Further evidence of human αa-l -fucosidase polymorphism

251

Clinica Chimica Acta, 123 (1982) 25 I-259 Etsevier Biomedical Press

CCA 2217

Further evidence of human cu+fucosidase polymorphism E.M.

Beyer

and G.Y.

~ieders~hain

*

Institute of 3io~~g~ca~and Medical Chemistry, Academy of Medicat Sciences of the USSR. Pogodina Street IO, Moscow, il9iZI (C’SSR,l (Received

October

14, 1981; revision April 14th, 1982)

Summary The results of a comparative study of multiple forms of cr-L-fucosidase from human kidney, liver, placenta and blood serum provide evidence for ar-r_-fucosidase polymorphism. On the basis of the data obtained the existence of certain phenotypic groups of cu-r.-fucosidase, dissimilar both in the quantitative and qualitative composition of the enzyme’s multiple forms, was revealed. Some possible reasons for a-L-fucosidase polyrno~~s~ are discussed. It is suggested that the data on cr-Lfucosidase polymorphism may be used in diagnosis and in the isolation and preparation of individual enzyme forms.

Introduction cr-L-Fucosidase (EC 3.2.1.51) is known to belong to a widely distributed group of glycosidases and to catalyze the hydrolytic splitting of fucose and fucose-related sugars from natural and synthetic compounds [l-3]. The deficiency of cu-L-fucosidase or the absence of some molecular forms of this enzyme lead to the development of the severe hereditary disease, fucosidosis [4]. Previous investigation of multiple forms of human and animal cu+fucosidase by isoelectrofocusin~ and electrophoresis revealed some intraspecific variations in quantities and ratios of rw-L-fucosidase components in enzyme preparations obtained from the same organs of different individuals. On the basis of these data some authors suggested the occurrence of cw-t-fucosidase polymorphism [5-71. This view, however, was challenged by other investigators (81. This paper presents new experimental evidence for cr-L-fucosidase polymorphism based on studies of composition of the enzyme’s multiple forms in human kidney, liver, placenta and blood serum of different individuals.

* To whom correspondence

0009-898 I /82/WO-OfX10/$02.75

shoufd he addressed.

Q 1982 Elsevier Biomedical

Press

252

Materials and methods Sephadex G-200 ‘Superfine’, Sephadex G-25 ‘Fine’ (Pharmacia, Sweden), carrier ampholines (LKB, Sweden), 4-methylumbelliferyl-a-r_-fucopyranoside (Koch-Light, UK) and p-nitrophenyl-a-L-fucopyranoside (Serva, FRG) were used. Kidney and liver specimens (20 and 10 samples, respectively) were obtained at autopsy from different individuals and were frozen at -2O’C. Placenta (chorionic villi) specimens (35 samples) were obtained at medical abortion in normal pregnancy and were frozen at -20°C also. Tissue homogenates were prepared using distilled water at 4°C in ratios 1: 3 for liver (or kidney), and 1 : 2 for placenta. Supernatants withdrawn after centrifugation at 20000 X g were used for isoelectric focusing. The blood serum samples from 35 healthy donors, prepared by centrifugation for 20 min at 20000 X g after clot formation at room temperature, were dialysed against distilled water for 48 h at 4°C. Enzyme assay a-L-Fucosidase activity was assayed by measuring the quantity of p-nitrophenol liberated from the corresponding substrate. The reaction mixture (final volume, 0.25 ml) contained 0.15 ml of the enzyme solution and 0.1 ml of the substrate (final concentration, 1 mmol/l) in 0.05 mol/l acetate buffer (pH 5.0) with 1 mmol/l EDTA. Incubation at 37°C was terminated by the addition of 0.25 ml 5% TCA. After the precipitation of the proteins by centrifugation at 600 X g the released p-nitrophenol was determined by adding 0.25 ml 0.25 mol/l NaOH to 0.25 ml of the supernatant. One unit of the activity was defined as the amount of the enzyme which hydrolyses 1 pmol of the p-nitrophenyl-a-L-fucopyranoside per hour. The specific activity was expressed as the number of units per milligram of protein. Protein was determined by the method of Lowry et al [9] with bovine serum albumin as standard. Isoelectric focusing in thin layer Isoelectric focusing was performed in a Desaga apparatus using glass plates with thin layer (0.6 ‘mm) of Sephadex G-200 ‘Superfine’ containing 1% ampholine solution with a pH range of 5-7 as described in [lo]. Ten samples of enzyme preparations were applied to the gel surface of a glass plate (20 X 20. cm) using automatic microliter pipettes (Brand); lo-30 ~1 of kidney, liver and placental extracts or 50-60 ~1 of serum were applied to each spot. Isoelectrofocusing was carried out at 4°C for 16 h using 300 V and then the voltage was increased stepwise to 600 V during 1.5 h. Identification of a-L-fucosidase components For identification of a+fucosidase we used a solution of 1 mmol/l umbelliferyl-cY-L-fucopyranoside in 0.05 mol/l acetate buffer, pH 5.0. 3-MM filter paper strips (19 X 0.5 cm) soaked in this solution were applied surface and the glass plates were incubated at 37°C for lo-15 min. incubation the bands of a-L-fucosidase were detected using long-wave

4-methylWhatman to the gel After the ultraviolet

253

light. For the intensification of the fluorescence the glass plates were placed in a chamber with steam of NH,. The pH gradient established in the gel was determined by direct measurement in the gel layer using a combined glass membrane electrode (Ingold) at room temperature. The measurements were done through each 1 cm. Isoelectric jhwing

on column

Enzyme preparations from human kidney and serum used for this purpose were isolated on a column with Sephadex G-25 ‘Fine’. lsoelectrof~using was performed using a 1lo-ml column of LKB 8100 electrof~using equipment at 4°C for 48 h. The carrier ampholyte concentration was 1% with a pH range from 4 to 6 in a sucorse gradient. Results

The experiments showed that a-L-fucosidase from different human organs and blood serum was separated into no less than 5-9 components during isoelectrofocusing in a pH gradient 5-7. The spectra of cu-r_-fucosidase from kidney extracts of nine individuals are presented in Fig. 1. As will be seen, the number of cu-L-fucosidase components is not

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?

2

F, F,

mmm

34567

89

F2 F3 F2 F; F;

F;

a

l

m a

F,

PI -6.35

l l l m a m -6.07 l m- 602 mm - 583 1.. 5.73

mmmmmm mm

Fig. 1. Isoelectric focusing of a-bfucosidase scheme.

from kidney of different ~~~du~s

l - 548

(n =9); right hand-

254

identical for different individuals, Taking into account the isoelectric point values of the most fluorogenic bands of the enzyme which are highly reproducible from experiment to experiment, the existence of at least five different phenotypes could be established. These were designated by us as phenotypes F,, F,, F,‘, Fj and F;. It should be noted that we did not find any correlation between the number of a-r.+-fucosidase components typical of one or another phenotype and total (Y-Lfucosidase activity in the initial enzyme preparation. Thus, we found individuals with phenotype Fz (the highest number of components) with 1555 units/ml total activity and some with F, phenotype having a total tu-r_-fucosidase activity of 2462 units/ml. Similar but not identical pictures were found in the isoelectric focusing of liver extracts from the same persons (Fig. 2). The isoenzyme spectra of a-r.-fucosidase preparations from the placenta of different individuals were aIso characterized by intraspecific variations (Fig. 3), and quantitative content of a-L-fucosidase multiple forms from placenta and kidney (or liver) was similar. However, p1 values of the apparently identical forms of a-~-

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0

Pr - 6.35

- 6.10 - 6.01

-5.80

-5.52

,5.20

Fig. 2. Isoelectric focusing of n+fucosidase

from liver of different individuals (n = 9).

255

fucosidase from placenta and kidney were different. Quite a different spectrum of cu+fucosidase isoenzymes was found when investigating the enzyme of human blood serum (Fig.4). The isoenzyme spectra of cu-L-fucosidase from most of the serum preparations were characterized by the existence of two major components with p1 4.6-4.8 and some minor components with more neutral isoelectric points. The essential differences observed between multiple forms of cw-L-fucosidase of parenchymatous organs (kidney, liver) and enzyme forms of blood serum corresponded to our data obtained previously upon isoelectrofocusing or-L-fucosidase preparations of kidney and serum in sucrose gradient on a column (Fig. 5), and to data obtained by other investigators obtained for cu+fucosidase from liver and serum f 113. It should be emphasized that there are some individual differences in a-L-fucosidase isoenzyme spectra of blood serum, but they were not so pronounced as in the case of parenchymatous organs. Thus, it is necessary to analyze a greater number of serum samples to be able to define more exactly the phenotypic differences of the enzyme from human blood serum.

0

PI

-E07

.5

Fig. 3. Isoelectric focusing of a+fucosidase

from placenta of different individuals (n = IO).

12

3

4

56789X

PI

-5

-4..80 -4

Fig. 4. Isoelectric

.60

focusing of blood serum cr-L-fucosidase from different individuals

(n = 10).

Discussion Analysis of the data obtained shows that in a variety of organs and biological fluids manifestation of differences in composition of a-L-fucosidase multiple forms may be either similar or quite different. Thus, as shown by our experiments, the compositions of a-L-fucosidase forms in kidney and liver from the same individuals were virtually identical (the other organs of these persons were not studied). The isoenzyme spectra of cy-L-fucosidase from placenta and parenchymatous organs were similar in the number of enzyme components. However, the pI values of placenta and kidney a+fucosidase components were different. In blood serum the phenotypic manifestation of differences in the number and ratio of the enzyme forms was less pronounced. As mentioned above, there were considerable differences in the composition of a+fucosidase forms in tissue and blood serum. It is possible that the differences observed are due to the presence of double the amount of sialic acid in serum cy-L-fucosidase, as compared with tissue enzyme [ 111. The data obtained confirm the existence of cY-L-fucosidase. polymorphism re-

257

15

23

47

39

31

55

7-I

63

FroctKm PH

60

22

30

38

46

54

62

70

70

Fructkrs

Fig. 5. Isoelectric focusing of a-L-fucosidase l), a-L-fucosidase activity; (0 -

(+---

from human kidney 0), pH gradient;

(A) and blood serum (B) on column; ), absorbance at 280 nm. (-

ported by Turner et al [6] and noted in our earlier papers [7,12]. Recently TrinchDinch-Khoi et al [ 131 described the polymorphism of a-r_-fucosidase in one of the regions of France. Butterworth and Guy also found the occurrence of individual variations in isoenzyme composition of cY-r_-fucosidase consisting in the presence or absence of the enzyme form with the greatest p1 value [14]. At present the causes of the polymorphism observed are not completely understood. It is quite possible that the polymorphism is caused by mutations of structural gene loci determining the formation of these enzyme subunits. Furthermore, the polymorphism may be a result of post-ribosomal modifications leading to formation of enzyme forms with a different charge of their molecules. The glycoprotein nature of ol-L-fucosidase [ 15,161 and the presence of sialic acid residues in its molecule causing anionic properties of the enzyme [ 11,171, indicate that post-ribosomal modifications do indeed take place. Turner et al [6] reported three a-L-fucosidase phenotypes, designated Fu 1, Fu 2 and Fu 2-1, among peripheral leukocytes from different individuals. The Fu 2 and Fu 2-l phenotypes can be reliably distinguished only after treatment with neuraminidase. The Fu 2-l phenotype seemed to represent a simple mixture of Fu 1 and

258

Fu2. On the basis of family data, the authors suggest that the polymorphism of ar-L-fucosidase may be explained by the existence of two common alleles, Fu’ and Fu’, at a single gene locus, In our experiments we found not three, but, at least, five ar-L-fucosidase phenotypes. We suggest that the variations in isoenzyme composition of a-L-fucosidase are not due to the limited proteolysis occurring during the enzyme isolation. As was reported by us 1181, leupeptin and pepstatin, inhibitors of thiol and acid proteinases, as well as phenylmethylsulphonylfl~oride, an inhibitor of serine proteinases, had no effect on the composition of multiple forms of placenta a-L-fucosidase. In addition, the use of almost optimal conditions for the action of acid proteinases (heating of the enzyme preparation at 55’C, pH 5.0) has not changed the picture of isoelectric separation typical of the initial enzyme preparation. Our data are in agreement with those of Thorpe and Robinson [ 191 who demonstrated the absence of any effect of phenylmethylsulphonylfluoride on the isoenzyme spectrum of human liver (Y-Lfucosidase. However, the question of participation of proteinases in the formation of cu-r_-fucosidase multiple forms in vivo still remains obscure, The data on polymorphism of cu-I--fucosidase were used by us in the isolation of individual enzyme forms by preparative isoelectrofocusing [ 181; by carrying out screening we were able to select the phenotypes most convenient for preparation of one or another well-separated a+fucosidase form. Furthermore, taking into account the possibility of the study of placenta at biopsy [20], the data obtained may be helpful in prenatal diagnosis of some types of fucosidosis caused by the absence of one of the cr+fucosidase forms. Acknowledgements We would like to thank Prof. IS. Rosovski and Dr. V.A. Bacharev (All-Union Research Center for Maternal and Child Health, Moscow) for the supply of placenta specimens. References I Wiederschain G, Kohbaba LG, Rosenfeld EL. Human a-r;fucosidases. Clin Chim Acta 1973; 46: 305~310. 2 Wiederschain GY, Beyer EM, Klyashchitsky BA, Shashkov AS. Specificity patterns of different types of human fucosidases: recognition of a certain region of pyranose ring in sugars by the enzymes. B&him Biophys Acta 1981; 659: 434-444. 3 Alhadeff JA. Human a+fucosidases and fucosidosis. In: Callahan JW, Lowden JA, eds. Lysosomal storage diseases. New York: Raven Press 1981: 299-314. 4 Alhadeff JA, O’Brien JS. Fucosidosis. In: Glew RH, Peters SP, Liss AR, eds. Practical enzymology of the sphingolipidoses, Vol. I. New York: 1977: 247-281. 5 Turner BM, Beratis NG, Turner VS, Hirschhorn K. Isoenzymes of human cx-L-fucosidase detectable by starch gel electrophoresis. Clin Chim Acta 1974; 57: 29-35. 6 Turner BM, Turner VS, Beratis NG, Hirschhorn K. Polymorphism of human a-fucosidase. Am J Hum Genet 1975; 27: 651-661. 7 Beyer EM, Wiederschain GY. Substrate specificity and some properties of free and immobilized n-r-fucosidase of animals. Biok~miya 1977: 42: 881-889.

259

8 Alhadeff JA, Tennant L, O’Brien JS. Altered isoenzymes patterns of human liver a-t-fucosidase during development. Dev Biol 1976; 47: 319-324. 9 Lowry OH, Rosebrough HJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193: 265-275. 10 Radola BJ. Isoelectric focusing in layers of granulated gels. I. Thin-layer isoelectric focusing of proteins. Biochim Biophys Acta 1973; 295: 412-428. 11 Alhadeff JA, Janowsky AJ. Human serum a-t-fucosidase. Clin Chim Acta 1978; 82: 133-140. 12 Beyer EM, Wiederschain GY. Individual differences in the isoenzyme spectrum of human a-r-fucosidase. Vopr Med Khim 1980; 26: 538-539. 13 Trinch-Dinch-Khoi I, Glaise D, Le Treut A, Fauchet R, Godin Y, Le Gall JY. Genetic polymorphism of a-t-fucosidase in Brittany (France). Hum Genet 1979; 51: 293-296. 14 Butterworth J, Guy GJ. Primary amniotic fluids cell, skin fibroblast and liver a-L-fucosidase and its relation to cystic fibrosis. Clin Chim Acta 1979; 92: 109-I 16. 15 Carlsen RB, Pierce JG. Purification and properties of an a-t-fucosidase from rat epidydimis. J Biol Chem 1972; 247: 23-32. 16 Alhadeff JA, Freeze H. Carbohydrate composition of purified human liver a-t_-fucosidase. Mol Cell B&hem 1977; 18: 33-37. 17 Alhadeff JA, Cimino G, Janowsky A. Isoenzymes of human liver a-L-fucosidase: chemical relationship, kinetic studies and immunochemical characterization. Mol Cell Biochem 1978; 19: 17 1- 180. 18 Bach NL, Beyer EM, Wiederschain GY, Bovin NV, Zurabyan SE. A study.of properties and substrate specificity of human n-t-fucosidase multiple forms using natural and synthetic fucose-containing oligosaccharides. Bioorgan Chem 1981; 7: 1024-1033. 19 Thorpe R, Robinson D. Isoelectric focusing of isoenzymes of human liver a-L-fucosidase. FEBS Lett 1975; 54: 89-92. 20 Scrimgeour JB. Other techniques for antenatal diagnosis. In: Emery AEH, ed. Antenatal diagnosis of genetic disease. Edinburgh and London: Churchill Livingstone, 1973: 40-57.