Purification and properties of human urinary β-d -mannosidase

BB

ELSEVIER

Biochi P~mic~a et BiophysicaA~ta Biochimica et Biophysica Acta 1293 (1996) 9-16

Purification and properties of human urinary fl-D-mannosidase Raffaella Guadalupi a, Maguy Bernard b, Antonio Orlacchio b, Marie Jos6 Foglietti b, Carla Emiliani a,* a Dipartimento di Eiologia Cellulare e Molecolare, Universit?t degli Studi di Perugia, via del Giochetto, 06126 Perugia, Italy b Universit£ Ren[ Descartes, Laboratoire de Biochimie et Glycobiologie, UFR des Sciences Pharmaceutiques et Biologiques, 4 Acenue de l'Obser~atoire, 75006 Paris, France

Received 8 May 1995; revised 3 October 1995; accepted 19 October 1995

Abstract

Two forms of the lysosomal enzyme /3-mannosidase were identified and purified from human urine. The purification strategy employed allowed sufficient quantities of both forms to be obtained for subunit analysis and for further characterizations. The two fl-mannosidases were identified as /3-mannosidase B and A, in order of their elution from an ion-exchange column. In all samples examined, the A form was predominant, and the B / A ratio was consistently 0.14. The two forms displayed the same optimum pH (i.e., 4.3) and both were retained by a Concanavalin-A Sepharose column, but showed different isoelectric points, molecular masses and subunit compositions. Native- and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses of pure fl-mannosidases B and A suggest that active protein B (160 kDa) consists of three subunits, one 75 kDa and two 49 kDa subunits. Protein A is smaller and appears to be composed of three subunits of 75 kDa, 49 kDa and 37 kDa. Two forms of/3-mannosidase, exhibiting a chromatographic behaviour comparable to the urinary forms, were also detected in human kidney. Nevertheless, in this tissue their relative distribution was different, the B / A ratio being 19. Keywords: /3-Mannosidase; Purification; Urine; Kidney; (Human)

I. Introduction

Lysosomal glycohydrolLase /3-mannosidase (/3-D-mannoside mannohydrolase, EC 3.2.1.25) is involved in the catabolism of mannose containing glycoconjugates (especially glycoproteins) in which it catalyses the hydrolysis of the fl-mannosidic linkage. Inherited deficiencies of flmannosidase, /3-mannosid,asis, have been described in Nubian goats [1-3], Saler cattle [4] and humans [5-9]. Disaccharide Man/3 1-4GlcNAc, trisaccharide Man/3 14GlcNac/3 1-4GlcNAc, and other more complex oligosaccharides have been seen to accumulate in the tissues and urine of affected animals [10]; in humans, disaccharide is the major storage produtct [11]. To date, /3-mannosidase has been a relatively unstudied enzyme and very little is known about its properties or subunit organization. The enzyme has been purified from guinea pig liver,

* Corresponding author. Fax: +39 75 5853443. 0167-4838/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 0 1 6 7 - 4 8 3 8 ( 9 5 ) 0 0 2 2 5 - 1

goat kidney and bovine kidney [12-14] and partially characterized from several other mammalian sources [15-18]. In all instances, the enzyme has been referred to as a unique acidic (lysosomal) form, with a molecular mass determined in the range of 80-120 kDa. Structural characterization of the purified enzyme led to the visualization of a single 110 kDa peptide in guinea pig liver [18], two 90 and 100 kDa peptides in goat kidney [13] and three 110, 100 and 84 kDa peptides in bovine kidney [14]. In humans, the only attempts to purify /3-mannosidase have been performed by using the placenta as the source of enzyme activity [19], but final preparations of the enzyme were found to be contaminated by other lysosomal glycohydrolase activities. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), purified placental fl-mannosidase showed several low-molecular weight peptides ranging from 98 to 57 kDa. Further characterizations of this enzyme are indispensable in humans in order to better understand the heterogeneity of /3-mannosidosis symptoms and perform precise enzymatic analyses for diagnostic purposes. To this end we

10

R. Guadalupiet al. / Biochimica et BiophysicaActa 1293 (1996) 9-16

found it necessary to establish a strategy to obtain sufficient quantities of pure enzyme from a readily available human source. In the present investigation we describe and characterize two acidic molecular forms of /3-mannosidase from normal human urine, which have been efficiently purified to homogeneity by an effective protocol of purification. Both forms are correlated to putative counterparts identified in human kidney.

2. Materials and methods

2.1. Materials 4-Methylumbelliferyl-fl-mannopyranoside (4MUffman), 4-methylumbelliferone, Sephacryl S-200 HR and gel filtration molecular weight markers were from Sigma (St. Louis, MO, USA). DE-52 DEAE-cellulose was from Whatman (Maidstone, Kent, UK). The Bio-Rad protein assay, ampholine and isoelectric point (pI) standards (pH range 4.6-9.6) for isoelectric focusing, SDS-PAGE molecular weight standards (10-100 kDa) and Silver Stain kit were from Bio-Rad (Richmond, CA, USA). Sephadex G-25 was from Pharmacia (Uppsala, Sweden). All other reagents were of analytical grade. 2.2. Urine collection Urine was collected daily, in sterile containers without preservatives, from healthy adults, mean age 30 (from 25 to 45) years, and stored at 4°C and processed no later than 1 day after collection. 2.3. Enzyme assay and protein determination Enzyme activity was measured using 3 mM 4MUff-man in 0.1 M citric acid/0.2 M disodium phosphate buffer (pH 4.3). Fluorescence of the liberated 4-methylumbelliferone was measured on a Perkin-Elmer LS 3 fluorimeter (excitation, 360 nm; emission, 446 nm). One unit (U) is the amount of enzyme that hydrolyses 1 /zmol of substrate/min at 37°C. The protein content was determined according to Bradford [20] by using crystalline bovine gamma globulin as the standard. Specific activity was expressed as enzyme units per mg of protein. 2.4. Separation and purification of urinary fl-mannosidases Solid ( N H 4 ) 2 S O 4 w a s added to urine (10 1 at a time) to give a final saturation of 25%, and stirred gently at 4°C for 16 h. The precipitate was removed by centrifugation at 4000 rpm for 30 min at 4°C and discarded. The supernatant was brought to 70% saturation w i t h ( N H 4 ) 2 S O 4 , stirred at 4°C for 7 h and the precipitate recovered by

centrifugation as before. This precipitate was dissolved in distilled water (about 100 ml) and loaded into a Sephadex G-25 (3 cm diam X 100 cm) column equilibrated with water. Fractions (10 ml) were collected. Enzymatically active fractions were pooled, adjusted to 0.01 M sodium phosphate buffer (pH 6.0) plus 0.1 M NaC1 and loaded into a Sephacryl S-200 HR column (1.6 cm diam. X 86 cm), equilibrated and eluted with the above buffer. Enzymatically active fractions arising from two partial purifications (20 litres of urine in total) were pooled at this stage and dialysed against 0.01 M sodium phosphate buffer (pH 6.0) then loaded into a DEAE-cellulose column (5 ml volume) equilibrated with the above buffer. After washing the column until the A%0 n m reached zero, elution was continued with 0.1 M NaC1 in the above buffer, again until the A 280 nm reached zero. This elution gave the first enzymatically active peak. The column was then developed with a linear gradient of NaCI (0.1-0.3 M) in 60 ml of buffer, and a second peak of activity was eluted at this stage. Finally the column was eluted with 1.0 M NaC1 in the same buffer. In preparation of preparative gel electrophoresis, the two enzymatically active peaks from DEAE-cellulose were separately concentrated to 0.5 ml and dialysed against water by vacuum-dialysis (Sartorius apparatus, Sartorius GmbH, GiSttingen, Germany). 2.5. Preparative gel electrophoresis in a gradient of polyacrylamide Electrophoresis was carried out in slab gels (16 cm × 20 cm X 3 cm) containing a gradient of polyacrylamide from 5 to 20% (Bio-Rad Protean II xi Slab Cell apparatus). The gels were pre-electrophoresed for 15 min at 125 V, followed by 20 rain at 70 V. The buffer consisted of 10.75 g of Tris, 5.04 g of boric acid and 0.93 g of EDTA per litre of distilled water. The two concentrated peaks of enzyme activity obtained as above and corresponding to /3-mannosidase A and B were made to 50% ( w / v ) compared to sucrose and applied to gradient polyacrylamide gels. Electrophoresis was carried out at 90 V until the samples were taken out of the sample wells and placed in the gradient gel, and was then continued at 125 V for 24 h. The gels were subsequently removed, incubated for 15 min at 4°C in 0.1 M citric acid/0.2 M disodium phosphate buffer (pH 4.3) and then stained briefly for/3-mannosidase activity by incubation at 37°C with the appropriate fluorogenic substrate 1 mM in the above buffer. In order to recover enzyme activity, the active bands were recovered and extracted by electroelution (Bio-Rad electroeluter, model 422) in 25 mM Tris, 192 mM glycine buffer (pH 8.3). The recovered enzyme was used for further characterizations. 2.6. Separation and partial purification of ~-mannosidases from human kidney A sample (10 g) of normal human kidney was obtained from autopsy and processed as previously described [21].

R. Guadalupi et al. / Biochimica et Biophysica Acta 1293 (1996) 9-16

Briefly, the tissue was homogenised, sonicated and treated with 0.02% Miranol H2M detergent. The pooled homogenate was centrifuged at 36 000 × g for 20 min. The supernatant was fractionated by 25-70% saturation with (NH4)2SO 4. The enzymatically active fraction was dissolved in water and dialysed against 0.01 M sodium phosphate buffer (pH 6.0) then loaded into a DEAE-cellulose column (2 ml volume) equilibrated with the above buffer. This column was developed as described above. The two enzymatically active peaks obtained were separately concentrated to 0.5 rnl and dialysed against water by vacuum dialysis (Sartorius apparatus, Sartorius, GiSttingen, Germany).

2.7. Polyacrylamide gel electrophoresis The purity of the two forms of t3-mannosidase was determined by non denatuFLng 5-20% polyacrylamide gradient gel electrophoresis (Bio-Rad mini-protean II apparatus). The gel was pre-electrophoresed for 15 min at 20 mA. The running buffer was 0.025 M Tris/0.129 M glycine (pH 8.3). Electrophoresis was carded out at 20 mA for 5 h. For /3-mannosidase activity staining, the gel was soaked in 0.1 M citric acid/0.2 M disodium phosphate buffer (pH 4.3), then incubated at 37°C for 30 rain in the above buffer containing 1 mM 4MU/3-man as substrate. The fluorescent bands were seen more clearly by spraying the gel, after incubation with the substrate, with 0.2 M glycine-NaOH buffer (pH 10.6) and photographing them immediately. Protein bands were detected by staining with 0.1% Coomassie brilliant blue in 40% methanol, 10% acetic acid. The gel was destained by repeated washing with 40% methanol, 10% acetic acid. Polyacrylamide gel electrophoresis in the presence of SDS was performed in 12% polyacrylamide gels (Bio-Rad mini-protean II apparatus) according to Laemmli [22]. Protein bands were detected by staining with Coomassie

11

brilliant blue as above or silver stained according to the manufacturer's instructions.

2.8. Determination of isoelectric point Isoelectric focusing was performed on mini gels, in the pH range 4.0 to 8.0, by using the Mini IEF Cell 111 Bio-Rad apparatus. Enzyme activity and proteins were stained as described above.

2.9. Molecular mass determination The molecular masses of the native proteins were determined by 5-20% polyacrylamide gradient gel electrophoresis as described above and using bovine serum albumin monomer (66 kDa) and dimer (132 kDa), /3amylase (200 kDa) and Jack bean urease (272 kDa) as standards.

2.10. Kinetic properties and thermal stability of /3-mannosidases pH Optima were determined in 0.1 M citric acid/0.2 M disodium phosphate buffers in the pH range 3.0 to 6.5 and in 0.1 M Na-phosphate buffers in the pH range 7.0 to 8.0. K m values were determined from Lineweaver-Burk plots [23]. Samples of the isoenzymes (50 /zl) were incubated with the fluorogenic substrate in the concentration range 0.27 to 2.27 raM. The buffer was 0.1 M citric acid/0.2 M disodium phosphate buffer (pH 4.3) and incubations were at 37°C. Thermal stability of /3-mannosidase isoenzymes was determined as previously described [24] by incubating samples of enzyme (50 /xl) in 0.01 M sodium phosphate buffer (pH 6.0) at various temperatures ranging from 37 to 70°C for various periods of time. Samples were then cooled on ice for 2 h and assayed for enzyme activity at

Table 1 Purification of urinary /3-manno,;idases Purification step

Total protein (mg)

Total activity (U)

Specific activity ( U / m g prot)

Yield (%)

Purification factor

Normal urine (201) (NH4)2SO 4 25-70% std. Sephadex G-25 chromatography Sephacryl S-200 chromatograph),

1857.14 1286.32 450.38 71.68

2.600 2.440 2.432 1.742

0.0014 0.0019 0.0054 0.0243

100 94 94 67

1.0 1.4 3.9 17.4

DEAE-Cellulose chromatography /3-Mannosidase B fl-Mannosidase A

1.12 4.76

0.200 1.448

0.1790 0.3040

8 56

127.4 217.1

Preparative gel fl-Mannosidase B fl-Mannosidase A

0.02 0.16

0.042 0.280

2.1000 1.7720

2 11

1500.0 1265.7

One unit (U) is the amount of enzyme that hydrolyzes 1 ~mole of substrate/min at 37°C. Both isoenzymes were co-purified up to the DEAE-cellulose chromatography step, then processed separately. For details see the text.

R. Guadalupi et al. / Biochimica et Biophysica Acta 1293 (1996) 9-16

12

37°C in the usual way. The results are the mean of three independent experiments and are expressed as the percentage of enzyme activity found in non-heated controls kept on ice.

G-25 and Sephacryl S-200 get filtration chromatographies (see Section 2 for details) was chromatographed on DEAE-cellulose column. This ion-exchange chromatography, under the conditions adopted by us, enabled the separation of two peaks of /3-mannosidase activity (Fig. 1). The first, identified as /3-mannosidase B, was eluted by 0.1 M NaCI and represented 12% of the activity loaded onto the column; the second, identified as /3-mannosidase A, was eluted by a gradient 0.1-0.3 M of NaCI and accounted for 83% of the loaded activity. This distribution of B and A activity was consistently found true for all samples examined (i.e., 10 healthy donors). DEAE-cellulose chromatography was also a useful purification step. Purification to homogeneity was achieved by preparative gel electrophoresis in a gradient of polyacrylamide. Probably because of the high instability of pure enzyme, the yield of this final step of purification was only 25% for /3-mannosidase B and 20% for A. The final preparations of both fl-mannosidase B and A were analyzed by analytical gel electrophoresis in a 5-20% gradient of polyacrylamide (Fig. 2). Single protein bands were detected with Coomassie brilliant blue (Fig. 2, panel a) which were found to be coincident with enzymatically active bands revealed by using the substrate 4MU fl-mann (Fig. 2 panel b). In addition, we observed that protein detection by the Silver stain method did not reveal contaminating bands. Single bands were also detected when the two preparations were analyzed by electrophoresis in 8% polyacrylamide, which again showed a single protein band for each preparation (data not shown).

2.11. Chromatography on Concanavalin-A Sepharose-4B Concanavalin-A Sepharose 4B chromatography was performed as before [25] using a 1 ml column equilibrated with 20 mM Tris-HC1 buffer (pH 7.4) containing 1 mM MnC12, 1 mM MgC12 and 1 mM CaC12. Partially purified fl-mannosidases A and B (after DEAE-cellulose chromatography) were dialysed against the same buffer and loaded onto the column. Glycoproteins retained by the column were eluted by a linear gradient of 0.0-0.5 M methyl a-mannopyranoside (20 ml in total), in 50 mM Tris-HC1 buffer (pH 7.4) containing l M NaC1.

3. Results

3.1. Separation and purification of two forms of fl-mannosidase from human urine The purification procedure adopted, summarized in Table 1, yielded pure /3-mannosidases B and A , with specific activities of 2.1 units/mg of protein and 1.8 units/mg of protein, and with recoveries of 2% and 11%, respectively. Urinary /3-mannosidase activity, after partial purification by ammonium sulfate fractionation, Sephadex

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Fig. 1. DEAE-Cellulose chromatography of human urinary /3-mannosidase. Enzymatically active fractions arising from previous purification steps were pooled and dialyzed against 0.01 M sodium phosphate buffer (pH 6.0), then loaded into a DEAE-cellulose column (5 ml volume) equilibrated with the above buffer. After washing the column until the A2sonm reached zero, the elution was continued with 0.1 M NaCI in the above buffer (arrow 1-2), again until the A28onm reached zero. The column was then developed with a linear gradient of NaCI (0.1-0.3 M) in 60 ml of buffer (arrow 2-3), and a second peak of activity was eluted at this stage. Finally the column was eluted with 1.0 M NaC1 in the same buffer (arrow 3 onwards). Fractions were assayed for activity towards 4MUff-man. B = fl-mannosidase B; A = fl-mannosidase A.

R. Guadalupi et al. / Biochimica et Biophysica Acta 1293 (1996) 9-16

(a) Mr (kDa)

13

of 160 kDa, whereas /3-mannosidase A had a lower molecular mass of 135 kDa.

Mr (kDa)

3.3. Subunit composition 272

-

200

-~ 160

132 -

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(b)

The subunit composition of pure fl-mannosidases B and A was determined by SDS-PAGE (Fig. 3). After silver staining, /3-mannosidase B exhibited two bands with apparent molecular masses of 75 kDa and 49 kDa, and /3-mannosidase A three bands of apparent molecular masses of 75 kDa, 49 kDa and 37 kDa. The revelation of the 49 kDa and 37 kDa bands in the latter preparation was strictly dependent on the sensitivity of the detection technique used. The patterns of the bands were unaffected either by using the reducing agent up to a concentration of 50 mM dithiothreitol or by increasing the time of heating. This 100,

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Fig. 2. Gel eleetrophoresis of purified /3-mannosidase B and A in a 5-20% gradient of polyacrylamide. The gel was stained for protein with Coomassie brilliant blue (panel a), and for/3-mannosidase activity (panel b). Lane 1, standard proteins; lane 2 and 3, fl-mannosidases B and A, respectively. .~

3.2. Estimation of molecular masses of fl-mannosidases A and B Analytical gradient gel electrophoresis (Fig. 2) suggested that native /3-mannosidase B had a molecular mass

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Fig. 3. Polyacrylamide gel electrophoresis of purified /3-mannosidase B and A in the presence of SDS. Samples of pure /3-mannosidases B and A (5 p.g) were analysed by electraphoresis in polyacrylamide (12%, w / v ) containing SDS. The gel was silver stained for protein. Lane 1: marker proteins; lane 2 and 3: /3-mannosidase A and B, respectively.

Fig. 4. Thermal inactivation of /3-mannosidases from human urine. Isoenzyme samples were heated at the indicated temperatures in 0.01 M sodium phosphate buffer (pH 6.0) for different time intervals, then kept at 4°C for 2 h and finally assayed for activity towards the substrate 4MUff-man in the usual way. The results are the averages of three experiments and are expressed as the percentages of enzyme activity found in untreated controls kept at 4°C. (©) /3-mannosidase B; ( Q ) /3-mannosidase A. (a) Partially purified isoenzymes, after DEAE-cellulose chromatography; (b) pure isoenzymes.

14

R. Guadalupi et al. / Biochimica et Biophysica Acta 1293 (1996) 9-16

suggests that reduction of the enzyme was complete under the conditions used.

with /3-mannosidase B being more stable than /3-mannosidase A (Fig. 4a). Both forms were rapidly inactivated at 70°C. Pure isoenzymes proved to be much more unstable when exposed to heating. Exposure to 40°C is enough to inactivate both forms (Fig. 4b). fl-Mannosidase A proved to be more unstable than B in this case too, and both forms displayed similar kinetics of inactivation. Both forms were only partially stabilized against temperature by the addition of human albumin. Neither EDTA nor metal ions such as Zn 2+, Mn z+ and Ca 2+ (in the concentration range 1 to 10 mM) had much effect on the enzyme activity of either forms.

3.4. Optimum pH, kinetic properties and thermal stability of/3-mannosidase isoenzymes pH Optima and kinetic parameters were calculated by employing partially purified isoenzymes (after DEAE-cellulose chromatography) and pure isoenzymes (after gel purifications). In both instances /3-mannosidase B and A had an optimum pH at 4.3 and also showed similar p H - a c tivity curves. K m (mlVI) values using 4MU/3-mann as substrate were 0.13 _+ 0.02 for fl-mannosidase B and 0.35 _+ 0.03 for /3mannosidase A. The two isoenzymes, when partially purified (after DEAE-Cellulose chromatography) were both almost stable when exposed for up to one hour at 37°C and 40°C. When exposed for different periods of time at 50°C and 60°C both isoenzymes displayed similar kinetics of inactivation,

3.5. Isoelectric point Broad bands of fl-mannosidase activity were observed after isoelectric focusing for both B and A forms. The most prominent activity was at pH 5.1 for B and at pH 4.8 for A.

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Fig. 5. (a) DEAE-cellulose chromatography of fl-mannosidase from human kidney. An extract of human kidney, performed as described in the experimental section, was loaded into a DEAE-cellulose column (2 ml volume). The chromatography was performed as in Fig. 1, with the exception that the NaC1 gradient total volume was 30 ml. (b) Electrophoresis in a 5-20% gradient of polyacrylamide of renal fl-mannosidases B and A. The two enzymatically active peaks arising from DEAE-cellulose chromatography and corresponding to renal fl-mannosidases B and A were pooled and analysed in a 5-20% gradient of polyacrylamide. The gel was stained for fl-mannosidase activity as described,

R. Guadalupi et al. / Biochimica et Biophysica Acta 1293 (1996) 9-16

3.6. Concanavalin-A Sepharose 4B chromatography Both /3-mannosidase B and A were retained on Concanavalin-A Sepharose 4B, and were eluted as single peaks of activity (90% of the activity loaded) by a linear gradient of methyl c~-mannopyranoside.

3. 7. [3-Mannosidases of human kidney When analysed by DEAE-cellulose chromatography, /3-mannosidase from an extract of human kidney was resolved into two peaks of activity, which required the same NaCI concentration to be eluted as the corresponding urinary ones (Fig. 5, panel a). In this case, the peak of activity corresponding to fl-mannosidase B accounted for 95% of the total activity :ecovered from the column, and the peak corresponding to /3-mannosidase A for 5%. When analysed by a 5-20% gradient gel electrophoresis (Fig. 5, panel b), renal /3-mannosidases B and A both consisted of single enzymatically active bands of different mobility, corresponding to a molecular weight of 155 kDa and 137 kDa, respectively.

4. Discussion

The occurrence of more than one molecular form of /3-mannosidase was hypothesised by Pearce et al. [3], who found multiple isoelectric points for goat /3-mannosidase. Additionally, a study by Frei et al. [17] suggested the presence of multiple enzyme forms, in goat kidney extracts, probably associated with different stages of enzyme processing or degrees of proteolysis. By employing normal human urine as a source of enzyme activity, in this paper we have reported the presence of two distinct forms of /3-mannosidase (A and B), which have been purified to homogeneity. Our protocol enabled us to obtain both /3-mannosidases free of contaminants, as shown by polyacrylamide gel electrophoresis. fl-Mannosidases A and 13 forms were purified 1265- and 1500-fold, respectively. Nevertheless, because of the instability of pure enzyme, the specific activities of both forms are probably underestimated. The instability of pure enzyme should also justify the relatively low yield obtained in the last purification step. The two fl-mannosidases differed in their molecular masses, isoelectric points, thermal stabilities and subunit compositions. After SDS-PAGE, fl-mannosidase B preparation exhibited two bands with apparent molecular mass of 75 kDa and 49 kDa, while /3-mannosidase A showed three bands of 75 kDa, .49 kDa and 37 kDa, respectively. However, the estimated molecular mass was 160 kDa and 135 kDa for the B and A forms, respectively, suggesting that active protein B may consist of three subunits, one 75 kDa and two 49 kDa subunits, whereas active protein A,

15

which is smaller, would have to contain the three subunits described above. Iwasaki et al. [19] described six subunits forming the enzyme in human placenta, with molecular masses ranging from 98 to 57 kDa, two of which appear to be coincident with the 75 and 49 kDa fragments. A 35 kDa peptide, immunologically related to larger forms (90 or 100 kDa) has already been described in goat fl-mannosidase [13]. This form, putatively comparable with the urinary 37 kDa fragment, has been discussed as an in vitro or in vivo proteolytic product of the larger peptide. However, since we only detected the 37 kDa peptide in /3-mannosidase A we conclude that low molecular mass peptide(s) should not be generated by the experimental procedures adopted for purification. The reproducibility of the data obtained from 10 healthy donors exclude doubts on the heterogeneity of the source. In any case more detailed studies on the biosynthesis and processing of these forms are needed to understand their origin definitely. For instance, protein A could represent a proteolysis product of protein B. Both A and B forms purified from human urine displayed an optimal activity at pH 4.3 and were retained by Concanavalin-A Sepharose column, suggesting that neither is the already described non-lysosomal form of the enzyme [3,15], which is characterized by a neutral optimum pH (5.0 to 8.0) and an inability to bind to a Concanavalin-A Sepharose column. Like human urine, human kidney also contains two forms of fl-mannosidase. Although their distribution in renal tissue was significantly different, with 95% of the total activity represented by /3-mannosidase B, both forms were found to exhibit the same behaviour on DEAE-cellulose and Concanavalin A-Sepharose chromatographies and display similar optimum pH and molecular masses. These findings are consistent with the renal origin of urinary /3-mannosidases. Furthermore, the renal origin of urinary /3-mannosidase is in agreement with the evidence that organs mainly involved in the clearance of tissue and serum forms of another lysosomal enzyme, /3-hexosaminidase, are the liver and spleen, but not the kidney [26]. Paigen et al. [27] found that several lysosomal enzymes are actively secreted into the urine in a coordinate fashion; in fact their secretion is unrelated to that of the cytosolic enzyme lactate dehydrogenase. Regarding /3-hexosaminidase Kress et al. [28] suggested a 'lysosomal' pathway leading to the secretion of the mature form of the enzyme, and a 'non-lysosomal' pathway leading to the direct secretion of the precursor form. Further studies are therefore necessary to establish whether or not the two urinary /3-mannosidases represent mature forms of the renal enzyme in order to shed light on the mechanism(s) controlling their secretion. This would be of great interest for diagnostic purposes. In fact preliminary results indicate that in urine from patients with drug induced renal damage, the /3-mannosidase isoenzymatic pattern is remarkably different from that observed in normal subjects' urine

16

R. Guadalupi et aL / Biochimica et Biophysica Acta 1293 (1996) 9-16

(R. Guadalupi et al., unpublished data), where the relative proportion between B and A forms is constant (all 10 healthy subjects' samples had the same A / B ratio). In conclusion, although further studies should be carried out to characterize /3-mannosidase A and B, we believe that their identification and separation may give new insight on /3-mannosidase biochemistry especially regarding its potential diagnostic value in renal associated damage and disease.

Acknowledgements Work supported by Italian Consiglio Nazionale delle Ricerche, Progetto Finalizzato A.C.R.O, SP 4, contract # 94.02208 PF39 and Progetto Finalizzato Ingegneria Genetica, contract # 93.000.32. A.O. is an Erasmus fellow (ICP 93-1-2078/13)

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