Int. J. Btochem. Vol. 19, No. 4, pp. 395-397, 1987 Printed in Great Britain. All fights reserved
0020-711X/87 $3.00+0.00 Copyright © 1987 Pergamon Journals Ltd
fl-D-MANNOSIDASE IN HUMAN POLYMORPHONUCLEAR LEUKOCYTES AND LYMPHOCYTES: A COMPARATIVE STUDY B. COLIN) M. BERNARD,I M. J. FOGLIETTI 2. and F. PERCHERON 1 ~Laboratoire Central de Biochimie Htpital de la Pitit, 83, Boulevard de l'Htpital, 75 013 Paris, France 2U.E.R. de Biologie Humaine et Exptrimentale, Uulversit6 Rent Descartes, 4, A. de l'Observatoire, 75 006 Paris, France [Tel. 4329-12-08]
(Received 28 July 1986) Al~lract--l. All lymphocytcs and polymorphonuclear leukocytes (PMNL) fl-D-mannosidas¢ activities are adsorbed on DEAE-Trisacryl column at pH 7.0. 2. Only one form is eluted with a 0,15 M linear gradient. 3. The two enzymes isolated from either type of cells exhibit similar properties. 4. The chromatographic profiles of fl-n-mannosidasc from leukemic lymphocytes (chronic lymphoid leukemia and hairy cells leukemia) differ from the normal ones by the presence of a more acidic minor form.
Cells were disrupted by successive congelation and decongelation, after which 0.02% Triton X-100 was added (vol/vol). After 30rain at +4°(2, the mixture was centrifuged at 800g for 10rain. The supernatant was used for enzyme assays. fl-D-Mannosidase activity. The enzymatic activity was determined with either p-nitrophenyl-~-D-mannopyranoside (Sigma) or 4-methylumbelliferyl-fl-n-mannopyranoside (Sigma) as substrates. p-Nitropbenyl-fl-D-mannopyranoside was used at a 1.5mM concentration in 0.1 M citrate-phosphate buffer, pH 4.5. After incubation at +37°C, the reaction was stopped by the addition of 0.1 M glycin-NaOH buffer, pH 10.7. p-Nitrophenol was estimated coiorimetrically at 405 nm. 4-Methylumbelliferyl-fl-D-mannopyranosidewas used at a 1 mM concentration in 0.1 M citrate-phosphate buffer, pH 4.5. After incubation at +37°C and alkalinization, the liberated 4-methylumbelliferone was estimated fluorimetrically at an excitation wavelength of 360 nm, and an emission wavelength of 450 nm. Results are expressed as nanomoles of substrate hydrolysed per rain per 104 cells.
INTRODUCTION
fl-D-Mannosidase (fl-D-mannoside mannohydrolase EC 3.2.1.25) is an acidic hydrolase involved in the catabolism of N-glycoprotein glycan moiety. Lysosomal enzymes are believed to participate in the cellular events preceding blast transformation and proliferation of normal h u m a n lymphocytes (Tanaka, 1979; Douglas et al., 1973). Theoretically, since lysosomal enzymes may alter the interactions of cell membrane with its environment, alterations in enzyme activity could reflect some of the changes observed in malignant transformation (Meusers et a/., 1976; Crockard et al., 1979). In the present report, characteristics of fl-D-mannosidase were investigated in normal polymorphonuclear leukocytes (PMNL) and lymphocytes. On the other hand, we attempted to determine the enzymatic profile of leukemic cells. MATERIALS AND METHODS Normal cells were isolated from fresh blood of healthy adults (Centre de Transfusion sanguine, H6pital PititSalp~tritre, Paris). Leukemic cells were obtained from patients with chronic lymphoid leukemia (CLL) and hairy cells leukemia (HCL).
Isolation of cells Lymphocytes. Normal lymphocytes were separated from deleukocytation pellets by centrifugation in Ficoll-Paque (Pharmacia, Fine Chemicals Uppsala, Sweden) according to the technique of B6yum (1968). The pellet is then suspended in a 0.9% NaCI solution (1 ml) and kept frozen until used. Leukemic lymphocytes were obtained from heparinized venous blood (2 ml) using the same technique as for normal ones.
Polymorphonuclear leukocytes (PMNL ). Normal PMNL were obtained from heparinized venous blood by centrifugation in Monopoly medium (Pharmacia Fine Chemicals, Uppsala, Sweden). *To whom all correspondence should be addressed.
DEAE-Trisacryl chromatography Enzymatic extract (1 ml corresponding to 125.106 cells for lymphocytes, and to 300.106 cells for PMNL) was chromatographed on a DEAE-Trisacryl (Industrie Biologique Franfaise, Villeneuve-La-Garenne, France) column (3.4 x 6cm) equilibrated with 0.005 M citrate--phosphate buffer, pH 7.0. The elution was performed with the same buffer until no absorbance at 280 nm was detected. Adsorbed proteins were eluted with a linear NaC1 gradient (0-0.25 M). The elution was run at +4°C at a 60mlh -t flow rate and 3 ml fractions were collected. The fractions containing fl-D-mannosidase activity were pooled and concentrated by ultrafiltration on Amicon gains. Isoelectric focusing Isoelectric focusing was performed using 5% acrylamide plates (LKB) containing 2.4% ampholytes (pH 3.5-9.5). The anodic and cathodic solutions were, respectively, 1 M phosphoric acid and 1 M sodium hydroxide. The starting power was 50 rnA at 200 V and reached 1000 V after 40 rain. Electrofocusing was conducted for 60 rain after which the gel was cut into 0.5 cm pieces.
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For each of these fractions, the pH was determined and fl-n-mannosidase activity was assayed with 4-methylumbelliferyl-fl-D-mannopyranoside as substrate after the gel was put respectively in distilled water and in 0.1 M citrate-phosphate buffer pH 4.2. Molecular weight determination Gel filtration. Mannosidase containing fractions from DEAE-Trisacryl chromatography were adsorbed on a Ultrogel ACA 34 (Industrie Biologique Fran(;aise) column (1.55 x 90 cm) equilibrated with 0.005 M citrate-phosphate buffer pH 7.0. The flow rate was 12mlh -I, and 3ml fractions were collected. Column calibration was performed with chymotrypsinogen A, albumin, aldolase, catalase and ferritin. SDS polyaerylamide gel electrophoresis. Gel electrophoresis was peformed according to the technique of Laemmli (1970) in the presence of sodium dodecyl sulphate (SDS). Gels contained 4-15% of acrylamide in 0.38M glycin 0.05 M Tris buffer pH 8.3. Proteins were stained with Coomassie blue. Molecular weight markers (mol. wt 20,100--94,000) were used for calibration. Enzymatic studies The optimum pH was determined with the p-nitrophenylfl-D-mannopyranoside in 0.1 M citrate-phosphate buffers of varying pH values (3.0-7.0). The apparent Michaelis constant (Kin) was determined graphically by the usual Lineweaver-Burk method using p-nitrophenyl-fl-D-mannopyranoside and 4-methylumbelliferyl-fl-D-mannopyranoside as substrates. For thermal stability studies, the enzymatic fraction was preincubated at +45 and +60°C for periods up to 45rain, after which the substrate (p-nitrophenyl-fln-mannopyranoside) was added and the samples were assayed as previously described.
RESULTS AND DISCUSSION Normal cells All lymphocyte and P M N L activities are adsorbed on DEAE-Trisacryl column at pH 7.0. Only one peak of activity is eluted with a 0.15 M NaCI concentration for both types of cells (Fig. 1). Similar results were reported with serum as well as urine by Chester and Ockerman (1981) and Bernard et al. (1986) using ion-exchange chromatography. The pH dependence of fl-D-mannosidase activity in P M N L and lymphocytes, as studied with the p nitrophenyl-fl-D-mannopyranoside, showed an unimodal pattern with a sharp peak between 4.0 and 4.5. These pH values are close to those reported by different authors for fl-o-mannosidase from various origins (Bartholomew and Perry, 1973; Chester and Ockerman, 1981; Panday et al., 1984; Bernard et al., 1986). We found no evidence for a neutral form of fl-D-mannosidase as reported in goat liver (Dawson, 1982). The substrate concentration dependence of fl-o-mannosidase activity shows Michaelis-Menten kinetics with an apparent Km of 0.95 mM for both P M N L and lymphocytes, which is in agreement with usual Km values for lysosomal hydrolases. The isoelectric profiles of both enzymes show only one peak at pH 4.7 (Fig. 2). This pI value can be compared to that of the fl-v-mannosidase from serum and urine (Bernard et al., 1986).
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Fraction No. Fig. I. DEAE-Trisacryl chromatography of normal lymphocytes (a) and P M N L (b) #-D-mannosidase. Elution was carried out with: 0.005 M citrate-phosphate pH 7.0 (A), and buffered NaCI linear gradient (B) (0-0.25 M). Activity was estimated with methyl-4-umbeUiferyl-fl-~mannopyranoside as substrate and expressed in arbitrary units (fluorescent intensity). - Proteins; • enzyme activity.
The molecular weight estimated by gel filtration is 71,000 +_ 3000 for both enzymes. On the other hand, the value estimated by SDS polyacrylamide electrophoresis performed without fl-mercaptoethanol is 70,000. The same value is obtained in the presence (a)
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Fig. 2. Isoclectric profiles of normal lymphocytes (a) and PMNL (b)/~-D-mannosidase. Enzyme activity is expressed in arbitrary units.
397
#-D-Mannosidas¢ A
B
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(Fig. 3), and characterized by a similar acidic isoelectric point (pI = 4.25) for the two types of pathological cells. To assess a diagnostic or prognostic value to this minor form, it will be necessary to extend our study to a greater number of cases, and to follow the patients during their treatment.
(a)
Acknowledgement--We are grateful to Dr J. J. Fournel m
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(Centre de Transfusion sanguine, Groupe Hospitalier PititSall~tritre, Paris) for the gift of blood samples.
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2o 30 40 Fraction No. Fig. 3. DEAE-Tris~ryi chromatography of LLC lympho-
cytes (a) and HCL cells Co) #-n-mannosidase. Elution was carried out with 0.005 M citrate-phosphate buffer pH 7.0 (A), and buffered NaCl linear gradient (B) (0-0.25 M). Activity was expressed in arbitrary units (fluorescent intensity). Proteins; • enzyme activity. of fl-mercaptoethanol. This could indicate that the enzyme is composed of only one polypeptide chain. In serum and urine, higher molecular weight values (100,000 + 10,000) were observed (Bernard et al., 1986). This discrepancy can be due to the predominance of the precursor form of the enzyme in serum and urine as reported for N-acetyl-#n-glucosaminidase by Zuhlsdorf et al. (1983), and a-giucosidase by Oude Elferink et al. (1984). Only immunological studies using monoclonal antibody could distinguish between precursor and mature forms. Thermal denaturation at two temperatures ( + 4 5 and + 60°C) leads to a linear decrease of activities which is in agreement with the existence of one enzymatic form. Moreover, profiles obtained show a comparable loss of activity of both enzymes. Leukemic cells
A difference in the chromatographic profiles of #-D-mannosidase enzymes between normal and leukemic lymphocytes (CLL and HCL) is observed. In addition to the normal form, leukemic cells exhibit a minor one, elutcd for 0.23 M NaCI concentration
Bartholomew B. A. and Perry A. L. (1973) The properties of synovial fluids p-mannosidase activity. Biochem. hiephys. Acta 315, 123-127. Bernard M., Sioud M., Percheron F. and Foglietti M. J. (1986) #-Mannosidasc in human serum and urine: a comparative study. Int. J. Biochem. 18, 1065-1068. B6yum A. (1968) Isolation of mononuclear cells and granulocytes from human blood. Isolation of mononuclear cells by one centrifugation and of granulocytes by combining centrifugation and sedimentation at 1 g. Scand. J. olin. Lab. Invest. Suppl. 21(97), 7%89. Chester A. M. and Ockerman P. A. (1981) Studies on human p-mannosidase. VI Int. Symposium on Glycoconjugates, Tokyo. Crockard A. D., Lewis M. H. R. and Bridges J. M. (1979) N-acetyl-#-D-glucosaminidase activity in normal and CLL lymphocytes. Clin. chim. Acta 93, 151-156. Dawson G. (1982) Evidence for two distinct forms of mammalian p-mannosidase. J. biol. Chem. 257, 3369--3371. Douglas S. D., Cohnen C., Konig E. and Brittinger C. (1973) Lymphocyte lysosomes and lysosomal enzymes in CLL. Blood 511-518. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. Meusers P., Konig E., Fink V. and Brittinger C. (1976) Lysosomal acid phosphatase, difference between normal acid CLL T and B lymphocytes. Blut 33, 313-318. Oude Elferink R. P. J., Brouwer-Kelder E. M., Surya I., Strijland A., Kroos M., Reuser A. J. J. and Tater J. M. (1984) Isolation and characterization of a precursor form of lysosomal a-glucosidase from human urine. Fur. J. Biochem. 139, 489--495. Panday R. J., Van Diggelen O. P., Kleijer E. W. J. and Niermeijer M. F. ( 198 I) # -Mannosidas¢ in human leukocytes and fibroblasts. J. Inher. Metab. D/~. 7, 155-156. Tanaka T. (1979) Biochemical activities of nine lysosomal enzymes in T and non T lymphocytes. FEBS Lett. 104, 161-164. Zuhlsdorf M., Imart M., Hasilik A. and Von Figura K. (1983) Molecular forms of #-hexosaminidase and cathepsin D in serum and urine of healthy subjects and patients with elevated activity of lysosomal enzymes. Biochem. J. 213, 733-740.