α-L-fucosidase in cultured skin fibroblasts from normal subjects and fucosidosis patients

Nicholas G. Beratis, Bryan M. Turner, Gundula Labadie, Kurt Hirschhorn

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Abstract

α-L-I'ucosidase (EC. 3.2.1.51) activity was studied in cul-tured skin nbroblasts obtained from 23 members of a family in which two cases of fucosidosis type 2 had occurred and in the Hbroblasts of a patient with fucosidosis type 1. Both the 4- metliylumbellifcryl glycoside and the p-nitrophenyl derivative were used as substrates. pH activity profiles showed two major peaks of activity. With the fluorogenic substrate pH optimum was at 4.5, whereas with the colorigenic substrate maximum activity was at pH 5.7. No activity was found in the nbroblasts of the three patients with the colorimetric assay. With the fluoronictric assay the mean activity in the two patients with fucosidosis type 2 was 4.1 and 2.8 (range 1.3-9.9); activity in the patient with fucosidosis type 1 was 4.6 nmol 4-inethylumbellifcr- one/mg protein/hr (range 1.0-8.6). The specific activity in the lysates of the patients' fibroblasts decreased as the amount of cellular protein used per assay increased. Fucosidosis fibroblasts cultured for 3 and 5 days in medium without fetal calf serum showed almost the same levels of apparent residual activity as fibroblasts cultured in medium containing fetal calf serum. Maximum activity in the deficient Fibroblasts was at pH 4.5-4.75. Mixing experiments between lysates of both types of fucosidosis and a normal fibroblast strain showed the expected enzymatic activities. The mean α-fucosidase activity in four heterozygotes for fu-cosidosis was 37.6 (range 24.1-48.7) and 30.3 (range 19.0- 44.1) nmol final product split/mg protein/hr with the fluorogenic and the colorigcnic substrate, respectively. In 12 normal fibro-blast strains the mean activity ± SD was 85.3 ± 24.4 (range 50.8-129.3) and 67.6 ± 21.f (range 31.1-118.3). However, in four family members in which the α-L-fucosidase phenotype (by isoelectric focusing) was type 2-1, and who should therefore be carrying two normal alleles, the activity was within the helerozygote range. This indicates that occasional overlap between normal subjects and carriers may be present in cultured skin fibroblasts. Increased specific activity of α-L-fucosidase at pH 3.2-4.0 was observed after incubation of cell lysates with neuraminidase. The α-L-fucosidase activity did not show any increase after fusion between fucosidosis fibroblasts types 1 and 2. Speculation: Although no biochemical evidence has been supplied indicating that the phenotypic differences between types 1 and 2 fucosi-dosis are genetically determined, it is possible that the two phenotypes are due to different mutations. The low residual activity found in the fibroblasts of both types of fucosidosis may represent either nonspecific hydrolysis of the fluorogenic substrate or genuine α-L-fucosidase activity. The use of natural substrates and immunologic studies may clarify this problem.

Original languageEnglish
Pages (from-to)862-866
Number of pages5
JournalPediatric Research
Volume11
Issue number7
DOIs
StatePublished - Jul 1977

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