Effect of elongation factor 1alpha promoter and SUMF1 over in vitro expression of N-acetylgalactosamine-6-sulfate sulfatase.
Morquio A is an autosomal recessive disease caused by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS), leading to the lysosomal accumulation of keratan-sulfate and chondroitin-6-sulfate. We evaluated in HEK293 cells the effect of the cytomegalovirus immediate early enhancer/promoter (CMV) or the elongation factor 1alpha (EF1alpha) promoters, and the coexpression with the sulfatase modifying factor 1 (SUMF1) on GALNS activity. Four days postransfection GALNS activity in transfected cells with CMV-pIRES-GALNS reached a plateau, whereas in cells transfected with EF1alpha-pIRES-GALNS continued to increase until day 8. Co-transfection with pCXN-SUMF1 showed an increment up to 2.6-fold in GALNS activity. Finally, computational analysis of transcription factor binding-sites and CpG islands showed that EF1alpha promoter has long CpG islands and high-density binding-sites for Sp1 compared to CMV. These results show the advantage of the SUMF1 coexpression on GALNS activity and indicate a considerable effect on the expression stability using EF1alpha promoter compared to CMV.
Alméciga-Díaz
Carlos J
CJ
Instituto de Errores Innatos del Metabolismo, Pontificia Universidad Javeriana, Bogota, D.C., Colombia.
Rueda-Paramo
Maria A
MA
Espejo
Angela J
AJ
Echeverri
Olga Y
OY
Montaño
Adriana
A
Tomatsu
Shunji
S
Barrera
Luis A
LA
D001665:Binding Sites; D002460:Cell Line; D002811:Chondroitinsulfatases / Q000235:genetics / Q000378:metabolism*; D019295:Computational Biology; D018899:CpG Islands / Q000235:genetics; D003587:Cytomegalovirus / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D050862:Oxidoreductases Acting on Sulfur Group Donors; D020648:Peptide Elongation Factor 1 / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D016329:Sp1 Transcription Factor / Q000378:metabolism; D013429:Sulfatases / Q000378:metabolism*; D014162:Transfection
D020648:Peptide Elongation Factor 1; D016329:Sp1 Transcription Factor; D050862:Oxidoreductases Acting on Sulfur Group Donors; D002811:Chondroitinsulfatases; C480483:SUMF1 protein, human; D013429:Sulfatases; C102295:GALNS protein, human