The conservative substitution Asp-645-->Glu in lysosomal alpha-glucosidase affects transport and phosphorylation of the enzyme in an adult patient with glycogen-storage disease type II.
Glycogen-storage disease type II (GSDII) is caused by the deficiency of lysosomal alpha-glucosidase (acid maltase). This paper reports on the analysis of the mutant alleles in an American black patient with an adult form of GSDII (GM1935). The lysosomal alpha-glucosidase precursor of this patient has abnormal molecular features: (i) the molecular mass is decreased, (ii) the phosphorylation is deficient and (iii) the proteolytic processing is impaired. Sequence analysis revealed four mutations leading to amino acid alterations: Asp-645-->Glu, Val-816-->Ile, Arg-854-->Stop and Thr-927-->Ile. By using allele-specific oligonucleotide hybridization on PCR-amplified cDNA we have demonstrated that the Arg-854-->Stop mutation is located in one allele that is not expressed, and that the other allele contains the remaining three mutations. Each of the mutations was introduced in wild-type cDNA and expressed in COS cells to analyse the effect on biosynthesis, transport and phosphorylation of lysosomal alpha-glucosidase. The Val-816-->Ile substitution appeared to have no significant effect in contrast with results [Martiniuk, Mehler, Bodkin, Tzall, Hirshhorn, Zhong and Hirschhorn (1991) DNA Cell Biol. 10, 681-687] and was therefore defined as a polymorphism. The Thr-927-->Ile substitution deleting one of the seven glycosylation sites was found to be responsible for the decrease in molecular-mass, but not for the deficient proteolytic processing and phosphorylation. It did not cause the enzyme deficiency either. The third mutation leading to the Asp-645-->Glu substitution was proven to account in full for the observed defects in transport, phosphorylation and proteolytic processing of the newly synthesized alpha-glucosidase precursor of the patient.
Hermans
M M
MM
Department of Cell Biology and Genetics, Erasmus University, Rotterdam, The Netherlands.
de Graaff
E
E
Kroos
M A
MA
Wisselaar
H A
HA
Willemsen
R
R
Oostra
B A
BA
Reuser
A J
AJ
D000328:Adult; D000483:Alleles; D001224:Aspartic Acid; D001483:Base Sequence; D001692:Biological Transport; D044383:Black People; D002478:Cells, Cultured; D003062:Codon; D004252:DNA Mutational Analysis; D005971:Glutamates; D018698:Glutamic Acid; D006003:Glycogen / Q000378:metabolism; D006009:Glycogen Storage Disease Type II / Q000235:genetics*; D006801:Humans; D008247:Lysosomes / Q000201:enzymology*; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D010641:Phenotype; D010766:Phosphorylation; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D012045:Regulatory Sequences, Nucleic Acid; D017422:Sequence Analysis, DNA; D014415:Tunicamycin / Q000494:pharmacology; D000520:alpha-Glucosidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001619:beta-N-Acetylhexosaminidases / Q000032:analysis
D003062:Codon; D005971:Glutamates; D014415:Tunicamycin; D001224:Aspartic Acid; D018698:Glutamic Acid; D006003:Glycogen; D000520:alpha-Glucosidases; D001619:beta-N-Acetylhexosaminidases