Urinary α-l -fucosidase in cystic fibrosis

Clinica Chimica Acta, 129 (1983) 263-269 Elsevier Biomedical Press

263

CCA 2455

Urinary cu+fucosidase in cystic fibrosis Claude Bourassa, Section de GbPtique

Hong Van Nguyen

and Michel Potier *

Medicale, Hspital Ste- Justine, Universitd de Montrkal, Mont&al, (Canada) (Received

June 10th; revision December

QuPbec H3T 1C5

lOth, 1982)

Summary The binding of the urinary lysosomal enzyme cu-L-fucosidase to free- and Sepharose 4B-bound concanavalin A has been compared in cystic fibrosis (CF) patients and normal controls. The concentration of methyl-cY-D-mannoside necessary to prevent 50% of total a-r_-fucosidase activity to bind to free and bound concanavalin respectively) and A (Ki,50%> was similar for CF (0.68 _+0.20 and 1.3 k 0.3 mmol/l, normal controls (0.53 f 0.18 and 1.9 k 0.5 mmol/l, respectively). The CF and normal urinary a+fucosidase also showed similar pH optima (4.8), K,,_r,, (0.071 and 0.074 mmol/l, respectively) and thermodenaturation curves at 44°C (t,,, = 108 min). We report that the kinetic and the concanavalin A-binding affinity of cY-r_-fucosidase are similar from urine of cystic fibrosis patients and controls.

Introduction Several reports have pointed to abnormalities of cu-L-fucosidase (EC 3.2.1.51) in cystic fibrosis (CF). This lysosomal enzyme has been purified to homogeneity from the liver of CF patients and controls [l]. The CF cu-r_-fucosidase was similar to normal when pH optimum, K_rr, subunit structure and antigenicity were compared, but gas-liquid chromatography analysis revealed altered carbohydrate composition. The three major sugars present in normal purified liver a-r_-fucosidase (mannose, N-acetylglucosamine and sialic acid) were reduced in CF a+fucosidase, to 518, 44% and 32%, respectively, of their normal amounts [ 11. Activities of cx-r_-fucosidase, both higher [2] and lower [3,4] than normal, were found in cultured CF fibroblasts, and reduced activity was found in cultured CF lymphoblasts [5]. It was also reported that CF serum contains lower than normal activity of o-L-fucosidase [6] but discordant findings were subsequently reported [7,8]. More recently, Alhadeff and Watkins [9] reported decreased binding to concanavalin A of CF liver

* Correspondence and reprint requests: Ste-Justine, 3175 Chemin &e-Catherine,

0009-8981/83/0000-0000/$03.00

Michel Potier, PhD, Section Montrkal, Qukbec, Canada,

0 1983 Elsevier Science Publishers

de GCnttique H3T ICS.

Mkdicale,

HBpital

264

cw-L-fucosidase compared to normal, and Maler et al [5] also obtained similar results with cultured lymphoblasts from CF patients. Urine is easily obtained and several lysosomal enzymes, including cu-L-fucosidase, have been detected in it [lo]. Small molecular weight inhibitors of the lysosomal glycosidases are removed by simple dialysis and the activity of these enzymes can then be determined under linear assay conditions [ 111. In view of discordant findings concerning the a-L-fucosidase in CF, we assayed this enzyme in CF and normal urine and also determined its binding affinity to concanavalin A.

Materials and methods Materials The concanavalin A was purchased from Miles Laboratories Ltd. (Elkhart Lake, IN, USA) and the concanavalin A-Sepharose 4B from Pharmacia Fine Chemicals Co. (Uppsala, Sweden). The 4-methylumbelliferyl derivative of a-L-fucose was obtained from Koch-Light Laboratories Ltd. (Colnbrook, Bucks, UK). The methyla-D-mannoside (grade II) was obtained from Sigma Chemical Co. (St. Louis, MO, USA). Urine collection and concentration Urine samples from 13 CF patients and 11 controls were collected from single micturitions, placed immediately at 0-4°C and made 1 mmol/l with NaN, to prevent bacterial growth. After removal of an aliquot for creatinine determination (alkaline picric acid method of MacFate et al [12]), solid ammonium sulfate was added to 50 or 75 ml of urine until a final saturation of 60% ammonium sulfate was reached. The precipitate was collected by centrifugation at 12000 X g for 15 min, dissolved in 8 ml of 0.03 M citrate buffer, pH 6, 0.1% (v/v) Triton X-100, 0.1 mol/l NaCl and dialyzed overnight against 1 liter of the same buffer at 4°C [ 111. This preparation was used for concanavalin A binding studies. The a-L-fucosidase activity was stable in both undialyzed urine (in the presence of 1 mmol/l NaN,) and dialyzed urine at 0-4°C for at least 4 days. In contrast, the enzyme lost most of its activity when frozen at - 2O’C. a-L.-Fucosidase assay cY-L-Fucosidase was assayed in the presence of 0.2 mol/l acetate buffer, pH 4.8 and 0.2 mmol/l 4-methylumbelliferyl-o-r_-fucoside as substrate in a total volume of 0.2 ml. The enzyme was incubated for 45 min at 37°C and the reaction was stopped by the addition of 1 ml 0.133 mol/l glycine buffer, pH 10.7,0.06 mol/l NaCl, 0.083 mol/l Na,CO,. Fluorescence was read with an Aminco-Bowman fluorometer (American Instruments Co.), excitation, 365 nm; emission, 450 nm. One unit of enzyme activity was the amount of enzyme that hydrolyzed 1 nmol of substrate per min. Concanavalin A-Se&rose 4B binding This study was performed essentially

as described

by Alhadeff

and Watkins

[9]

265

except that concanavalin A bound to Sepharose 4B was used instead of free concanavalin A. To 0.1 ml of a 50% (v/v) suspension of concanavalin A-Sepharose 4B in 0.01 mol/l phosphate buffer, pH 7, 0.5 mol/l NaCl, 0.02% (w/v) NaN,, were added various amounts of methyl-a-~-mannoside to obtain final concentrations between 0.25 and 40 mmol/l in the same buffer after the addition of urine. After 10 min at room temperature, 0.50 ml of concentrated urine prepared as described above and containing between 0.1 and 0.8 unit of a-L-fucosidase activity was added. Bovine serum albumin was then added to a final concentration of 40 mg/ml and after 60 min at 4”C, the gel was centrifuged at 100 x g for 3 min. The supernatant was assayed for rr-r_-fucosidase activity. The reciprocal of cu-r_-fucosidase activity versus the reciprocal of methyl-rw-D-mannoside concentration was plotted and the K ,,50w value was determined graphically [9]. Free concanavalin A binding This experiment was carried out as with the concanavalin A-Sepharose 4B except that the methyl-ff-D-mannoside con~ntration was varied between 0.3 and 10 mmol/l (final concentrations after the addition of urine). Dialyzed and concentrated urine (0.20-0.22 units of a-L-fucosidase activity) was incubated with free concanavalin A (0.6 mg in a total volume of 0.4 ml) for 2 h at 37’C and the precipitate was centrifuged at 13000 X g for 45 min at 22°C. a-r_-Fucosidase activity was determined in the supematant fraction. Determination of apparent MichaZ;lis-Menten constant K m.app was determined under standard assay conditions with 4-methylumbelliferyl-cu-t-fucoside as substrate except that substrate concentration was varied between 0.04 and 0.2 mmol/l. A Lineweaver-Burk plot was used for graphical deter~nation of Km,+_,. The substrate concentrations were selected to ensure even distribution of points in the reciprocal plot. Heat inactivation of a-t-fucosidase Dialyzed urine containing 0.1-0.2 unit of a+fucosidase activity in 0.03 mol/l citrate buffer, pH 6, 0.1 mol/l NaCl, 0.1% (v/v) Triton X-100, and 10 mg/ml bovine serum albumin was pre~ncubated at 44°C. At intervals of 60 min, 0.1 ml aliquots were taken and immediately cooled at 0°C. The aliquots were then equilibrated at 37°C for 3 min and cY+fucosidase activity was assayed as described above. Results

Great care was taken to ensure that the cY-r_-fucosidaseactivity was assayed under linear conditions with respect to time and amount of enzyme. The urine samples must be dialyzed to remove small molecular weight inhibitors of a+fucosidase. After dialysis, reasonably linear relationships with the volume of urine in the incubation medium (up to 100 ~1 of dialyzed urine) was obtained for a-L-fucosidase. However, we cannot rule out the possibility that high molecular weight undialyzable

266

___ Normal -

0

2

1

3

a

Cystic fibrosis

5

TIME (Hours) Fig. 1. Thermodenaturation urinary a-L-fucosidase.

TABLE

curves at 44°C and pH 6 of normal

(O-

*)

I

URINARY a-~-FUCOSIDASE TION OF WL-FUCOSIDASE CANAVALIN A

ACTIVITY BINDING

AND Ki,sos FOR METHYL-a-D-MANNOSIDE INHIBITO FREEAND SEPHAROSE 4B-BOUND CON-

Controls

Cystic fibrosis

Age, sex *

Free concanavalin 7, M 9, F 10, F 16, F 19, F Mean 12.2 SD 5.1 Sepharose 6, M 8, F 10, F 9, F 14, F 18, F

(O---O) and cystic fibrosis

4B-bound

Enzyme activity (units/mg creatinine)

Ki,scs (mmol/l)

Age, sex *

Emyme activity (units/mg creatinine)

Ki,ssa (mmol/l)

0.30 0.21 0.13 0.14 0.19 0.19 0.07

0.41 0.44 0.83 0.57 0.41 0.53 0.18

7, F 12, M 15, M 17, F 19, F 14.0 4.2

0.09 0.23 0.12 0.13 0.22 0.16 0.06

0.75 0.49 0.56 1.00 0.61 0.68 0.20

0.18 0.15 0.04 0.15 0.15 0.07

1.9 2.4 2.6 1.5 2.0 1.2

5, F 8, M 8, M 10, F

0.12 0.06

1.9 0.5

0.18 0.11 0.15 0.10 0.09 0.09 0.04 0.15 0.11 0.04

1.3 1.6 1.0 1.2 1.0 1.3 1.2 1.8 1.3 0.3

A

concanavalin

Mean 10.8 SD 4.4 *Age in years. M, male; F, female.

A

9, M 13, M 15, F 16,M 10.1 4.0

267

2

I

0

I/(METHYL-u-O-MANNOSIDE)

1

2

3

(~io~t4-~)

Fig. 2. Double reciprocal plot of a+fucosidase activity in the supematant fraction versus methyl-o-nmannoside concentration. A constant amount of free concanavalin A was used in the medium (0.6 mg). The urine from a normal control was concentrated by 60% saturated (NH4)aS04 precipitation as described in ‘Materials and methods’. A typical plot is represented. The error on enzyme assays in the presence of methyl-o-D-mannoside was determined to be less than 3%.

inhibitors may still be present after dialysis. The urinary cY+fucosidase activity was efficiently concentrated by (NH4)2S04 precipitation since about 97% of activity was recovered. The Triton X-100 added to the buffer helped to resolubilize the precipitated enzyme without affecting the activity. Kinetic properties of a+fucosidase in three CF urines and three controls were very similar: K,,,. app was 0.071 f 0.018 and 0.074 k 0.019 mmol/l (mean f SD), respectively, and pH optimum was 4.8 in both CF urine and control. Fig. 1 shows denaturation curves of cY+fucosidase at 44°C in CF urine and control. The half-life of the enzyme was about 108 min in both urine samples. Conditions for optimum binding of urinary cy-L-fucosidase with both free- and Sepharose 4B-bound concanavalin A were established. In concentrated urine, it was found that the cx-r_-fucosidase activity was stable for at least 2 h at 37°C in the presence of 40 mg/ml bovine serum albumin. The optimal NaCl concentration for precipitation with free concanavalin A was 0.22 mol/l. The optimum reaction time between concanavalin A and cu+fucosidase was 2 h with both normal and cystic fibrosis urine in the presence of 1 or 4 mmol/l methyl-cY-D-mannoside. It was also found that maximum cu+fucosidase activity was precipitated with 0.2-0.6 mg concanavalin A in the incubation medium. In both control and CF urine, about lo-17% of total a+fucosidase activity cannot be precipitated even with higher

268

quantities of concanavalin A. It is not known whether this ‘non-reactive’ (Y-Lfucosidase activity represents a different form of the enzyme with altered sugar content. However, no difference in this amount was observed between control and CF urine. All of the a-L-fucosidase activity precipitated with concanavalin A can be recovered in the supernatant by addition of methyl-a-o-mannoside to a final concentration of 40 mmol/l. Even at this relatively high methyl-a-D-mannoside concentration, the a-L-fucosidase activity was not inhibited. The double reciprocal plot of a-L-fucosidase activity in the supernatant fraction versus methyl-a-D-mannoside concentration, used to determine Ki,SO’g, is shown in Fig. 2. The methyl-a-o-mannoside concentrations used were selected to ensure even distribution of points in the reciprocal plot. Table 1 gives results of a-L-fucosidase activity and Ki,50S values in a series of CF and control urines. a-L-Fucosidase activities and K,,5,,W values were not significantly different in CF and control urine (Student’s t test, p < 0.01). Discussion Alhadeff and Watkins [9] reported decreased binding of CF liver a-L-fucosidase to concanavalin A and Maler et al [5] obtained similar results with a+fucosidase of CF cultured lymphoblasts. This paper indicates that binding of urinary a+fucosidase to free and Sepharose 4B-bound concanavalin A is unaffected in CF patients (Table I). There is also no statistically significant difference between a-L-fucosidase activity in CF and control urine ( p c 0.01). From all the data recently collected suggesting abnormalities of glycoprotein structure in CF, two questions can be raised: (1) are these abnormalities found in all glycoproteins? and (2) are they generalized to all organs? In this report, we used an a-L-fucosidase-concanavalin A binding test, the lectin being used as a probe to detect changes in the carbohydrate portion of a-r_-fucosidase. The results presented in this paper suggest no change in the a-r.,-fucosidase binding affinity to concanavalin A in CF urine. It is believed that the major portion of urinary lysosomal enzymes originate from the kidney [ll]. It is possible that the abnormality may not be expressed in this organ or may not be detected by the concanavalin A binding test. was 2- to 3-fold lower with It is interesting that the KiqSOWfor methyl-a-D-mannoside free than with Sepharose 4B-bound concanavalin A. Apparently, the attachment to Sepharose 4B modifies the binding properties of concanavalin A either by increasing the affinity for a+fucosidase, or by decreasing that for methyl-a-D-mannoside or both. Acknowledgements This work is supported by a research grant, and a studentship to C.B., from the Canadian Cystic Fibrosis Foundation. The authors would like to thank Drs. Jean-Guy Lapierre and Roger Lasalle, the Cystic Fibrosis Clinic of this Hospital, and the patients and parents attending the Clinic for their cooperation.

269

References 1 Alhadeff JA, Watkins P, Freeze H. Purification and characterization of altered cystic fibrosis liver a-t_-fucosidase, Clin Genet 1978; 13: 417-424. 2 Scanlin, TF. Matacic SS, Pace M, Santer UV, Glick MC. Abnormal distribution of cr-L-fucosidase in cystic fibrosis: increased activity in skin fibroblasts. Biochem Biophys Res Commun 1977; 79: 868-876. 3 Hosli P, Vogt E. Cystic fibrosis: leakage of lysosomal enzymes and of alkaline phosphatase into the extracellular space. Biochem Biophys Res. Commun 1977; 79: 741-748. 4 Butterworth J, Guy GJ. Primary amniotic fluid cell, skin fibroblast and liver a-L-fucosidase and its relation to cystic fibrosis. Clin Chim 1979; Acta 92: 109- 116. 5 Maler T, Duthie M, Alon M, Riordan JR. a+Fucosidase is quantitatively reduced in cultured lymphoblasts from patients with cystic fibrosis. J Biol Chem 1981; 256: 1420-1427. 6 Scanlin TF, Matacic SS, Glick MC. Abnormal distribution of o-L-fucosidase in cystic fibrosis: decreased activity in serum. Clin Chim Acta 1979; 91: 197-202. 7 Alhadeff JA, Watkins P. Cystic fibrosis serum a-L-fucosidase: confirmation of normal activity levels and normal kinetic and isoelectric focusing properties. Clin Chim Acta 1980; 105: 131- 135. 8 Riordan JR, Alon N, Duthie M, Maler T, Buchwald M. Hydrolytic enzymes as markers for cystic fibrosis. In: Sturgess JM, ed. Perspectives in cystic fibrosis. Toronto: Imperial Press, 1980: 313-321. 9 Alhadeff JA, Watkins P. Differential concanavalin A binding of cystic fibrosis and normal liver a-L-fucosidase. Biochem Biophys Res Commun 1979; 86: 787-792. 10 Tappel AL. Lysosomal enzymes and other components. In: Dingle JT, Fell HB, eds. Lysosomes in biology and pathology, Vol. 2. Amsterdam: North-Holland Publishing Co., 1969: 209-213. 11 Paigen K, Peterson J. Coordinacy of lysosomal enzyme excretion in human urine. J Clin Invest 1978; 61: 751-762. 12 MacFate RP, Cohn C, Eichelberger L, Cooper J. Symposium on azotemia. Am J Clin Path01 1954: 24: 511-571.