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1.
Glycobiology ; 20(3): 356-65, 2010 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-19917668

RESUMEN

Enzyme enhancement therapy, utilizing small molecules as pharmacological chaperones, is an attractive approach for the treatment of lysosomal storage diseases that are associated with protein misfolding. However, pharmacological chaperones are also inhibitors of their target enzyme. Thus, a major concern with this approach is that, despite enhancing protein folding within, and intracellular transport of the functional mutant enzyme out of the endoplasmic reticulum, the chaperone will continue to inhibit the enzyme in the lysosome, preventing substrate clearance. Here we demonstrate that the in vitro hydrolysis of a fluorescent derivative of lyso-GM2 ganglioside, like natural GM2 ganglioside, is specifically carried out by the beta-hexosaminidase A isozyme, requires the GM2 activator protein as a co-factor, increases when the derivative is incorporated into anionic liposomes and follows similar Michaelis-Menten kinetics. This substrate can also be used to differentiate between lysates from normal and GM2 activator-deficient cells. When added to the growth medium of cells, the substrate is internalized and primarily incorporated into lysosomes. Utilizing adult Tay-Sachs fibroblasts that have been pre-treated with the pharmacological chaperone Pyrimethamine and subsequently loaded with this substrate, we demonstrate an increase in both the levels of mutant beta-hexosaminidase A and substrate-hydrolysis as compared to mock-treated cells.


Asunto(s)
Técnica del Anticuerpo Fluorescente/métodos , Gangliósido G(M2)/análisis , Gangliósido G(M2)/metabolismo , Células Cultivadas , Gangliósido G(M2)/análogos & derivados , Humanos , Hidrólisis , Cinética , Liposomas/metabolismo , Espectrometría de Masas , Enfermedad de Tay-Sachs
2.
Anal Biochem ; 381(2): 276-8, 2008 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-18619939

RESUMEN

The low levels of human lysosomal glucocerebrosidase activity expressed in transiently transfected Chinese hamster ovary (CHO) cells were investigated. Reverse transcription PCR (RT-PCR) demonstrated that a significant portion of the transcribed RNA was misspliced owing to the presence of a cryptic splice site in the complementary DNA (cDNA). Missplicing results in the deletion of 179 bp of coding sequence and a premature stop codon. A repaired cDNA was constructed abolishing the splice site without changing the amino acid sequence. The level of glucocerebrosidase expression was increased sixfold. These data demonstrate that for maximum expression of any cDNA construct, the transcription products should be examined.


Asunto(s)
Empalme Alternativo , Glucosilceramidasa/genética , Sitios de Empalme de ARN , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células CHO , Cricetinae , Cricetulus , Análisis Mutacional de ADN , ADN Complementario , Enfermedad de Gaucher/etiología , Enfermedad de Gaucher/genética , Glucosilceramidasa/aislamiento & purificación , Glucosilceramidasa/metabolismo , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
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