Expression and functional characterization of mutated glucocerebrosidase alleles causing Gaucher disease in Spanish...

Gaucher disease (GD) is a heterogeneous disease characterized by an impaired activity of the lysosomal glucocerebrosidase. This heterogeneity is attributed in part to the existence of a large number of mutations in the corresponding gene.

To establish genotype-phenotype relationships, we analyzed the residual enzyme activities of six naturally occurring mutations found in Spanish population in the glucocerebrosidase gene [c.160G > A (V15M), c.485T>C (M123T), c.914C>T (P266L), c.1124T>C (L336P), c.1207A>C (S364R) and c.1510-1512delTCT (S465del)]. The mutated genes were subcloned into the mammalian expression vector pCR 3.1 and expressed by transient transfection in COS cells. The enzymatic activity of the expressed protein were measured and compared with the wild-type human glucocerebrosidase cDNA. Also, two previously alleles, c.1226A>G (N370S) and c.1448T>C (L444P), were used for comparative purposes.

The residual activity of the expressed proteins using the synthetic substrate (4-methylumbelliferyl-beta-D-glucopyranoside, 4MU-Glu) ranged from 5.5% (for the 3-bp deletion) to 42.7% (for S364R mutation) of the activity of the wild-type enzyme.

The present analyses may help to better understand the molecular basis and the pathogenesis of Gaucher disease. However, results of expression of mutated enzymes are necessary but not sufficient to explain the ultimate clinical outcome of Gaucher disease.