Characterization of beta-galactosidase mutations Asp(332) -> Asn and Arg(148) -> Ser, and a polymorphism, Ser(532) -> Gly, in a case of G(M1) gangliosidosis

We have identified and characterized three missense mutations in a patient with type 1G(M1) gangliosidosis, namely a substitution of G for A at nucleo tide position 1044 (G1044 --> A; in exon 10) on one allele, which converts Asp(332) into asparagine, and both a mutation (C492 --> A in exon 4, leadin g to the amino acid change of Arg(148), Ser) ana a polymorphism (A1644 --> G in exon 15, leading to a change of Ser(532) --> Gly) on the other allele. This patient had less than 1% residual beta-galactosidase activity and min imally detectable levels of immunoreactive P-galactosidase protein in fibro blasts. To account for the above findings, a series of expression and immun olocalization studies were undertaken to assess the impact of each mutation . Transient overexpression in COS-1 cells of cDNAs encoding Asp(332)Asn, Ar g(148)Ser and Ser(532)Gly mutant beta-galactosidases produced abundant amou nts of precursor beta-galactosidase, with activities of 0, 84 and 81% compa red with the cDNA clone for wild-type beta-galactosidase (GP8). Since the l evel of vector-driven expression is much less in Chinese hamster ovary (CHO ) cells than in COS-I cells, and we knew that exogenous P-galactosidase und ergoes lysosomal processing when expressed in these cells, transient expres sion studies were performed of Arg(148)Ser and Ser(532)Gly, which yielded a ctive forms of the enzyme. In this case, the Arg(148)Ser and Ser(532)Gly pr oducts gave rise to 11% and 86% of the control activity respectively. These results were not unexpected, since the Arg(148)Ser mutation introduced a m ajor conformational change into the protein, and we anticipated that it wou ld be degraded in the endoplasmic reticulum (ER), whereas the polymorphism was expected to produce near-normal activity. To examine the effect of the Asp(332)Asn mutation on the catalytic activity, we isolated CHO clones perm anently transfected with the Asp(332)Asn and Asp(332)Glu constructs, purifi ed the enzymes by substrate-analogue-affinity chromatography, and determine d their kinetic parameters. The V-max values of both mutant recombinant enz ymes were markedly reduced (less than 0.9 % of the control), and the K-m va lues were unchanged compared with the corresponding wild-type enzyme isolat ed at the same time. Both the Arg(148)Ser beta-galactosidase in CHO cells a nd Asp(332)Asn beta-galactosidases (in COS-1 and CHO cells) produced abunda nt immunoreaction in the perinuclear area, consistent with localization in the ER. A low amount was detected in lysosomes. Incubation of patient fibro blasts in the presence ofleupeptin, which reduces the rate of degradation o f lysosomal beta-galactosidase by thiol proteases, had no effect on residua l enzyme activity, and immunostaining was again detected largely in the per inuclear area (localized to the ER) with much lower amounts in the lysosome s. In summary, the Arg(148)Ser mutation has no effect on catalytic activity , whereas the Asp(332)Asn mutation seriously reduces catalytic activity, su ggesting that Asp(332) might play a role in the active site. Immunofluoresc ence studies indicate the expressed mutant proteins with Arg(148)Ser and As p(332)Asn 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 cu ltured fibroblasts. Our results imply that most of the missense mutations described in G(M1) ga ngliosidosis to date have little effect on catalytic activity, but do affec t protein conformation such that the resulting protein cannot be transporte d 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 G(M1) gangliosi dosis patient fibroblasts.