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Titolo:
Glycosidase active site mutations in human alpha-L-iduronidase
Autore:
Brooks, DA; Fabrega, S; Hein, LK; Parkinson, EJ; Durand, P; Yogalingam, G; Matte, U; Guigliani, R; Dasvarma, A; Eslahpazire, J; Henrissat, B; Mornon, JP; Hopwood, JJ; Lehn, P;
Indirizzi:
Womens & Childrens Hosp, Dept Chem Pathol, Lysosomal Dis Res Unit, Adelaide, SA 5006, Australia Womens & Childrens Hosp Adelaide SA Australia 5006 de, SA 5006, Australia Hop Robert Debre, INSERM U458, F-75019 Paris, France Hop Robert Debre Paris France F-75019 INSERM U458, F-75019 Paris, France Univ Paris 06, CNRS UMR 7590, Lab Mineral Cristallog, F-75252 Paris, France Univ Paris 06 Paris France F-75252 ral Cristallog, F-75252 Paris, France Univ Aix Marseille 1, CNRS UMR 6098, F-13402 Marseille 20, France Univ AixMarseille 1 Marseille France 20 8, F-13402 Marseille 20, France Univ Aix Marseille 2, CNRS UMR 6098, F-13402 Marseille 20, France Univ AixMarseille 2 Marseille France 20 8, F-13402 Marseille 20, France
Titolo Testata:
GLYCOBIOLOGY
fascicolo: 9, volume: 11, anno: 2001,
pagine: 741 - 750
SICI:
0959-6658(200109)11:9<741:GASMIH>2.0.ZU;2-I
Fonte:
ISI
Lingua:
ENG
Soggetto:
MUCOPOLYSACCHARIDOSIS-TYPE-I; SEQUENCE-BASED CLASSIFICATION; HYDROPHOBIC CLUSTER-ANALYSIS; GLYCOSYL HYDROLASES; SCHEIE SYNDROMES; ACID-HYDROLASES; HURLER SYNDROME; CDNA ISOLATION; PROTEIN; IDENTIFICATION;
Keywords:
active site residues; catalytic machinery; alpha-L-iduronidase; Hurler syndrome; mucopolysaccharidosis I;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
42
Recensione:
Indirizzi per estratti:
Indirizzo: Brooks, DA Womens & Childrens Hosp, Dept Chem Pathol, Lysosomal Dis Res Unit, King William Rd, Adelaide, SA 5006, Australia Womens & Childrens Hosp King William Rd Adelaide SA Australia 5006
Citazione:
D.A. Brooks et al., "Glycosidase active site mutations in human alpha-L-iduronidase", GLYCOBIOLOG, 11(9), 2001, pp. 741-750

Abstract

Mucopolysaccharidosis type I (NIPS I; McKusick 25280) results from a deficiency in alpha -L-iduronidase activity. Using a bioinformatics approach, wehave 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 a-L-iduronidase. The E299A mutantprotein 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 removalof key catalytic residues. In addition, we have identified a NIPS 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 E182A and E299 in the catalytic mechanism of a-L-iduronidase and we propose that the mutation of either of these residues would contribute to a very severe clinical phenotype in a NIPS I patient.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 22/01/20 alle ore 18:40:03