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Technical Briefs |
1
Department of Clinical Chemistry, Päijät-Häme Central Hospital, 15850 Lahti, Finland, and
2
Department of Clinical Chemistry, Kuopio University Hospital, P.O.Box 1777, 70211 Kuopio, Finland;
a author for correspondence: fax 358-17-173186, e-mail ilkka.mononen{at}uku.fi
Glycosylasparaginase (GA; EC 3.5.1.26) is a lysosomal enzyme
that cleaves the N-glycosidic bond between asparagine and
N-acetylglucosamine residues in the degradation of
glycoproteins (1). Deficient enzyme activity leads to the
lysosomal storage disease aspartylglycosaminuria (McKusick 208400).
Human GA is synthesized as a single, enzymatically inactive precursor
polypeptide. The single-chain precursor is thought to be activated on
its way to lysosomes (2)(3) by cleavage into two
N-glycosylated subunits of 24 kDA (
) and 18 kDA (ß) and
association of these subunits to form enzymatically active heterodimer
or heterotetramer structures (4). GA is actively transported
into human lysosomes via mannose-6-phosphate receptor-mediated
endocytosis (5).
I-Cell disease (mucolipidosis II, McKusick 252500) and a clinically milder, form pseudo-Hurler polydystrophy (mucolipidosis III, McKusick 252600), are autosomal, recessively inherited lysosomal storage diseases in which the transport of newly synthesized lysosomal enzymes into lysosomes is affected (6). The defect is in the enzyme, UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (EC 2.7.8.17), which adds the phosphate trafficking signal to lysosomal enzymes. The activity of phosphotransferase can be measured using radioactive UDP-N-acetylglucosamine as the donor substrate and endogenous lysosomal enzymes or artificial carbohydrate structures as the assay acceptors (7). The defective synthesis of mannose-6-phosphate recognition markers on the carbohydrate moieties of lysosomal enzymes leads to the leakage of newly synthesized enzymes out of cells instead of their transport into the lysosomes through mannose-6-phosphate receptor-mediated endocytosis (8). Almost normal activity of certain lysosomal enzymes such as ß-glucocerebrosidase (EC 3.2.1.45) and lysosomal acid phosphatase (EC 3.1.3.2) remains in fibroblasts from patients with I-cell disease and pseudo-Hurler polydystrophy (9). These enzymes continue to be transported to lysosomes as transmembrane proteins because this process does not involve mannose-6-phosphate receptors (10)(11). We now report our findings on GA activity in serum, urine, and fibroblasts from I-cell disease patients, which suggest that the measurement of GA activity can be used as a marker in the detection of I-cell disease.
The fibroblast cell lines were from two different children, both with phenotypes typical of I-cell disease (8). The cultured skin fibroblasts contained pleomorphic material in enlarged lysosomes and very low ß-galactosidase (EC 3.2.1.23) and arylsulfatase (EC 3.1.6.1) activities. Highly increased ß-galactosidase activity was detected in the serum of both patients. The GA activity was determined fluorometrically using L-aspartic acid ß-(7-amino-4-methylcoumarin), from Bachem AG, as substrate (12). The GA activity in cultured fibroblasts and serum was assayed as described previously (12)(13). The GA activity in urine was determined by incubating 100 µL of sample with 20 µL of 10 mmol/L (in ethylene glycol) L-aspartic acid ß-(7-amino-4-methylcoumarin) and 80 µL of Tris-HCl buffer (50 mmol/L, pH 7.5) for 2–3 h at 37 °C.
For ß-galactosidase or arylsulfatase activity assays, harvested fibroblasts were resuspended in 1.0 mL of distilled water and sonicated for 1 min in ice. The fibroblast homogenate or serum was used to determine the activity of ß-galactosidase and arylsulfatase, using 4-methylumbelliferyl ß-D-galactopyranoside (9) and 4-methylumbelliferyl sulfate (14), respectively, as substrates (Sigma Chemical Co.). The protein concentration was determined with a protein assay kit (Protein Assay kit; Bio-Rad).
In fibroblasts from the patients with I-cell disease, the GA activity
was 8–11%, the ß-galactosidase activity was 0.1–4%, and the
arylsulfatase activity was 5–12% of the mean activity of the
corresponding enzymes in control fibroblasts (Table 1
A). The GA activity in serum from the patients with I-cell
disease was 9- to 12-fold higher than the mean value in controls. The
serum activity of ß-galactosidase in the patients with I-cell disease
was 14- to 17-fold higher than in healthy controls (Table 1B
). The
activity of GA in urine of the patients with I-cell disease was 9- to
17-fold higher than the mean activity in control urine (Table 1C
).
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The highly increased GA activity in serum and urine from patients with I-cell disease shows that active GA is truly formed in this disease. Considerable residual GA activity in I-cell disease fibroblasts compared with that in GA-deficient aspartylglycosaminuria fibroblasts (12) indicates that the autoproteolytic cleavage of the precursor polypeptide of the enzyme into subunits and association of at least one of each subunit to form the active enzyme in humans (4)(15) takes place inside the cells before leakage into extracellular fluids. The activation thus occurs before transfer of the enzyme into lysosomes, probably in the endoplasmic reticulum, as predicted experimentally (3).
The GA activity in I-cell fibroblasts was <11% of that in control cells, and its activity in serum from patients with I-cell disease was at least ninefold higher than in control serum. The magnitude of the changes in GA activity resembles those reported previously for ß-galactosidase and arylsulfatase in patients with I-cell disease (8) and clearly differs from those reported for the membrane-bound enzymes ß-glucocerebrosidase and lysosomal acid phosphatase, which continue to be transported to lysosomes in the disease (6)(9). Both ß-galactosidase and arylsulfatase are targeted into lysosomes predominantly via mannose-6-phosphate receptor-mediated endocytosis (8), which the available evidence suggests is the major transport route of GA into lysosomes in humans as well. This is also supported by in vitro experiments with purified recombinant human GA, in which the enzyme was actively transported from the culture medium into lysosomes of GA-deficient fibroblasts. Enzymatic removal of either the phosphate residues or the carbohydrate chains, however, abolished the uptake of the enzyme into the cells (5). The major increase in GA activity in serum and urine and the markedly reduced activity in cultured fibroblasts can be used as a diagnostic aid for detecting I-cell disease. These findings also suggest that newborn babies affected by I-cell disease could be detected on the basis of high GA values in neonatal screening of aspartylglycosaminuria (16). However, this assay, like others for increased lysosomal enzymes in serum, cannot distinguish between mucolipidosis II and mucolipidosis III, which usually must be differentiated on the basis of events seen in clinical progression.
Acknowledgments
This work was supported in part by grants from the Sigrid Juselius Foundation and the Ulla Hjelt Memorial Fund awarded by the Foundation for Pediatric Research (I.M.). We thank Aili Sormunen for technical assistance.
References
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