Characterization of β-galactosidase mutations Asp332→Asn and Arg148→Ser, and a polymorphism, Ser532→Gly, in a case of GM1 gangliosidosis

We have identified and characterized three missense mutations in a patient with type 1 GM1 gangliosidosis, namely a substitution of G for A at nucleotide position 1044 (G1044 → A; in exon 10) on one allele, which converts Asp332 into asparagine, and both a mutation (C492 → A in exon 4, leading to the amino acid change of Arg148 → Ser) and a polymorphism (A1644 → G in exon 15, leading to a change of Ser532 → Gly) on the other allele. This patient had less than 1% residual β-galactosidase activity and minimally detectable levels of immunoreactive β-galactosidase protein in fibroblasts. To account for the above findings, a series of expression and immunolocalization studies were undertaken to assess the impact of each mutation. Transient overexpression in COS-1 cells of cDNAs encoding Asp332Asn, Arg148Ser and Ser532Gly mutant β-galactosidases produced abundant amounts of precursor β-galactosidase, with activities of 0, 84 and 81% compared with the cDNA clone for wild-type β-galactosidase (GP8). Since the level of vector-driven expression is much less in Chinese hamster ovary (CHO) cells than in COS-1 cells, and we knew that exogenous β-galactosidase undergoes lysosomal processing when expressed in these cells, transient expression studies were performed of Arg148Ser and Ser532Gly, which yielded active forms of the enzyme. In this case, the Arg148Ser and Ser532Gly products gave rise to 11% and 86% of the control activity respectively. These results were not unexpected, since the Arg148Ser mutation introduced a major conformational change into the protein, and we anticipated that it would be degraded in the endoplasmic reticulum (ER), whereas the polymorphism was expected to produce near-normal activity. To examine the effect of the Asp332Asn mutation on the catalytic activity, we isolated CHO clones permanently transfected with the Asp332Asn and Asp332Glu constructs, purified the enzymes by substrate-analogue-affinity chromatography, and determined their kinetic parameters. The V max values of both mutant recombinant enzymes were markedly reduced (less than 0.9% of the control), and the K m values were unchanged compared with the corresponding wild-type enzyme isolated at the same time. Both the Arg148Ser β-galactosidase in CHO cells and Asp332Asn β-galactosidases (in COS-1 and CHO cells) produced abundant immunoreaction in the perinuclear area, consistent with localization in the ER. A low amount was detected in lysosomes. Incubation of patient fibroblasts in the presence of leupeptin, which reduces the rate of degradation of lysosomal β-galactosidase by thiol proteases, had no effect on residual enzyme activity, and immunostaining was again detected largely in the perinuclear area (localized to the ER) with much lower amounts in the lysosomes. In summary, the Arg148Ser mutation has no effect on catalytic activity, whereas the Asp332Asn mutation seriously reduces catalytic activity, suggesting that Asp332 might play a role in the active site. Immunofluorescence studies indicate the expressed mutant proteins with Arg148Ser and Asp332Asn mutations are held up in the ER, where they are probably degraded, resulting in only minimum amounts of the enzyme becoming localized in the lysosomes. These results are completely consistent with findings in the cultured fibroblasts. Our results imply that most of the missense mutations described in GM1 gangliosidosis to date have little effect on catalytic activity, but do affect protein conformation such that the resulting protein cannot be transported out of the ER and fails to arrive in the lysosome. This accounts for the minimal amounts of enzyme protein and activity seen in most GM1 gangliosidosis patient fibroblasts.