title: Mucolipidosis II-related mutations inhibit the exit from the endoplasmic reticulum and proteolytic cleavage of GlcNAc-1-phosphotransferase precursor protein (GNPTAB).
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.
authors.affiliation: Section Biochemistry, Children's Hospital, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
authors.forename: Raffaella
authors.initials: R
authors.lastname: De Pace
authors.forename: Maria Francisca
authors.initials: MF
authors.lastname: Coutinho
authors.forename: Friedrich
authors.initials: F
authors.lastname: Koch-Nolte
authors.forename: Friedrich
authors.initials: F
authors.lastname: Haag
authors.forename: Maria João
authors.initials: MJ
authors.lastname: Prata
authors.forename: Sandra
authors.initials: S
authors.lastname: Alves
authors.forename: Thomas
authors.initials: T
authors.lastname: Braulke
authors.forename: Sandra
authors.initials: S
authors.lastname: Pohl
mesh_terms: D000015:Abnormalities, Multiple / Q000235:genetics*; D000483:Alleles; D000818:Animals; D016466:CHO Cells; D002648:Child; D002675:Child, Preschool; D003412:Cricetulus; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D005838:Genotype; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D008297:Male; D009081:Mucolipidoses / Q000235:genetics*; D020125:Mutation, Missense; D010641:Phenotype; D059748:Proteolysis; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017855:Transferases (Other Substituted Phosphate Groups) / Q000235:genetics*
chemical_list: D012333:RNA, Messenger; D017855:Transferases (Other Substituted Phosphate Groups); C511050:GNPTAB protein, human
keywords: GNPTAB; Golgi; lysosomal storage disorder; mannose 6-phosphate; site-1 protease