Document Detail


Glycosidase active site mutations in human alpha-L-iduronidase.
MedLine Citation:
PMID:  11555618     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Mucopolysaccharidosis type I (MPS I; McKusick 25280) results from a deficiency in alpha-L-iduronidase activity. Using a bioinformatics approach, we have previously predicted the putative acid/base catalyst and nucleophile residues in the active site of this human lysosomal glycosidase to be Glu182 and Glu299, respectively. To obtain experimental evidence supporting these predictions, wild-type alpha-L-iduronidase and site-directed mutants E182A and E299A were individually expressed in Chinese hamster ovary-K1 cell lines. We have compared the synthesis, processing, and catalytic properties of the two mutant proteins with wild-type human alpha-L-iduronidase. Both E182A and E299A transfected cells produced catalytically inactive human alpha-L-iduronidase protein at levels comparable to the wild-type control. The E182A protein was synthesized, processed, targeted to the lysosome, and secreted in a similar fashion to wild-type alpha-L-iduronidase. The E299A mutant protein was also synthesized and secreted similarly to the wild-type enzyme, but there were alterations in its rate of traffic and proteolytic processing. These data indicate that the enzymatic inactivity of the E182A and E299A mutants is not due to problems of synthesis/folding, but to the removal of key catalytic residues. In addition, we have identified a MPS I patient with an E182K mutant allele. The E182K mutant protein was expressed in CHO-K1 cells and also found to be enzymatically inactive. Together, these results support the predicted role of E182 and E299 in the catalytic mechanism of alpha-L-iduronidase and we propose that the mutation of either of these residues would contribute to a very severe clinical phenotype in a MPS I patient.
Authors:
D A Brooks; S Fabrega; L K Hein; E J Parkinson; P Durand; G Yogalingam; U Matte; R Giugliani; A Dasvarma; J Eslahpazire; B Henrissat; J P Mornon; J J Hopwood; P Lehn
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Glycobiology     Volume:  11     ISSN:  0959-6658     ISO Abbreviation:  Glycobiology     Publication Date:  2001 Sep 
Date Detail:
Created Date:  2001-09-13     Completed Date:  2001-12-07     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  9104124     Medline TA:  Glycobiology     Country:  England    
Other Details:
Languages:  eng     Pagination:  741-50     Citation Subset:  IM    
Affiliation:
Lysosomal Diseases Research Unit, Department of Chemical Pathology, Women's and Children's Hospital, King William Road, North Adelaide, SA 5006, Australia.
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MeSH Terms
Descriptor/Qualifier:
Amino Acid Sequence
Animals
Antibodies, Monoclonal / chemistry,  immunology
Base Sequence
Binding Sites
Blotting, Western
CHO Cells
Cricetinae
DNA Primers
Epitope Mapping
Glycoside Hydrolases / genetics,  immunology,  metabolism*
Humans
Iduronidase / genetics,  immunology,  metabolism*
Mucopolysaccharidosis I / enzymology*
Mutation*
Recombinant Proteins / genetics,  immunology,  metabolism
Subcellular Fractions / enzymology
Chemical
Reg. No./Substance:
0/Antibodies, Monoclonal; 0/DNA Primers; 0/Recombinant Proteins; EC 3.2.1.-/Glycoside Hydrolases; EC 3.2.1.76/Iduronidase

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