Protective protein/cathepsin A rescues N-glycosylation defects in neuraminidase-1 |
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Authors: | Dongning Wang Slava Zaitsev Garry Taylor Alessandra d'Azzo Erik Bonten |
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Institution: | 1. Department of Genetics and Tumor Cell Biology, St. Jude Children''s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105-2794, USA;2. Centre for Biomolecular Sciences, University of St Andrews, St Andrews, Fife KY16 9UA, Scotland |
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Abstract: | BackgroundNeuraminidase-1 (NEU1) catabolizes the hydrolysis of sialic acids from sialo-glycoconjugates. NEU1 depends on its interaction with the protective protein/cathepsin A (PPCA) for lysosomal compartmentalization and catalytic activation. Murine NEU1 contains 4 N-glycosylation sites, 3 of which are conserved in the human enzyme. The expression of NEU1 gives rise to differentially glycosylated proteins.MethodsWe generated single-point mutations in mouse NEU1 at each of the 4 N-glycosylation sites. Mutant enzymes were expressed in NEU1-deficient cells in the presence and absence of PPCA.ResultsAll 4 N-glycosylation variants were targeted to the lysosomal/endosomal compartment. All N-glycans, with the exception of the most C-terminal glycan, were important for maintaining stability or catalytic activity. The loss of catalytic activity caused by the deletion of the second N-glycan was rescued by increasing PPCA expression. Similar results were obtained with a human NEU1 N-glycosylation mutant identified in a sialidosis patient. The N-terminal N-glycan of NEU1 is indispensable for its function, whereas the C-terminal N-glycan appears to be non-essential. The omission of the second N-glycan can be compensated for by upregulating the expression of PPCA.General significanceThese findings could be relevant for the design of target therapies for patients carrying specific NEU1 mutations. |
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Keywords: | D aspartic acid DMEM Dulbecco's modified Eagle's medium DTT dl-dithio-threitol FACS fluorescence activated cell sorting FBS fetal bovine serum GFP green fluorescent protein LSD lysosomal storage disease N asparagine NEU1 lysosomal neuraminidase-1 PCR polymerase chain reaction PPCA protective protein/cathepsin A YFP yellow fluorescent protein |
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