In vivo and in vitro function of human UDP-galactose 4'-epimerase variants

Autor: Thomas J. McCorvie, Jamie Wasilenko, Ying Liu, Judith L. Fridovich-Keil, David J. Timson
Rok vydání: 2011
Předmět:
Galactosemias
ANS
1-anilinonaphthalene-8-sulphonic acid
UDP-galactose 4′-epimerase
Saccharomyces cerevisiae
Blotting
Western
Endogeny
Type III galactosemia
GALE
UDP-galactose 4′-epimerase
Biology
medicine.disease_cause
Biochemistry
03 medical and health sciences
chemistry.chemical_compound
UDPglucose 4-Epimerase
0302 clinical medicine
In vivo
Enzyme Stability
medicine
gal-1P
galactose 1-phosphate
Humans
hGALE
human GALE
Disease-associated mutation
Escherichia coli
Alleles
030304 developmental biology
Yeast model
chemistry.chemical_classification
0303 health sciences
GuHCl
guanidine hydrochloride
Galactosemia
RBC
red blood cells
General Medicine
medicine.disease
biology.organism_classification
In vitro
GALE
Enzyme
chemistry
030220 oncology & carcinogenesis
Galactose
MeOH
methanol
Research Paper
Zdroj: Biochimie
ISSN: 1638-6183
Popis: Type III galactosemia results from reduced activity of the enzyme UDP-galactose 4′-epimerase. Five disease-associated alleles (G90E, V94M, D103G, N34S and L183P) and three artificial alleles (Y105C, N268D, and M284K) were tested for their ability to alleviate galactose-induced growth arrest in a Saccharomyces cerevisiae strain which lacks endogenous UDP-galactose 4′-epimerase. For all of these alleles, except M284K, the ability to alleviate galactose sensitivity was correlated with the UDP-galactose 4′-epimerase activity detected in cell extracts. The M284K allele, however, was able to substantially alleviate galactose sensitivity, but demonstrated near-zero activity in cell extracts. Recombinant expression of the corresponding protein in Escherichia coli resulted in a protein with reduced enzymatic activity and reduced stability towards denaturants in vitro. This lack of stability may result from the introduction of an unpaired positive charge into a bundle of three α-helices near the surface of the protein. The disparities between the in vivo and in vitro data for M284K-hGALE further suggest that there are additional, stabilising factors present in the cell. Taken together, these results reinforce the need for care in the interpretation of in vitro, enzymatic diagnostic tests for type III galactosemia.
Highlights ► GALE activity and ability to compensate a yeast deletion are correlated. ► The correlation does not hold for an artificial allele, M284K. ► M284K was able to compensate in vivo, but had near zero activity in cell extracts. ► M284K exhibits reduced Vmax and increased Km compared to wild type. ► M284K is highly unstable and probably largely unfolded in dilute solution.
Databáze: OpenAIRE
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