Analysis of normal and mutant iduronate-2-sulphatase conformation.
Mammalian sulphatases (EC 3.1.6) are a family of enzymes that have a high degree of similarity in amino acid sequence, structure and catalytic mechanism. IDS (iduronate-2-sulphatase; EC 3.1.6.13) is a lysosomal exo-sulphatase that belongs to this protein family and is involved in the degradation of the glycosaminoglycans heparan sulphate and dermatan sulphate. An IDS deficiency causes the lysosomal storage disorder MPS II (mucopolysaccharidosis type II). To examine the structural alterations in heat-denatured and mutant IDS, a panel of four monoclonal antibodies was raised to the denatured protein and used as probes of protein conformation. The linear sequence epitope reactivity of a polyclonal antibody raised against the native protein and the monoclonal antibodies were defined and mapped to distinct regions on the IDS protein. The antigenicity of native IDS was higher in regions without glycosylation, but reactivity was not restricted to protein surface epitopes. One monoclonal epitope was relatively surface accessible and in close proximity to an N-linked glycosylation site, while three others required additional thermal energy to expose the epitopes. The monoclonal antibodies demonstrated the capacity to differentiate progressive structural changes in IDS and could be used to characterize the severity of MPS type II in patients based on variable denatured microstates.
Parkinson-Lawrence
Emma
E
Lysosomal Diseases Research Unit, Department of Genetic Medicine, Women's and Children's Hospital, 72 King William Rd, North Adelaide, South Australia 5006, Australia.
Turner
Christopher
C
Hopwood
John
J
Brooks
Doug
D
D017668:Age of Onset; D019943:Amino Acid Substitution; D000818:Animals; D000911:Antibodies, Monoclonal; D016466:CHO Cells / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism; D002460:Cell Line; D002490:Central Nervous System / Q000201:enzymology / Q000473:pathology; D002675:Child, Preschool; D006224:Cricetinae; D003412:Cricetulus; D042783:Endothelial Cells / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism; D018604:Epitope Mapping / Q000379:methods; D000939:Epitopes / Q000235:genetics / Q000276:immunology; D006358:Hot Temperature; D006801:Humans; D007066:Iduronate Sulfatase / Q000097:blood / Q000737:chemistry* / Q000235:genetics* / Q000276:immunology; D007962:Leukocytes / Q000737:chemistry / Q000378:metabolism; D008099:Liver / Q000166:cytology; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D016532:Mucopolysaccharidosis II / Q000097:blood / Q000201:enzymology / Q000235:genetics; D009154:Mutation / Q000235:genetics*; D010641:Phenotype; D011487:Protein Conformation; D011489:Protein Denaturation; D012756:Sheep
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007066:Iduronate Sulfatase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase