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

Authors

  • Raffaella De Pace,

    1. Section Biochemistry, Children's Hospital, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
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    • These authors contributed equally to this work.

  • Maria Francisca Coutinho,

    1. Research and Development Unit, Department of Genetics, CGMJM, INSA, Faculty of Sciences, Porto, Portugal
    2. IPATIMUP, Faculty of Sciences, Porto, Portugal
    3. Department of Biology, Faculty of Sciences, Porto, Portugal
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    • These authors contributed equally to this work.

  • Friedrich Koch-Nolte,

    1. Institute of Immunology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
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  • Friedrich Haag,

    1. Institute of Immunology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
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  • Maria João Prata,

    1. IPATIMUP, Faculty of Sciences, Porto, Portugal
    2. Department of Biology, Faculty of Sciences, Porto, Portugal
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  • Sandra Alves,

    1. Research and Development Unit, Department of Genetics, CGMJM, INSA, Faculty of Sciences, Porto, Portugal
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  • Thomas Braulke,

    1. Section Biochemistry, Children's Hospital, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
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  • Sandra Pohl

    Corresponding author
    1. Section Biochemistry, Children's Hospital, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
    • Correspondence to: Sandra Pohl, Section Biochemistry, Children's Hospital, University Medical Center Hamburg-Eppendorf, Hamburg, Germany. E-mail: s.pohl@uke.de

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  • Contract grant sponsors: Fundação para a Ciência e a Tecnologia (FCT) (PIC/IC/83252/2007, SFRH/BD/48103/2008); Deutsche Forschungsgemeinschaft (GRK1459, SFB877).

  • Communicated by Hans R. Waterham

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.

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