α-l -Fucosidase activity in normal human lymphocytes

Clinica Chimica Acia, 180 (1989) 303-310 Elsevier

303

CCA 04414

ar+Fucosidase activity in normal human lymphocytes Liliane Roger, Marguerite-Anne Bernard, Fran@ and Marie-Jose Foglietti

Percheron

Luboratoire de Chimie Biologique, U. E. R. de Biologie Ifumaine et Expbimentale, Paris (France)

Universit& Red

Descartes

(Received 20 July 1988; revision received 24 December 1988; accepted 13 January 1989) Key words: a+Fucosidase;

Lymphocytes

After DEAE-Trisacryl chromatography two forms of a+fucosidase have been characterized in normal human lymphocytes. These enzymatic forms were different with respect to their optimum pH, kinetic properties and isoelectric behaviour. After neuraminidase treatment two forms are still observed with a neutral shift in the pZ values. These results suggest that at least two structurally different a-~fucosidase units exist.

Introduction

a+Fucosidase (EC 3.2.1.51) is a lysosomal enzymes that splits off non-reducing terminal L-fucose from glycoproteins and glycolipids [l]. It has been observed in various human tissues and in serum [2-41. It is also present in polymorphonuclear leukocytes [5]. Lysosomal enzymes are believed to be involved in the cellular events preceding blast transformation and proliferation of normal human lymphocytes [6]. Therefore changes in enzymic activity could reflect changes in the biological properties of tumours [7]. In the present report the a-L-fucosidase isoenzyme characteristics were investigated in normal lymphocytes prior to studies on leukemic cells. Correspondence to: Professor M.-J. Foglietti, Laboratoire de Chimie Biologique, U.E.R. de Biologie Humaine et Experimentale, Univexsitt Rend Descartes, 4, Avenue de l’Observatoire, 75 006, Paris, France.

0009-8981/89/$03.50

6 1989 Elsevier Science Publishers B.V. (Biomedical Division)

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Materials and methods 4-Methyhunbelliferyl-cu-L-fucopyranoside was purchased from Sigma Chemical Co. (St Louis, MO, USA); DEAE-Trisacryl from Industrie Biologique Frangaise (Villeneuve-la-Garenne, France); concanavalin A-Sepharose, polybuffer exchange PBE-94, polybuffer 74, ampholines PAG plates and Ficoll-Paque were from Pharmacia-LKB (Bois d’Arcy, France).

Cell separation and enzyme extraction Lymphocytes were isolated from fresh blood of healthy adults (Centre de Transfusion Sanguine, Hapital la PitiC-SalpCtriere, Paris, France). White cells were obtained by passing blood through a cell separator (CS 3000 FENWAL). Separation of lymphocytes from this pellet was achieved by centrifugation in Ficoll-Paque according to the technique of Biiyum [8]. The lymphocytes were then suspended in a 0.15 mol/l NaCl solution (1 ml) and kept frozen at -20°C until used. Cells were disrupted by successive freezing and unfreezing, after which 0.02% Triton X-100 was added (v/v). After 30 min at + 4” C, the mixture was centrifuged at 800 X g for 10 min. The supematant was used as crude enzymatic extract. Prior to chromatographic separations, this extract (1 ml) was dialyzed against a 0.005 mol/l sodium citrate-sodium phosphate, pH 7.6 (100 ml), buffer.

cr-L-Fucosidase activity The enzymatic activity was determined with 4-methylumbelliferyl a-~fucopyranoside at a 1 mmol/l concentration in 0.1 mol/l sodium citrate-sodium phosphate buffer, pH 5.4. After incubation at + 37 o C and alkalinization with a 0.2 mol/l glycine-NaOH, pH 10 buffer, the liberated 4-methylumbelliferone was measured fluorimetrically at an excitation wavelength of 360 nm, and an emission wavelength of 450 nm. Results were expressed as nmol of substrate hydrolyzed/min per 1O’cells or per mg proteins.

DEA E- Trisacryl chromatography Enzymatic extract (1 ml corresponding to 2 X lo8 cells) was chromatographed on a DEAE-Trisacryl column (2.5 X 20 cm) successively equilibrated with 0.5 mol/l and then with 0.005 mol/l sodium citrate-sodium phosphate buffers, pH 7.6. Elution was performed with the last equilibrating buffer until no absorbance at 280 nm was detected. Adsorbed proteins were eluted with a linear NaCl gradient (O-O.25 mol/l). The elution was run at +4”C at a 15 ml/h flow rate, and 2.5 ml fractions were collected. The fractions containing a-r.-fucosidase activity were pooled and freeze-dried or concentrated on an Amicon ultrafiltration system.

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Isoelectrojocusing Isoelectric focusing was performed using 5% acrylamide (245 x 110 x 1 mm) plates (LKB) containing 2.4% ampholytes (pH 3.5-9.5). The anodic and cathodic solutions were respectively 1 mol/l phosphoric acid and 1 mol/l sodium hydroxide. The starting power was 50 mA at 200 V and reached 1000 V after 40 min. Electrofocusing was conducted for 60 min after which the gel was cut into 0.5-cm strips. For each of these strips, the pH was determined and a-L-fucosidase activity was assayed after the gel was put respectively in distilled water for pH determination and in a buffered substrate solution for enzyme activity determinations. Neuraminidase treatment Each a-L-fucosidase fraction isolated after DEAE-Trisacryl chromatography was treated for 4 h at + 37 o C with Clostridium perjringens neuraminidase (EC 3.2.1.18) (Sigma, type VI) prepared in 0.01 mol/l mono-, disodium phosphate buffer, pH 5.6, and then subjected to electrofocusing as described above. A ratio of 1 U of neuraminidase (1 U will liberate 1 nmol of N-acetylneuraminic acid/mm at pH 5.0 and + 37 o C) to 20 U of a-r_.-fucosidase was used. Control samples were treated identically except that buffer was substituted for neuraminidase. Chromatojocusing Chromatofocusing was performed at room temperature on an anion exchanger PBE-94 column (1 x 6 cm) equilibrated with a 0.025 mol/l imidazole buffer, pH 7.4. The continuous gradient (pH 7.4 to pH 4.0) was generated with a PB-74 buffer pH 4.0 (1.5-fold the column volume). After the sample was adsorbed, elution was performed with the PB-74 buffer at a 20 ml h flow rate. 2.5 ml fractions were collected at 4°C.

a-L-Fucosidase separation All lymphocyte a+fucosidase activity was adsorbed on DEAE-Trisacryl column at pH 7.6. When the linear NaCl gradient was applied, two peaks of activity were separated. The first was eluted with 0.06 mol/l NaCl and the other with 0.12 NaCl (Fig. 1). The fractions isolated by ion-exchange chromatography were then characterized. Physicochemical properties The effect of pH on the activity of the two enzymatic forms was studied using 4-methylumbelliferyl-a-r.-fucopyranoside in 0.1 mol/l sodium citrate-sodium phosphate buffers ranging from pH 4-7. The two pH-activity profiles indicate only one maximum respectively at pH 6.2 for fraction I and pH 5.6 for fraction II (Fig. 2). The effect of substrate concentration on the reaction rate was studied with varying amounts of 4-methylumbelliferyl-a-L-fucopyranoside. The apparent K,

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E : 2 ; : 4

0.5

Fig. 1. DEAE-Trisacryl chromatography of normal lymphocytes a+fucosidase. Elution was carried out with: 0.005 mol/l citrate-phosphate buffer, pH 7.6 (A) and buffered NaCl linear gradient (B) (O-O.25 mol/l). Activity was estimated with Cmethylumbelliferyl a-r-fucopyranoside as substrate and expressed *) enzyme activity. ) proteins; (* in arbitrary units. (-

for hydrolysis of the substrate are respectively 0.5 mmol/l and 0.9 mmol/l for fractions I and II (Fig. 3). The two enzymatic forms were further characterized by their isoelectric profiles. The isoelectric profiles of both forms each showed only one peak respectively at pH 6.2 for fraction I and at pH 5.7 for fraction II (Fig. 4). values

Chromatofocusing behaviour To confirm the presence of two cY+fucosidase forms in human lymphocytes, the crude extract was submitted to chromatofocusing. The cu-r_-fucosidase activity was

i

L

* 4

Fig. 2. Effects (0 -0)

5

6

7

PH

of pH on the hydrolysis of Cmethylumbelliferyl a-r-fucopyranoside by fraction and fraction II (A- - -A) obtained after DEAE-Trisacryl chromatography.

I

307

1

2

4

8 1/(4MU-Fuc)mmol/l

Fig. 3. Lineweaver-Burk chromatography (O-

plot of the two a-L-fucosidase fractions obtained after DEAE-Trisacryl 0) fraction I, (A- - -A) fraction II. Enzyme activity was expressed in arbitrary units (fluorescent intensity).

fractionated in two forms (Fig. 5). Their elution pH was respectively pH 6.6 and pH 5.3, very close to the values obtained when the two forms isolated by anion exchange chromatography were electrofocused. These results confirm the presence of two forms of cy-r_-fucosidase in normal lymphocytes. Neuraminidase

treatment

a+Fucosidase is a glycoprotein and the differences observed in pl values of the two forms is likely the results from their sialic acid content. Accordingly the two enzymatic forms were submitted to the action of neuraminidase. This treatment results in a slight neutral shift for the two fractions (~1 6.9 for fraction I and pZ 6.5 for fraction II) (Fig. 4). Discussion

After ion-exchange chromatography, two forms of a+fucosidase have been characterized in normal human lymphocytes. Similarly, two forms of a+fucosidase have been identified from various tissues using different anion exchangers [9-111. These enzymatic forms were different with respect to their optimum pH, kinetic properties and isoelectric behaviour. The optimum pH of the two forms of human leukocyte a+fucosidase are very close to that reported for the enzyme present in several tissues [10,12,13]. In some experiments with crude cu+fucosidase from human polymorphonuclear leukocytes, it~was found a biphasic.curve with optimum pH values at 5.2 and 5.6 [5], but the authors on the basis of their experiment were unable to confirm the presence of two forms as reported in several other human tissues.

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a

6

2 PH

4

6

b t

Fractions

n’

Fig. 4. I@ectric focusing profiles of the two u-t_-fucosidase fractions obtained after DEAE-Trisacryl chromatography without and after neuraminidase treatment. a. Fraction I (0 -a) without and after neuraminidase treatment. b. Fraction II (A- - -A) without and (A- - -A) after (0 -0) neuraminidase treatment. Enzyme activity was expressed in arbitrary units (fluorescent intensity).

Broadhead ,[14] reported that lymphocyte ly-L-fucosidase normally appeared in a single peak at ~16.0-6.5. On the other hand, the author observed in leukemic cells, the apparition of a second form at p1 5-O-5.5. It is difficult to compare our own results to these experiments wheie the ‘normal’ profiles were obtained using cells from subjects in remission. Nevertheless the reported pl values are similar to those ebtaiaed ia the present study.

309

PH

lb

Practionc

Fig. 5. Chromatofocusing on PBE-94 of normal lymphocytes a-L-fucosidase. The pH gradient (- - - - - -) (pH 7.4 to 4.0) was generated by a PB-74 buffer pH 4.0 (1.5 fold the column volume). Enzyme activity was expressed in arbitrary units (fluorescent intensity).

In a recent study, Orlacchio [15] also reported in lymphocytes on two forms of a+fucosidase obtained by automated chromatofocusing. The B form was eluted at pH 6.2, but the pH of elution of the A form was not specified. The difference in pl values observed with primary forms cannot be attributed only to the sialic acid content since after neuraminidase treatment they are still different. These results suggest that at least two structurally different a-L-fucosidase units exists. These data are in accordance to other works. In a study on electrophoretic patterns of cultured lymphocytes a+fucosidase, Turner [16] reported that after neuraminidase treatment two or three bands are still observed. In a same way, two different forms of liver a+fucosidase are observed after neuraminidase treatment [171* These data suggest that the normal human lymphocytes contain two forms of a+fucosidase as reported in several other human tissues [9,10,11,14,15,18]. References 1 Alhadeff JA, Miller AL, Wenass H, Vedvick T, O’Brien JS. Human liver a+fucosidase. 1975:250:7106-7113.

J Biol Chem

310 2 AIhadeff JA, Tennant L, O’Brien JS. Isoenxyme patterns of human liver a+fucosidase during development. Dev Bid 1975;47:319-324. 3 Turner BM. Purification and ch~acte~sation of a-x;fucosidase from human placenta. pH dependent changes in molecular size. Biochim Biophys Acta 1979;578:325-336. 4 Di Ciocco RA, Barlow JJ, Mattei KJ. Substrate specificity and other properties of a-r_-fucosidase from human serum. J Biol Chem 1982;167:714-718. 5 Avila JL, Convit J Studies on human polymorphonuclear leukocyte enzymes. IV. Intracellular distribution and properties of a+fucosidase. B&him Biophys Acta 1974;358:308-318. 6 Tanaka T. Biochemical activities of urine lysosomal. enzymes in T and non T lymphocytes. FEBS Lett 1979;104:161-164. 7 Mensers P, K&ig E, Fink V, Bridges JM. ~-A~tyI-~D-~u~~nida~ activity in normaI and CLL lymphocytes. CIin Chim Acta 1979;93:151-156. 8 myurn A. Isolation of mononucIear &Is and grantdocytes from human blood. Isolation of mononuclear celIs by one centrifugation and of granulocytes by combining centrifugation and sedimentation at 1 g Stand J Clin Lab Invest 1%8;21:77-89. 9 Wiederschain GYa, Kolibaba LG, Rosenfefd EL. Human n-L-fucosidases. CIin Chim Acta 1973;46:305-310. 10 Robinson D, Thorpe R. Human liver a-L-fucosidases. Clin Cbim Acta 1973;47:~3-~7. 11 Bernard M, Foglietti MJ, Percheron F. Properties of two forms of a-r;fucosidase isolated from normal human sera with Iow and high enzymatic activity. Chn Chim Acta 1981;116:91-99. 12 AIhadeff JA, Andrews-Smith GL. Purification and characterization of a+fucosidase from the liver of a fucosidosis patient. Biochem J 1980;187:45-51. 13 WiUems PJ, Romeo E, Den Tandt W, Van EIsen A, Leroy J. pH dependent association-dissociation of high and low activity pIasma n-tfucosidase. Hum Genet 1981;59:115-118. 14 Broadbead DM, Be&y GTN, Moss SE, Bain AD, Eden OB, Sainsbury CPQ. Recognition of abnormal lysosomai enzyme patterns in chiIdhood leukaemia by isoelectric focusing, with speciaI reference to some properties of abnormally expressed components. Leukemia Res 1981;5:29-40. 15 Odacchio A, Maffei C, Rambotti P, Davis S. u-L-Fucosidase activity and &enzyme characteristics anaIyr.ed by chromatofocusing in normal and leukemic Iymphocytes. Anticancer Res 1984,4:431-434. 16 Turner BM, Be&is NG, Turner VS, Him&horn K. Isoenzymes of human a-r-fucosidase detectable by starch gel eiectrophoresis. CIin Chim Acta 1974;57:29-35. 17 Chester MA, Huftberg 8, SjobIad S. A comparison of the n+fucosidase activities of human Liver and serum. Biochim Biophys Acta 1977;485:14?-155. 18 Beier EM, KIyashchitskii BA, Vindershain GYa. Heterogeneity of a+fucosidase from the human liver in affinity chromatography. Biokbimiya 1979;44:1936-1943.