Gaucher disease: functional expression of the normal glucocerebrosidase and Gaucher T1366G and G1604A alleles in Baculovirus-transfected Spodoptera frugiperda cells.

Gaucher disease is an inherited sphingolipidosis resulting from deficient glucocerebrosidase activity. Three clinical forms of Gaucher disease have been described: type 1 as non-neuronopathic, type 2 as acute neuronopathic, and type 3 as subacute neuronopathic. We recently identified a rare mutation (G-->A at glucocerebrosidase cDNA nucleotide position 1604) [Choy et al., 1994a, Am J Med Genet 51:156-160] and a novel mutation (T-->G at glucocerebrosidase cDNA nucleotide position 1366) in two type 1 Gaucher patients by sequence analysis of the entire glucocerebrosidase coding region [Choy et al., 1994a, 1994b, Hum Mol Genet 3:821-823]. To demonstrate that these are deleterious and not neutral mutations, we cloned the full-length glucocerebrosidase cDNA of patients and of a normal control in the plasmid vector pAcUW1, recombined the human gene into the Baculovirus genome downstream of its polyhedron p10 promoter, and expressed the inserted gene in cultured cells of Spodoptera frugiperda transfected by recombinant Baculovirus. The levels of residual glucocerebrosidase activity determined in transfected cells with the Gaucher G1604A and T1366G alleles are 6.9% and 2.9% of that expressed by the normal allele (normal = 352.0 nmol/hr/mg protein or 100%). By comparison, the enzyme-specific activity expressed in transfected cells by 2 known Gaucher alleles, A1226G and T1448C, that are prevalent in type 1 and type 2 Gaucher disease are 23.4% and 3.3% of normal. No endogeneous glucocerebrosidase activity was detected in cultured cells transfected by either the wild-type Baculovirus or Baculovirus with the pAcUW1 plasmid vector without the glucocerebrosidase cDNA insert. These findings show that the Baculovirus expression system in cultured Spodoptera frugiperda cells is a suitable system for the functional expression and characterization of the normal and mutant glucocerebrosidase alleles. Moreover, the use of this expression system demonstrates that the G1604A and T1366G mutations are both deleterious mutations resulting in profoundly deficient glucocerebrosidase activity and subsequent Gaucher disease.