title: Analyses of disease-related GNPTAB mutations define a novel GlcNAc-1-phosphotransferase interaction domain and an alternative site-1 protease cleavage site.
abstract: Mucolipidosis II (MLII) and III alpha/beta are autosomal-recessive diseases of childhood caused by mutations in GNPTAB encoding the α/β-subunit precursor protein of the GlcNAc-1-phosphotransferase complex. This enzyme modifies lysosomal hydrolases with mannose 6-phosphate targeting signals. Upon arrival in the Golgi apparatus, the newly synthesized α/β-subunit precursor is catalytically activated by site-1 protease (S1P). Here we performed comprehensive expression studies of GNPTAB mutations, including two novel mutations T644M and T1223del, identified in Brazilian MLII/MLIII alpha/beta patients. We show that the frameshift E757KfsX1 and the non-sense R587X mutations result in the retention of enzymatically inactive truncated precursor proteins in the endoplasmic reticulum (ER) due to loss of cytosolic ER exit motifs consistent with a severe clinical phenotype in homozygosity. The luminal missense mutations, C505Y, G575R and T644M, partially impaired ER exit and proteolytic activation in accordance with less severe MLIII alpha/beta disease symptoms. Analogous to the previously characterized S399F mutant, we found that the missense mutation I403T led to retention in the ER and loss of catalytic activity. Substitution of further conserved residues in stealth domain 2 (I346 and W357) revealed similar biochemical properties and allowed us to define a putative binding site for accessory proteins required for ER exit of α/β-subunit precursors. Interestingly, the analysis of the Y937_M972del mutant revealed partial Golgi localization and formation of abnormal inactive β-subunits generated by S1P which correlate with a clinical MLII phenotype. Expression analyses of mutations identified in patients underline genotype-phenotype correlations in MLII/MLIII alpha/beta and provide novel insights into structural requirements of proper GlcNAc-1-phosphotransferase activity.
authors: Department of Genetics, Universidade Federal do Rio Grande do Sul, Avenida Paulo Gama 110, 90040-060 Porto Alegre, Brazil.
authors: Renata Voltolini
authors: RV
authors: Velho
authors: Department of Biochemistry, Children's Hospital, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany.
authors: Raffaella
authors: R
authors: De Pace
authors: Department of Biochemistry, Children's Hospital, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany.
authors: Sarah
authors: S
authors: Klünder
authors: Laboratory of Basic Research and Advanced Investigations in Neurosciences, Hospital de Clínicas de Porto Alegre, Rua Ramiro Barcellos 2350, 90035-903 Porto Alegre, Brazil and.
authors: Fernanda
authors: F
authors: Sperb-Ludwig
authors: Neurogenetics Unit, Department of Neurology, School of Medicine of Ribeirao Preto, University of São Paulo, Avenida Bandeirantes 3900, 14050-260 Ribeirão Preto, Brazil.
authors: Charles Marques
authors: CM
authors: Lourenço
authors: Department of Genetics, Universidade Federal do Rio Grande do Sul, Avenida Paulo Gama 110, 90040-060 Porto Alegre, Brazil, Laboratory of Basic Research and Advanced Investigations in Neurosciences, Hospital de Clínicas de Porto Alegre, Rua Ramiro Barcellos 2350, 90035-903 Porto Alegre, Brazil and.
authors: Ida V D
authors: IV
authors: Schwartz
authors: Department of Biochemistry, Children's Hospital, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany.
authors: Thomas
authors: T
authors: Braulke
authors: Department of Biochemistry, Children's Hospital, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany, s.pohl@uke.de.
authors: Sandra
authors: S
authors: Pohl
mesh_terms: D000818:Animals; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004789:Enzyme Activation; D015870:Gene Expression; D056726:Genetic Association Studies; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D008297:Male; D009154:Mutation; D043484:Proprotein Convertases / Q000235:genetics / Q000378:metabolism*; D054730:Protein Interaction Domains and Motifs; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport; D059748:Proteolysis; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D017855:Transferases (Other Substituted Phosphate Groups) / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D021122:Protein Subunits; D017855:Transferases (Other Substituted Phosphate Groups); C505859:alpha-beta GlcNAc-1-phosphotransferase, human; C511050:GNPTAB protein, human; D043484:Proprotein Convertases; D012697:Serine Endopeptidases; C115575:membrane-bound transcription factor peptidase, site 1