Clinical Genetics 1979: 16: 357-363
Human amniotic fluid
a-L-fucosidase
JACK A. ALHAIYEFF AND GRAIL. ANDREWS-Sm
Department of Neurosciences, School of Medicine, University of California, San Diego, La Jolla, California, U S A . The activity and properties of a-L-fucosidase in 24 samples of amniotic fluid have been investigated using the 4-methylumbelliferyl substrate. A wide range of fucosidase specific activity (0.20-1.45 nmoVmin/mg protein) was found with an average value of 0.62 nmoV min/mg protein. Although no clear-cut correlations exist, there appear to be trends of decreasing fucosidase specific activity with increasing maternal age of donor and increasing gestational time. a-L-Fucosidase activity from four amniotic fluids was characterized kinetically and immunochemically and found not to differ in its properties due to maternal age of donor or gestational time. Isoelectric focusing revealed multiple forms between pH 5.0 and 6.8, with the majority of activity present between pH 5.0 and 6.0. The pH optimum is near pH 5.0 with a second optimum suggested at pH 6.2. The apparent Michaelis constant (Km) for the 4-methylumbelliferyl substrate is 0.05 k 0.01 mM. The enzyme is completely thermostable at 37°C for at least 4 h but loses 95 % of its activity after preincubation at 45°C for 1 h. Double immuncdiffusion and immunoprecipitation experiments using anti-liver a-L-fucosidase antibody suggest that the amniotic fluid a-L-fucosidase is antigenically similar, if not identical, to the human liver enzyme. Received 17 April, accepted f o r publication I1 June 1979 Key words: Amniotic fluid; a-L-fucosidase; fucosidosis; prenatal diagnosis.
Fucosidosis is a rare neurovisceral storage disease characterized by progressive mental and motor retardation, decorticate rigidity, emaciation, bony deformities, progressive spasticity and respiratory difficulties (Van Hoof & Hers 1968, Durand et al. 1969). Biochemically this genetic disease involves a severe deficiency of activity of the lysosoma1 hydrolase a-L-fucosidase (fucosidefucohydrolase EC 3.2.1.51) coupled with accumulation of fucose-containing glycoconjugates (Loeb et al. 1969, Van Hoof 1973, Tsay & Dawson 1976). The absence of fucosidase activity in cultured skin fibroblasts in fucosidosis patients (Zielke et al. 1972) and the presence of fucosidase activity in amniotic fluid and
cells (Wiederschain e t al. 1971) indicate that this disorder should be diagnosable prenatally by assay of amniotic fluid and/or cultured amniotic fluid cells (Matsuda et al. 1975, Poenaru et al. 1976). Little is known about the activity and properties of a-Lfucosidase in amniotic fluid and cultured amniotic cells. Wiederschain et al. (1971) have shown that fucosidase activity is present in amniotic fluid at various stages of pregnancy (20-30 weeks). Since the forms of a-L-fucosidase have been shown to change during fetal development (Alhadeff et al. 1975b), further baseline studies on amniotic fluid and cultured amniotic fluid cells are necessary for development of an accurate prenatal diagnostic test for fucosi-
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ALHADEFF AND ANDREWS-SMITH
dosis. In the present study we have investigated the activity and properties of a-Lfucosidase in 24 amniotic fluids obtained between 14 and 22 weeks of gestation. Material and Methods
General All procedures were carried out at 0-4°C unless otherwise stated. a-L-Fucosidase activity was assayed using 4-methylumbelliferyl-a-L-fucopyranoside (Koch-Light, Colnbrook, Buckinghamshire, U.K.), as previcusly described (Alhadeff & O’Brien 1977). Enzyme assays were performed under conditions where enzyme activity was linear with respect to time (20-60 min) and amount of protein (20-40 p1 amniotic fluid). A unit of fucosidase activity is defined as the amount of enzyme that hydrolyses 1 nmol of substrate per min at 37°C. Protein was determined by the Lowry method (Lowry et al. 1951) using human serum albumin as standard. Agarose-E-aminocaproylfucosamine was purchased from MilesYeda Ltd. (Elkhart, IN, USA). The procedures which involved human tissues were approved by the Committee on Jnvestigations/Activities Involving Human Subjects of the School of Medicine, University of California, San Diego. Preparation of Amniotic Fluid Amniotic fluid was prepared from the material (10-30 ml) obtained on transabdominal amniocentesis by centrifuging at 2,500 X g for 10 min (Young et al. 1976). The supernatants (which were free of cellular material) were assayed for a-L-fucosidase activity, for amount of protein, and were stored frozen (-20°C) until used. Freezing did not alter the kinetic and immunochemical properties of a-L-fucosidase. The gestational age of the fetus at the time of amniocentesis was determined by ultrasonography.
Purification o f Amniotic Fluid a-L-Fucosidase a-L-Fucosidase was purified from 225 ml of combined amniotic fluid samples by affinity chromatography on agarose-c-aminocaproylfucosamine, as previously described (Alhadeff et al. 2975a). This partially purified a-L-fucosidase was used in the double immunodiffusion experiments (see below). Isoelectric Focusing Isoelectric focusing was performed on four samples of amniotic fluid a-L-fucosidase (1.0 ml) using a 40 ml column, as previously described (Alhadeff & Andrews-Smith 1979). One percent ampholytes (pH 5-8) from LKB Produkter (Stockholm, Swedeill were used in a gradient of 0-67 % (w/v) sucrose. Electrofocusing was conducted for 17-20 h at a starting amperage of 2.5 mA and 600 V. Kinetic Studies Apparent Michaelis constants (Km) of four preparations of amniotic fluid a-L-fucosidase for the 4-methylumbelliferyl substrate were determined graphically by the Lineweaver-Burk method (Lineweaver & Burk 1934). For these studies, enzyme sample (20 pl) was incubated in duplicate for 30 min at 37°C in substrates of varying concentration in 100 mM citric acid-sodium citrate buffer (pH 5.0). The pH optima profiles were determined for four preparations of amniotic fluid a-L-fucosidase in 100 mM buffers of varying p H values (citric acid-sodium citrate for p H values 3.1 to 5.8; KH2P04-NapHP04 for pH values 6.0 to 8.0). Incubations were carried out on 30 pl aliquots of amniotic fluid in duplicate for 30 min at 37°C. Thermal stability studies were performed on three preparations of amniotic fluid a-L-fucosidase by preincubation of 40 PI aliquots of amniotic fluid at 37°C or 45°C for up to 4 h. After preincubation, substrate was added and the
HUMAN AMNIOTIC FLUID a-L-FUCOSIDASE
samples were assayed in duplicate for 30 min at 37°C. Inimunochemicat Studies The IgG fraction of antibodies made against purified human liver a-L-fucosidase in a previous study (Alhadeff et al. 1975a) was used for the immunochemical studies. Amniotic fluid a-L-fucosidase and human liver a-L-fucosidase (diluted to contain an amount of activity comparable to that in the amniotic fluid) were separately preincubated with 0-70 p1 of anti-liver a-Lfucosidase IgG in 400 pl conical plastic centrifuge tubes (Cole Scientific, Calabasas, CA, USA) for 20 h at 2 ' 4 ° C . The preincubated samples were centrifuged at 48,000 X g for 30 min and the resulting supernatants were assayed in duplicate for a-L-fucosidase activity. The IgG fraction (8 pl) was also used in double immunodiffusion experiments on Hyland immunoplates (Costa Mesa CA, USA) against both purified liver a-L-fucosidase (15-244 units) and the amniotic fluid a-L-iucosidase (9 units) which had been purified by affinity chromatography. The plates were spotted with samples, placed at 0-4°C and read after 2-3 days.
359
Table 1 Amniotic fluid a-L-fucosidase activity
Patient
Maternal age 'years)
1 2 3 4 5 6 7 8 9 10
34.5 35 39 34 42 35 40 36 43 35 42
11
?
12 13 14 15 16 17 18 19
39 29 37 35 34 21 38 25 34.5
20
21 22 23 24
?
Range Average
u-L-Fucosidase Time Of activity amniocentesis (weeks of units/mg gestation)' units/ml protein
19.4 15 19.5 16.5 17.5 19.0 22.0 17.0 18.0 14 18 ? 17.5
0.90
3.50 1.75 2.00 1.25 3.35 3.30 1.35 2.63 3.27 2.30 2.25 4.40 1.40 3.85 2.50
?
16 20 16 17 19 16.5 ? ?
35 40
18.5 15.5
21-43
14-22
35.7
17.6
0.45 0.49 0.29 1.36 0.20 0.60 0.39 0.48 0.26 1.45 0.61 0.56 0.28 0.60 0.82 0.41 0.51 1.11 0.50 0.63 0.93 0.48 0.78 0.72
2.00 1.40 1.25 3.10 1.05 2.65 2.30 2.05
0.9&4.40 0.20-1.45 2.33
0.62
* Determined by ultrasonography.
Results
Table 1 summarizes the a-L-fucosidase activity (unitdml), specific activity (unitdmg protein), the time of amniocentesis in weeks gestation and the age of the 24 women who provided samples for this study. A wide range of fucosidase specific activity (0.201.45 units/mg protein) was found for these amniotic fluids, with an average value of 0.62 units/mg protein. When the amniotic fluid a-L-fucosidase specific activities are plotted versus either maternal age (Fig. 1) or gestation time (Fig. 2), no clear-cut correlations are seen. However, for both plots, amniotic fluid a-L-fucosidase specific activi-
..
"i. 12
-* 21
23 25
27 29
31
33
..
35 37 39 41 43
MATERNAL AGE (YEARS)
Fig. 1. Amniotic fluid 0-L-fucosidase specific activity vs. maternal age of donor.
360
ALHADEFF AND ANDREWS-SMITH
'7-
.
1.4{
14
48
15
16
17
18
19
20
21
r
22
TIME OF AMNIOCENTESIS (WEEKS GESTATION) Flg. 2. Amniotic fluid a-L-fucosidase specific activity vs. time of amniocentesis (weeks gestation).
ty appears to decrease with increasing maternal age and increasing gestational times. These trends should be interpreted with caution, since a considerable amount of variabilitv in fucosidase activitv can be seen for one maternal age (e.g., 35 years) or one gestational time (e.g., 16.5 weeks). Isoelectric focusing of amniotic fluid a-Lfucosidase revealed the pattern depicted in Figure 3. Multiple forms are found with isoelectric points (PI'S) between p H 5.0 and 6.8, with the majority of activity present between pH 5.0 and 6.0. No major differences were seen in the a-L-fucosidase isoelectric profiles of the four amniotic fluids which were focused.
-
6.0
7.0
8.0
PH
Michaelis constants of a-L-fucosidase for the 4-methylumbelliferyl substrate were determined on four amniotic fluids. The Lineweaver-Burk double reciprocal plots yielded straight lines with similar apparent Km's (ranging from 0.04-0.06 mM) with
2
1
5.0
4.0
Fig. 4. pH Optimum curve of amniotic fluid a-l-fucosidase activity. (See Materials and Methods section for details.)
t
30 r a0 -
40 tI
t; a
100
--.-
=
=
-3
7 d
-
J
70 -
-
:I 60
i - "v\
30
2o
I
t
30
40
In
'\ 60
50
70
80
PH Flg. 3. lsoelectric focusing (pH 5-8) of amniotic fluid a-L-fucosidase activity. (See Materials and Methods section for details.)
arr PREINCUBATION TIME (HAS) Fig. 5. Thermostability curves of amniotic fluid a-Lfucosidase activity at 37" and 45°C. (See Materials and Methods section for details.)
HUMAN AMNIOTIC FLUID a-L-FUCOSIDASE
361
Figure 6 indicates that the antibody against liver a-L-fucosidase crossreacts with the purified amniotic fluid a-L-fucosidase (wells A and B) to give a single precipitin line coincident with that from liver (wells C and D). When the same amounts of liver and amniotic fluid a-L-fucosidase activity were preincubated separately with the IgG fraction of liver antiserum, comparable amounts of activity were immunoprecipitated . Flg. 6. Double immunodiffusion (Ouchterlony) plate of anti-liver a-L-fucosidase antibody (center well) against purified amniotic fluid a-L-fucosidase (wells A and B) and purified human liver a-L-fucosidase (wells C and D). (See Materials and Methods section for details.)
an average of 0.05 +. 0.01 mM. Figure 4 depicts the p H optimum curve for a-L-fucosidase, which was similar for the four amniotic fluids investigated. The optimum is near pH 5.0, with a second optimum suggested at pH 6.2. The biphasic nature of the pH optimum curve does not appear to be an artifact of using two buffer systems (citrate and phosphate), since the ascent of the second optimum was seen when the citrate buffer was employed beyond pH 53-6.1. Amniotic fluid a-L-fucosidase activity is completely thermostable at 37°C with no loss of activity after preincubation for 4 h (Fig. 5). However, at 45°C the activity is very thermolabile with 70 % and 95 % losses of activity after 30 and 60 min preincubations, respectively. The thermostability curves depicted in Figure 5 for both 37" and 45°C were typical of the three amniotic fluid a-L-fucosidases investigated. The IgG fraction of antiserum made against pure liver a-L-fucosidase was used in double immunodiffusion experiments against both the purified liver enzyme and the purified amniotic fluid a-L-fucosidase.
Discussion
There are currently two reports in the literature of attempted prenatal diagnoses for families at risk for fucosidosis. In one case the fetus was diagnosed correctly as being free from the disease by assay of amniotic fluid and amniotic fluid cell fucosidase activity (Poenaru et al. 1976). In the other case, amniotic fluid obtained between 14 and 18 weeks of gestation was found t o contain 10 % of control fucosidase activity. Assay of cultured amniotic fluid cells indicated the presence of about 30 % of normal fucosidase activity. On the basis of these assays the fetus was diagnosed as having a carrier state of the disease. The diagnosis was shown to be incorrect after delivery when a-L-fucosidase activity was absent in white blood cells from the identical twins (Matsuda et al. 1975). This report indicates that great care must be taken in the prenatal diagnosis of fucosidosis and suggests that further studies on amniotic fluid cell a-Lfucosidase are warranted. In the present investigation we have done baseline studies on the activity and properties of a-L-fucosidase in amniotic fluid. Three other laboratories have provided limited data on the activity of a-L-fucosidase in a few samples of control amniotic fluid (Wiederschain et al. 1971, Matsuda et al. 1975, Poenaru et al. 1976), but n o characterization studies of amniotic fluid a-L-
362
ALHADEFF AND ANDREWS-SMITH
fucosidase have been published. We have found similar or increased fucosidase activity (when compared to previous studies) in the 24 amniotic fluids studied. Although no strong correlations exist, there appear to be trends of decreasing fucosidase specific activity with increasing maternal age and increasing gestational time. However, the isoelectric profiles and kinetic properties (pH curves, Km’s, thermostability curves) were similar for the three to four amniotic fluid a-L-fucosidases characterized, despite varying maternal age of donor (35-43 years) and varying gestational age (14-22 weeks). The isoelectric profiles are similar to those previously reported for human a-L-fucosidases (Thorpe & Robinson 1975, DiMatteo et al. 1976, Alhadeff & O’Brien 1977) with a multiplicity of peaks from p H 4.8-6.8. The amniotic fluid profile is most similar to the human serum pattern which contains an abundance of activity of the more acidic forms (Alhadeff & Janowsky 1978). However, amniotic fluid a-L-fucosidase is unlike the thermostable Serum enzyme & Andrews-Smith 1979) in that it displays greater thermolability at 45°C. This increased thermolability may be due to the low protein concentration of amniotic fluid when compared to human serum. The amniotic fluid a-L-fucosidase pH curves are very to those reported for Other human a-L-fucosidase (Ockerman 1969, Robinson & Thorpe 1973, Troost et al. 1976, Alhadeff & O’Brien 1977) with an optimum near or a t pH 5.0. The apparent Km for the 4-methylumbelliferyl substrate is lower than that reported for human liver (Alhadeff et al. 1975a) and placenta (Di Matteo et al. 1976), but comparable to that of human serum (Alhadeff & AndrewsSmith 1979). Immunochemical characterization of amniotic fluid a-L-fucosidase suggests that it is antigenically similar, if not identical to, human liver a-L-fucosidase, as has been shown for the human brain (Alha-
deff & Janowsky 1977), spleen (Thorpe & Robinson 1975), and serum (Thorpe & Robinson 1978, Alhadeff & Janowsky 1978) enzymes. In prenatal diagnosis it is often difficult to determine accurately levels and/or ratios of enzymatic activity, particularly in the difficult job of differentiating heterozygotes from homozygotes. It would be useful if the presence of an altered enzyme could be demonstrated for an affected fetus. If a mutant a-L-fucosidase is present in fucosidosis, then prenatal diagnosis could be based not only on decreased levels of a-L-fucosidase activity but also on a demonstrable, altered property of the residual enzymatic activity. The studies reported here on the activity and properties of amniotic fluid a-L-fucosidase should provide useful information for the prenatal diagnosis of fucosidosis.
Acknowledgments
This research was supported by grants from the National Foundation-March of Dimes (1-395) and NIH (1 RO1 AM 20409). J.A.A. is recipient of NIH Research Career Development Award 1 KO4 A M 0025701~1 from NIAMDD. We gratefully acknowledge the Division of Medical Genetics, UCSD, for the generous supply of amniotic fluids.
References
Alhadeff, J. A. & G. Andrews-Smith (1979). Kinetic and immunochemical characterization of low-activity serum a-L-fucosidase from a phenotypically normal individual. Biochem. Med. 20, 357-363. Alhadeff, J. A. & A. Janowsky (1977). Purification and properties of human brain a-Lfucosidase. J . Neurochem. 28, 423427. Alhadeff, J. A. & A. Janowsky (1978). Human serum a-L-fucosidase. Clin. chim. Acta 82, 133-140.
H U M A N A M N I O T I C FLUID a - L - F U C O S I D A S E
Alhadeff, J. A., A. L. Miller, H. Wenaas, T. Vedvick & J . S. O’Brien (1975a). Human liver a-L-fucosidase. Purification, characterization and immunochemical studies. J . biol. Chem. 250, 7106-7113. Alhadeff, J . A. & J. S. O’Brien (1977). Fucosidosis. Practical Enzymology o f the Sphingolipidoses, ed. R. H. Glew & S. P. Peters. Alan R. Liss, Inc., New York, pp. 247-281. Alhadeff, J . A., L. Tennant & J. S. O’Brien (1975b). Isoenzyme patterns of human liver a-L-fucosidase during development. Develop. Biol. 47, 319-324. DiMatteo, G., M. A. Orfeo & G. Romeo (1976). Human a-L-fucosidase: Purification and properties. Biochim. biophys. Acta (Arnst.) 429, 527-537. Durand, P., C. Borrone & G. Della Cella (1969). Fucosidosis. J . Pediat. 75, 665-674. Lineweaver, H.& D. Burk (1934). The determination of enzyme dissociation constants. J . Arner. Chem. Soc. 56, 658-666. Loeb, H., M. Tonduer, G. Jonniaux, S. Mockel-Pohl & E. Vamos-Hurwitz (1969). Biochemical and ultrastructural studies in a case of Mucopolysaccharidosis F. Helv. Pediat. Acta 24, 519-537. Lowry, 0.H., N. J. Rosebrough, A. L. Farr & R. J. Randall (1951). Protein measurements with the folin phenol reagent. J . biol. Chem. 193, 265-275. Matsuda, I., S. Arashima, Y . Oka, T. Mitsuyama, S. Ariga, T. Ikeuchi & T. Ichida (1975). Prenatal diagnosis of fucosidosis. Clin. chim. Acta 63, 55-60. Ockerman, P. A. (1969). Acid hydrolases in human skin. Acta Derrn.-venereol. (Stockh.) 49, 139-141. Poenaru, L., J.-C. Dreyfus, J. Boue, H. Nicolesco, N. Ravise & J. Bamberger (1976). Prenatal diagnosis of fucosidosis. Clin. Genet. 10, 266264. Robinson, D.& R. Thorpe (1973). Human liver a-L-ficosidases. Clin. chirn. Acta 47, 403407.
363
Thorpe, R. & D. Robinson (1978). Purification and serological studies of human a-L-fucosidase in the normal and fucosidosis states. Clin. chim. Acta 86, 21-30. Thorpe, R. & D. Robinson (1975). Isoelectric focusing of isoenzymes of human liver a-Lfucosidase. F.E.B.S. Lett. 54, 89-92. Troost, J., M. C. M. van der Heijden & G. E. J. Staal (1976). Human leukocyte a-L-fucosidase. Clin. chim. Acta 73, 321-327. Tsay, G. C . & G. Dawson (1976). Structure of the accumulating oligosaccharide in fucosidosis. J . biol. Chem. 251, 5852-5859. Van Hoof, F. (1973). “Fucosidosis” in Lysosomes and Storage Diseases, ed. H. G. Hers & F. Van Hoof. New York, Academic Press, pp. 277-290. Van Hcof, F. & H. G. Hers (1968). The abnormalities of lysosomal enzymes in mucopolysaccharides. Europ. J . Biochern. 7. 34-44. Wiederschain, G. Y., E. L. Rosenfeld, A. I. Brusilovsky .& L. G. Kolibaba (1971). a-LFucosidase and other glycosidases in human placenta, foetus, liver and amniotic fluid at various stages of gestation. Clin. chim. Acta 35, 99-107. Young, P. E., M. R. Matson & 0. W. Jones (1976). Amniocentesis for antenatal diagnosis. Review of problems and outcomes in a large series. Arner. J . Obstet. Gynec. 125, 495-500. Zielke, K., M. L. Veath & J. S. O’Brien (1972). Fucosidosis: Deficiency of a-L-fucosidase in cultured skin fibroblasts. J . exp. Med. 136, 197-199.
Address: Jack A . Alhadeff (M-024), Ph.D. Department o f Neurosciences School of Medicine University of California, Sun Diego La Jolla C A 92093 U.S.A.
Human amniotic fluid
a-L-fucosidase
JACK A. ALHAIYEFF AND GRAIL. ANDREWS-Sm
Department of Neurosciences, School of Medicine, University of California, San Diego, La Jolla, California, U S A . The activity and properties of a-L-fucosidase in 24 samples of amniotic fluid have been investigated using the 4-methylumbelliferyl substrate. A wide range of fucosidase specific activity (0.20-1.45 nmoVmin/mg protein) was found with an average value of 0.62 nmoV min/mg protein. Although no clear-cut correlations exist, there appear to be trends of decreasing fucosidase specific activity with increasing maternal age of donor and increasing gestational time. a-L-Fucosidase activity from four amniotic fluids was characterized kinetically and immunochemically and found not to differ in its properties due to maternal age of donor or gestational time. Isoelectric focusing revealed multiple forms between pH 5.0 and 6.8, with the majority of activity present between pH 5.0 and 6.0. The pH optimum is near pH 5.0 with a second optimum suggested at pH 6.2. The apparent Michaelis constant (Km) for the 4-methylumbelliferyl substrate is 0.05 k 0.01 mM. The enzyme is completely thermostable at 37°C for at least 4 h but loses 95 % of its activity after preincubation at 45°C for 1 h. Double immuncdiffusion and immunoprecipitation experiments using anti-liver a-L-fucosidase antibody suggest that the amniotic fluid a-L-fucosidase is antigenically similar, if not identical, to the human liver enzyme. Received 17 April, accepted f o r publication I1 June 1979 Key words: Amniotic fluid; a-L-fucosidase; fucosidosis; prenatal diagnosis.
Fucosidosis is a rare neurovisceral storage disease characterized by progressive mental and motor retardation, decorticate rigidity, emaciation, bony deformities, progressive spasticity and respiratory difficulties (Van Hoof & Hers 1968, Durand et al. 1969). Biochemically this genetic disease involves a severe deficiency of activity of the lysosoma1 hydrolase a-L-fucosidase (fucosidefucohydrolase EC 3.2.1.51) coupled with accumulation of fucose-containing glycoconjugates (Loeb et al. 1969, Van Hoof 1973, Tsay & Dawson 1976). The absence of fucosidase activity in cultured skin fibroblasts in fucosidosis patients (Zielke et al. 1972) and the presence of fucosidase activity in amniotic fluid and
cells (Wiederschain e t al. 1971) indicate that this disorder should be diagnosable prenatally by assay of amniotic fluid and/or cultured amniotic fluid cells (Matsuda et al. 1975, Poenaru et al. 1976). Little is known about the activity and properties of a-Lfucosidase in amniotic fluid and cultured amniotic cells. Wiederschain et al. (1971) have shown that fucosidase activity is present in amniotic fluid at various stages of pregnancy (20-30 weeks). Since the forms of a-L-fucosidase have been shown to change during fetal development (Alhadeff et al. 1975b), further baseline studies on amniotic fluid and cultured amniotic fluid cells are necessary for development of an accurate prenatal diagnostic test for fucosi-
0OO9-9163/79/110357--O7$02.50/0 0 1979 Munksgaard, Copenhagen
358
ALHADEFF AND ANDREWS-SMITH
dosis. In the present study we have investigated the activity and properties of a-Lfucosidase in 24 amniotic fluids obtained between 14 and 22 weeks of gestation. Material and Methods
General All procedures were carried out at 0-4°C unless otherwise stated. a-L-Fucosidase activity was assayed using 4-methylumbelliferyl-a-L-fucopyranoside (Koch-Light, Colnbrook, Buckinghamshire, U.K.), as previcusly described (Alhadeff & O’Brien 1977). Enzyme assays were performed under conditions where enzyme activity was linear with respect to time (20-60 min) and amount of protein (20-40 p1 amniotic fluid). A unit of fucosidase activity is defined as the amount of enzyme that hydrolyses 1 nmol of substrate per min at 37°C. Protein was determined by the Lowry method (Lowry et al. 1951) using human serum albumin as standard. Agarose-E-aminocaproylfucosamine was purchased from MilesYeda Ltd. (Elkhart, IN, USA). The procedures which involved human tissues were approved by the Committee on Jnvestigations/Activities Involving Human Subjects of the School of Medicine, University of California, San Diego. Preparation of Amniotic Fluid Amniotic fluid was prepared from the material (10-30 ml) obtained on transabdominal amniocentesis by centrifuging at 2,500 X g for 10 min (Young et al. 1976). The supernatants (which were free of cellular material) were assayed for a-L-fucosidase activity, for amount of protein, and were stored frozen (-20°C) until used. Freezing did not alter the kinetic and immunochemical properties of a-L-fucosidase. The gestational age of the fetus at the time of amniocentesis was determined by ultrasonography.
Purification o f Amniotic Fluid a-L-Fucosidase a-L-Fucosidase was purified from 225 ml of combined amniotic fluid samples by affinity chromatography on agarose-c-aminocaproylfucosamine, as previously described (Alhadeff et al. 2975a). This partially purified a-L-fucosidase was used in the double immunodiffusion experiments (see below). Isoelectric Focusing Isoelectric focusing was performed on four samples of amniotic fluid a-L-fucosidase (1.0 ml) using a 40 ml column, as previously described (Alhadeff & Andrews-Smith 1979). One percent ampholytes (pH 5-8) from LKB Produkter (Stockholm, Swedeill were used in a gradient of 0-67 % (w/v) sucrose. Electrofocusing was conducted for 17-20 h at a starting amperage of 2.5 mA and 600 V. Kinetic Studies Apparent Michaelis constants (Km) of four preparations of amniotic fluid a-L-fucosidase for the 4-methylumbelliferyl substrate were determined graphically by the Lineweaver-Burk method (Lineweaver & Burk 1934). For these studies, enzyme sample (20 pl) was incubated in duplicate for 30 min at 37°C in substrates of varying concentration in 100 mM citric acid-sodium citrate buffer (pH 5.0). The pH optima profiles were determined for four preparations of amniotic fluid a-L-fucosidase in 100 mM buffers of varying p H values (citric acid-sodium citrate for p H values 3.1 to 5.8; KH2P04-NapHP04 for pH values 6.0 to 8.0). Incubations were carried out on 30 pl aliquots of amniotic fluid in duplicate for 30 min at 37°C. Thermal stability studies were performed on three preparations of amniotic fluid a-L-fucosidase by preincubation of 40 PI aliquots of amniotic fluid at 37°C or 45°C for up to 4 h. After preincubation, substrate was added and the
HUMAN AMNIOTIC FLUID a-L-FUCOSIDASE
samples were assayed in duplicate for 30 min at 37°C. Inimunochemicat Studies The IgG fraction of antibodies made against purified human liver a-L-fucosidase in a previous study (Alhadeff et al. 1975a) was used for the immunochemical studies. Amniotic fluid a-L-fucosidase and human liver a-L-fucosidase (diluted to contain an amount of activity comparable to that in the amniotic fluid) were separately preincubated with 0-70 p1 of anti-liver a-Lfucosidase IgG in 400 pl conical plastic centrifuge tubes (Cole Scientific, Calabasas, CA, USA) for 20 h at 2 ' 4 ° C . The preincubated samples were centrifuged at 48,000 X g for 30 min and the resulting supernatants were assayed in duplicate for a-L-fucosidase activity. The IgG fraction (8 pl) was also used in double immunodiffusion experiments on Hyland immunoplates (Costa Mesa CA, USA) against both purified liver a-L-fucosidase (15-244 units) and the amniotic fluid a-L-iucosidase (9 units) which had been purified by affinity chromatography. The plates were spotted with samples, placed at 0-4°C and read after 2-3 days.
359
Table 1 Amniotic fluid a-L-fucosidase activity
Patient
Maternal age 'years)
1 2 3 4 5 6 7 8 9 10
34.5 35 39 34 42 35 40 36 43 35 42
11
?
12 13 14 15 16 17 18 19
39 29 37 35 34 21 38 25 34.5
20
21 22 23 24
?
Range Average
u-L-Fucosidase Time Of activity amniocentesis (weeks of units/mg gestation)' units/ml protein
19.4 15 19.5 16.5 17.5 19.0 22.0 17.0 18.0 14 18 ? 17.5
0.90
3.50 1.75 2.00 1.25 3.35 3.30 1.35 2.63 3.27 2.30 2.25 4.40 1.40 3.85 2.50
?
16 20 16 17 19 16.5 ? ?
35 40
18.5 15.5
21-43
14-22
35.7
17.6
0.45 0.49 0.29 1.36 0.20 0.60 0.39 0.48 0.26 1.45 0.61 0.56 0.28 0.60 0.82 0.41 0.51 1.11 0.50 0.63 0.93 0.48 0.78 0.72
2.00 1.40 1.25 3.10 1.05 2.65 2.30 2.05
0.9&4.40 0.20-1.45 2.33
0.62
* Determined by ultrasonography.
Results
Table 1 summarizes the a-L-fucosidase activity (unitdml), specific activity (unitdmg protein), the time of amniocentesis in weeks gestation and the age of the 24 women who provided samples for this study. A wide range of fucosidase specific activity (0.201.45 units/mg protein) was found for these amniotic fluids, with an average value of 0.62 units/mg protein. When the amniotic fluid a-L-fucosidase specific activities are plotted versus either maternal age (Fig. 1) or gestation time (Fig. 2), no clear-cut correlations are seen. However, for both plots, amniotic fluid a-L-fucosidase specific activi-
..
"i. 12
-* 21
23 25
27 29
31
33
..
35 37 39 41 43
MATERNAL AGE (YEARS)
Fig. 1. Amniotic fluid 0-L-fucosidase specific activity vs. maternal age of donor.
360
ALHADEFF AND ANDREWS-SMITH
'7-
.
1.4{
14
48
15
16
17
18
19
20
21
r
22
TIME OF AMNIOCENTESIS (WEEKS GESTATION) Flg. 2. Amniotic fluid a-L-fucosidase specific activity vs. time of amniocentesis (weeks gestation).
ty appears to decrease with increasing maternal age and increasing gestational times. These trends should be interpreted with caution, since a considerable amount of variabilitv in fucosidase activitv can be seen for one maternal age (e.g., 35 years) or one gestational time (e.g., 16.5 weeks). Isoelectric focusing of amniotic fluid a-Lfucosidase revealed the pattern depicted in Figure 3. Multiple forms are found with isoelectric points (PI'S) between p H 5.0 and 6.8, with the majority of activity present between pH 5.0 and 6.0. No major differences were seen in the a-L-fucosidase isoelectric profiles of the four amniotic fluids which were focused.
-
6.0
7.0
8.0
PH
Michaelis constants of a-L-fucosidase for the 4-methylumbelliferyl substrate were determined on four amniotic fluids. The Lineweaver-Burk double reciprocal plots yielded straight lines with similar apparent Km's (ranging from 0.04-0.06 mM) with
2
1
5.0
4.0
Fig. 4. pH Optimum curve of amniotic fluid a-l-fucosidase activity. (See Materials and Methods section for details.)
t
30 r a0 -
40 tI
t; a
100
--.-
=
=
-3
7 d
-
J
70 -
-
:I 60
i - "v\
30
2o
I
t
30
40
In
'\ 60
50
70
80
PH Flg. 3. lsoelectric focusing (pH 5-8) of amniotic fluid a-L-fucosidase activity. (See Materials and Methods section for details.)
arr PREINCUBATION TIME (HAS) Fig. 5. Thermostability curves of amniotic fluid a-Lfucosidase activity at 37" and 45°C. (See Materials and Methods section for details.)
HUMAN AMNIOTIC FLUID a-L-FUCOSIDASE
361
Figure 6 indicates that the antibody against liver a-L-fucosidase crossreacts with the purified amniotic fluid a-L-fucosidase (wells A and B) to give a single precipitin line coincident with that from liver (wells C and D). When the same amounts of liver and amniotic fluid a-L-fucosidase activity were preincubated separately with the IgG fraction of liver antiserum, comparable amounts of activity were immunoprecipitated . Flg. 6. Double immunodiffusion (Ouchterlony) plate of anti-liver a-L-fucosidase antibody (center well) against purified amniotic fluid a-L-fucosidase (wells A and B) and purified human liver a-L-fucosidase (wells C and D). (See Materials and Methods section for details.)
an average of 0.05 +. 0.01 mM. Figure 4 depicts the p H optimum curve for a-L-fucosidase, which was similar for the four amniotic fluids investigated. The optimum is near pH 5.0, with a second optimum suggested at pH 6.2. The biphasic nature of the pH optimum curve does not appear to be an artifact of using two buffer systems (citrate and phosphate), since the ascent of the second optimum was seen when the citrate buffer was employed beyond pH 53-6.1. Amniotic fluid a-L-fucosidase activity is completely thermostable at 37°C with no loss of activity after preincubation for 4 h (Fig. 5). However, at 45°C the activity is very thermolabile with 70 % and 95 % losses of activity after 30 and 60 min preincubations, respectively. The thermostability curves depicted in Figure 5 for both 37" and 45°C were typical of the three amniotic fluid a-L-fucosidases investigated. The IgG fraction of antiserum made against pure liver a-L-fucosidase was used in double immunodiffusion experiments against both the purified liver enzyme and the purified amniotic fluid a-L-fucosidase.
Discussion
There are currently two reports in the literature of attempted prenatal diagnoses for families at risk for fucosidosis. In one case the fetus was diagnosed correctly as being free from the disease by assay of amniotic fluid and amniotic fluid cell fucosidase activity (Poenaru et al. 1976). In the other case, amniotic fluid obtained between 14 and 18 weeks of gestation was found t o contain 10 % of control fucosidase activity. Assay of cultured amniotic fluid cells indicated the presence of about 30 % of normal fucosidase activity. On the basis of these assays the fetus was diagnosed as having a carrier state of the disease. The diagnosis was shown to be incorrect after delivery when a-L-fucosidase activity was absent in white blood cells from the identical twins (Matsuda et al. 1975). This report indicates that great care must be taken in the prenatal diagnosis of fucosidosis and suggests that further studies on amniotic fluid cell a-Lfucosidase are warranted. In the present investigation we have done baseline studies on the activity and properties of a-L-fucosidase in amniotic fluid. Three other laboratories have provided limited data on the activity of a-L-fucosidase in a few samples of control amniotic fluid (Wiederschain et al. 1971, Matsuda et al. 1975, Poenaru et al. 1976), but n o characterization studies of amniotic fluid a-L-
362
ALHADEFF AND ANDREWS-SMITH
fucosidase have been published. We have found similar or increased fucosidase activity (when compared to previous studies) in the 24 amniotic fluids studied. Although no strong correlations exist, there appear to be trends of decreasing fucosidase specific activity with increasing maternal age and increasing gestational time. However, the isoelectric profiles and kinetic properties (pH curves, Km’s, thermostability curves) were similar for the three to four amniotic fluid a-L-fucosidases characterized, despite varying maternal age of donor (35-43 years) and varying gestational age (14-22 weeks). The isoelectric profiles are similar to those previously reported for human a-L-fucosidases (Thorpe & Robinson 1975, DiMatteo et al. 1976, Alhadeff & O’Brien 1977) with a multiplicity of peaks from p H 4.8-6.8. The amniotic fluid profile is most similar to the human serum pattern which contains an abundance of activity of the more acidic forms (Alhadeff & Janowsky 1978). However, amniotic fluid a-L-fucosidase is unlike the thermostable Serum enzyme & Andrews-Smith 1979) in that it displays greater thermolability at 45°C. This increased thermolability may be due to the low protein concentration of amniotic fluid when compared to human serum. The amniotic fluid a-L-fucosidase pH curves are very to those reported for Other human a-L-fucosidase (Ockerman 1969, Robinson & Thorpe 1973, Troost et al. 1976, Alhadeff & O’Brien 1977) with an optimum near or a t pH 5.0. The apparent Km for the 4-methylumbelliferyl substrate is lower than that reported for human liver (Alhadeff et al. 1975a) and placenta (Di Matteo et al. 1976), but comparable to that of human serum (Alhadeff & AndrewsSmith 1979). Immunochemical characterization of amniotic fluid a-L-fucosidase suggests that it is antigenically similar, if not identical to, human liver a-L-fucosidase, as has been shown for the human brain (Alha-
deff & Janowsky 1977), spleen (Thorpe & Robinson 1975), and serum (Thorpe & Robinson 1978, Alhadeff & Janowsky 1978) enzymes. In prenatal diagnosis it is often difficult to determine accurately levels and/or ratios of enzymatic activity, particularly in the difficult job of differentiating heterozygotes from homozygotes. It would be useful if the presence of an altered enzyme could be demonstrated for an affected fetus. If a mutant a-L-fucosidase is present in fucosidosis, then prenatal diagnosis could be based not only on decreased levels of a-L-fucosidase activity but also on a demonstrable, altered property of the residual enzymatic activity. The studies reported here on the activity and properties of amniotic fluid a-L-fucosidase should provide useful information for the prenatal diagnosis of fucosidosis.
Acknowledgments
This research was supported by grants from the National Foundation-March of Dimes (1-395) and NIH (1 RO1 AM 20409). J.A.A. is recipient of NIH Research Career Development Award 1 KO4 A M 0025701~1 from NIAMDD. We gratefully acknowledge the Division of Medical Genetics, UCSD, for the generous supply of amniotic fluids.
References
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H U M A N A M N I O T I C FLUID a - L - F U C O S I D A S E
Alhadeff, J. A., A. L. Miller, H. Wenaas, T. Vedvick & J . S. O’Brien (1975a). Human liver a-L-fucosidase. Purification, characterization and immunochemical studies. J . biol. Chem. 250, 7106-7113. Alhadeff, J . A. & J. S. O’Brien (1977). Fucosidosis. Practical Enzymology o f the Sphingolipidoses, ed. R. H. Glew & S. P. Peters. Alan R. Liss, Inc., New York, pp. 247-281. Alhadeff, J . A., L. Tennant & J. S. O’Brien (1975b). Isoenzyme patterns of human liver a-L-fucosidase during development. Develop. Biol. 47, 319-324. DiMatteo, G., M. A. Orfeo & G. Romeo (1976). Human a-L-fucosidase: Purification and properties. Biochim. biophys. Acta (Arnst.) 429, 527-537. Durand, P., C. Borrone & G. Della Cella (1969). Fucosidosis. J . Pediat. 75, 665-674. Lineweaver, H.& D. Burk (1934). The determination of enzyme dissociation constants. J . Arner. Chem. Soc. 56, 658-666. Loeb, H., M. Tonduer, G. Jonniaux, S. Mockel-Pohl & E. Vamos-Hurwitz (1969). Biochemical and ultrastructural studies in a case of Mucopolysaccharidosis F. Helv. Pediat. Acta 24, 519-537. Lowry, 0.H., N. J. Rosebrough, A. L. Farr & R. J. Randall (1951). Protein measurements with the folin phenol reagent. J . biol. Chem. 193, 265-275. Matsuda, I., S. Arashima, Y . Oka, T. Mitsuyama, S. Ariga, T. Ikeuchi & T. Ichida (1975). Prenatal diagnosis of fucosidosis. Clin. chim. Acta 63, 55-60. Ockerman, P. A. (1969). Acid hydrolases in human skin. Acta Derrn.-venereol. (Stockh.) 49, 139-141. Poenaru, L., J.-C. Dreyfus, J. Boue, H. Nicolesco, N. Ravise & J. Bamberger (1976). Prenatal diagnosis of fucosidosis. Clin. Genet. 10, 266264. Robinson, D.& R. Thorpe (1973). Human liver a-L-ficosidases. Clin. chirn. Acta 47, 403407.
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Thorpe, R. & D. Robinson (1978). Purification and serological studies of human a-L-fucosidase in the normal and fucosidosis states. Clin. chim. Acta 86, 21-30. Thorpe, R. & D. Robinson (1975). Isoelectric focusing of isoenzymes of human liver a-Lfucosidase. F.E.B.S. Lett. 54, 89-92. Troost, J., M. C. M. van der Heijden & G. E. J. Staal (1976). Human leukocyte a-L-fucosidase. Clin. chim. Acta 73, 321-327. Tsay, G. C . & G. Dawson (1976). Structure of the accumulating oligosaccharide in fucosidosis. J . biol. Chem. 251, 5852-5859. Van Hoof, F. (1973). “Fucosidosis” in Lysosomes and Storage Diseases, ed. H. G. Hers & F. Van Hoof. New York, Academic Press, pp. 277-290. Van Hcof, F. & H. G. Hers (1968). The abnormalities of lysosomal enzymes in mucopolysaccharides. Europ. J . Biochern. 7. 34-44. Wiederschain, G. Y., E. L. Rosenfeld, A. I. Brusilovsky .& L. G. Kolibaba (1971). a-LFucosidase and other glycosidases in human placenta, foetus, liver and amniotic fluid at various stages of gestation. Clin. chim. Acta 35, 99-107. Young, P. E., M. R. Matson & 0. W. Jones (1976). Amniocentesis for antenatal diagnosis. Review of problems and outcomes in a large series. Arner. J . Obstet. Gynec. 125, 495-500. Zielke, K., M. L. Veath & J. S. O’Brien (1972). Fucosidosis: Deficiency of a-L-fucosidase in cultured skin fibroblasts. J . exp. Med. 136, 197-199.
Address: Jack A . Alhadeff (M-024), Ph.D. Department o f Neurosciences School of Medicine University of California, Sun Diego La Jolla C A 92093 U.S.A.
