Signal sequence and DNA-mediated expression of human lysosomal α-galactosidase A

Authors

  • Shoji TSUJI,

    1. Molecular Neurogenetics Unit, Clinical Neuroscience Branch, National Institute of Mental Health, Alcohol, Drug Abuse and Mental Health Administration, Bethesda, Maryland
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  • Brian M. MARTIN,

    1. Molecular Neurogenetics Unit, Clinical Neuroscience Branch, National Institute of Mental Health, Alcohol, Drug Abuse and Mental Health Administration, Bethesda, Maryland
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  • David C. KASLOW,

    1. Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, Maryland
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  • Barbara R. MIGEON,

    1. Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, Maryland
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  • Prabhakara V. CHOUDARY,

    1. Centers for Biotechnology and for Advanced Research in Molecular Genetics, Jawaharlal Nehru University, New Delhi
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  • Barbara K. STUBBLEFLIED,

    1. Molecular Neurogenetics Unit, Clinical Neuroscience Branch, National Institute of Mental Health, Alcohol, Drug Abuse and Mental Health Administration, Bethesda, Maryland
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  • June A. MAYOR,

    1. Molecular Neurogenetics Unit, Clinical Neuroscience Branch, National Institute of Mental Health, Alcohol, Drug Abuse and Mental Health Administration, Bethesda, Maryland
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  • Gary J. MURRAY,

    1. Laboratory of Molecular Genetics, National Institute of Neurological and Communicative Disorders, and Stroke, National Institutes of Health, Bethesda, Maryland
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  • John A. BARRANGER,

    1. Division of Medical Genetics, Children's Hospital of Los Angeles, California
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  • Edward I. GINNS

    Corresponding author
    1. Molecular Neurogenetics Unit, Clinical Neuroscience Branch, National Institute of Mental Health, Alcohol, Drug Abuse and Mental Health Administration, Bethesda, Maryland
      Correspondence to E. I. Ginns, Molecular Neurogenetics Unit, Clinical Neuroscience Branch, Bldg. 10, Rm. 3D16, National Institute of Mental Health, Bethesda, Maryland, USA 20892
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Correspondence to E. I. Ginns, Molecular Neurogenetics Unit, Clinical Neuroscience Branch, Bldg. 10, Rm. 3D16, National Institute of Mental Health, Bethesda, Maryland, USA 20892

Abstract

Twelve complementary DNA clones for human lysosomal α-galactosidase A were isolated from an Okayama-Berg library constructed from SV40-transformed human fibroblasts. The identity of these clones was confirmed by complete colinearity of the nucleotide-deduced amino acid sequence with that determined by direct chemical sequencing of human placental α-galactosidase A. Hybridization of the α-galactosidase A cDNA to genomic DNA from individuals with varying numbers of X chromosomes as well as from interspecies somatic-cell hybrids showed only a single locus in the genome at Xq 13.1 – Xq 22. One cDNA clone (pcD-AG210) contained the complete coding sequence for both the signal peptide and mature α-galactosidase A. The signal peptide of 31 amino acids contains the expected hydrophobic domains consisting of Leu-Gly-Cys-Ala-Leu-Ala-Leu and Phe-Leu-Ala-Leu-Val and has Ala at the signal peptidase cleavage site. Twelve out of fifteen G residues flanking the 5′ end of the cDNA in pcD-AG210 were removed and the truncated fragment was ligated into the original vector. This construct, pcD-AG502, encoded enzymatically active human α-galactosidase A in monkey COS cells.

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