG mRNA level were not detected. A different electrophoretic mobility of the 97 kDa GNPTG dimer suggested posttranslational modification abrogating the compartmentalization of GNPTG in the Golgi apparatus. A nucleotide substitution in the GNPTA gene (c.3707A>T) was identified altering the predicted C-terminal transmembrane anchor of the phosphotransferase subunit. The data demonstrate that defective GNPTA not only impairs lysosomal enzyme targeting but also the availability of intact GNPTG required for phosphotransferase activity and assembly of subunits.

Molecular characterization of 22 novel UDP-N-acetylglucosamine-1-phosphate transferase α- and β-subunit (GNPTAB) gene mutations causing mucolipidosis types IIα/β and IIIα/β in 46 patients

“…Mutations in the GNPTAB gene, which encodes the α/β-subunits, are known to be responsible for MLII and MLIIIA [Tiede et al, 2005a and2005b;Kudo et al, 2005;Steet et al, 2005;Paik et al, 2005;Bargal et al, 2006;Tiede et al, 2006;Lam et al, 2007;Otomo et al, 2009]. Mutations in the γ-subunit, encoded by the GNPTG gene (MIM# 607838; chromosome 16p13.3), have been reported to be associated with MLIIIC [Raas-Rothschild et al, 2000Tiede et al, 2004].…”

Transport of the GlcNAc-1-phosphotransferase α/β-Subunit Precursor Protein to the Golgi Apparatus Requires a Combinatorial Sorting Motif

“…Two homozygous missense mutations, K4Q and K1236M, changing amino acids in the N-and C-terminal domains of the ␣/␤-subunit precursor protein, respectively, have been identified in GNPTAB MLII/III patients (23,24). Here, we show that the K4Q mutation reduced the proteolytic processing of the ␣/␤-subunit precursor protein by 60 -70%, which most likely impaired the 5 LL 6 -dependent ER export rather than directly the proteolytic cleavage in the luminal domain between residues Lys-928-Asp-929.…”

“…Because our data excluded 1236 KRK 1238 as active ER export signal, it is unlikely that the K1236M mutation affects the transport of the PT ␣/␤-subunit precursor protein to the Golgi apparatus and subsequently allows its proteolytic cleavage. The intermediate phenotype of a patient who survived to the age of 14 years (24) suggests that the mutant GlcNAc-1-phosphotransferase exhibits residual activity. Further studies are needed to examine whether K1236M substitution impairs the assembly of ␣ 2 ␤ 2 tetramers or ␣ 2 ␤ 2 ␥ 2 hexamer complexes in the ER, which might be important for proper phosphorylation of all lysosomal hydrolases (7).…”

“…Protein-To analyze whether mutations in cytosolic domains found in MLIII ␣/␤ patients with milder phenotype affect ER export and proteolytic cleavage of the PT ␣/␤-subunit precursor protein, we introduced missense mutations K4Q (23) and K1236M (24) into the PT ␣/␤-Myc cDNA. COS-7 cells were transfected with wild-type and mutant PT ␣/␤-Myc K4Q or K1236M, and cell extracts were analyzed by Western blotting (Fig.…”

Proteolytic Processing of the γ-Subunit Is Associated with the Failure to Form GlcNAc-1-phosphotransferase Complexes and Mannose 6-Phosphate Residues on Lysosomal Enzymes in Human Macrophages

“…in premature translational termination and mRNA decay (p.V176GfsX18, p.G204VfsX17, or p.Q203HfsX3), lead to the loss of the M6P recognition marker on lysosomal enzymes (24,39). 6 In addition mutations in GNPTAB encoding the ␣/␤-subunit precursor result in a secondary degradation of the ␥-subunit (40,41) and the associated loss of the M6P marker. Whereas proteolytic modifications of the ␤-subunit can be excluded in macrophages, no data are available on the ␣-subunit polypeptide because specific antibodies are lacking.…”