Comp. Biochem. Physiol. Vol. 99B, No. 2, pp. 395-398, 1991 Printed in Great Britain
0305-0491/91 $3.00+0.00 © 1991 Pergamon Press plc
THE BASIC ISOELECTRIC FORM OF ~-L-FUCOSIDASE FROM THE HEPATOPANCREAS OF THE SHRIMP P E N A E U S M O N O D O N (CRUSTACEA: DECAPODA) NIN-NIN CHUANG,*CHE-CHUNGYEH and KUNG-SHIH LXN Division of Biochemistry and Molecular Science, Institute of Zoology, Academia Sinica, Nankang, Taipei, Taiwan, Republic of China
(Received 22 November 1990) Abstract--1. ~-L-Fucosidase was purified ca 10,889-fold to homogeneity from Penaeus monodon, with a final spec. act. of 31,250 U/mg of protein. 2. By using SDS-polyacrylamide gel electrophoresis, the monomers of shrimp ~t-L-fucosidase were discovered to have mol. wts of 63,000 and those of human placental enzyme, 46,000 and 20,000. Since the active shrimp ~t-L-fucosidase was found to have a mol. wt of 233,000 by SuperoseTM 12 FPLC, it was concluded that the purified shrimp enzyme was tetrameric. 3. In contrast to the discovery of thermolability with human placental Ct-L-fucosidase, the shrimp enzyme was found to be stable to heating at 65°C for 10 min. 4. The shrimp Ct-L-fucosidase has an isoelectric point (pI) of 8.5, but the human placental enzyme has a pI of 4.0. The shrimp enzyme was sialyated. 5. The shrimp Ct-L-fucosidase has a pH optimum at 5.5 and its K~ was 22.2pM with 4-methylumbelliferyl-a-L-fucopyranoside as substrate. The human enzyme has a broad pH optimum between 5.0 and 6.5.
INTRODUCTION ~t-L-Ftr,..osidase (~-L-fucoside ftr, ohydrolase, EC 3.2.1.51) is an ubiquitous lysosomal enzyme that has been purified and characterized from various mammalian tissues and cells, including placenta (Turner, 1979; Di Matteo et al., 1976; Alhadeff et al., 1974), liver (Fisher and Aronson, 1989; White et al., 1987; Kress et al., 1980), epididymis (Wright et al., 1976; Jain et al., 1977), spleen (Chien and Dawson, 1980), thyroid (Grove and Serif, 1981), brain (Hopfer et al., 1990; Hopfer and Alhadeff, 1985), seminal plasma (Srivastava et al., 1986), serum (Alhadeff and Janowsky, 1978; Di Cioccio et al., 1982) and lymphocytes (Roger et al., 1989). This enzyme is essential for the degradation of fucoglyco-conjugates, and deficiency of its activity results in the rare and fatal neurovisceral storage disease, fucosidosis (Van Hoof, 1973). In most mammalian species, Ct-L-fucosidase is present in four to eight electrophoretic forms with isoelectric points (pls) ranging from 4.5 to 7.5 (Alhadeff and O'Brien, 1977). In addition to these usual acidic and neutral forms of ~-L-fucosidase, mouse tissues (Laury-Kleintop et al., 1987), camel and zebra livers (Watkins and Alhadeff, 1980) contain unusual basic forms of the enzyme. All these isoelectric forms of ~t-L-fucosidase appear to be developmentally regulated, but no phylogenetic trend among them has been found. The aim o f the present study is to prepare the purified Ct-L-fucosidases from a species with distant evolutionary development from mammals, such as *Author to whom correspondence should be addressed.
Penaeus monodon, and use them in investigating the nature and extent of differences from the human enzyme. It is hoped that from this comparison, an index can be born which can be applied to set up the evolutionary relationship among different isoelectric forms of Ct-L-fucosidase. MATERIALS AND METHODS
Materials All reagents used were of the highest grade available commercially. Sialidase (Clostridium perfringens) was obtained from Calbiochem, UK. 4-Methylumbelliferyl-ct-Lfucopyranoside was purchased from Sigma, USA. Reagents for isoelectric focusing were from Pharmacia, Sweden. Experimental animals Shrimps (Panaeus monodon, 1 kg), collected off the coast of Taiwan, were kept at 18°C for less than 3 days in a recirculating sea-water system. Hepatopancreases were dissected out immediately after death. Human placentas were removed from the body not more than 1 hr after delivery and stored at -20°C for up to a week at most before use. Purification of ~-L-fucosidase Hepatopancreases were chopped into small fragments and homogenized in 30 ml of 50 mM sodium citrate (pH 5.5) for 5min with a Polytron unit. After centrifugation at 6000g for 20rain, the precipitate was discarded and the supernatant was adjusted to pH 3.0 by adding 1 M sodium citrate and centrifuged at 6000g for 20 min. The supernatant was adjusted to pH 5.5 with 0.5 M sodium hydroxide and acetone (-20°C) was added with stirring to bring the concentration of acetone to 40% (v/v). After centrifugation at 6000g for 20min, the precipitate was discarded and the acetone (-20°C) was added to the supernatant to a concentration of 70% (v/v). The mixture 395
396
NIN-NIN CHUANGet al. Table 1. Purification of a-L-fucosidasefrom Penaeus monodon Specific Total protein Total activity Recovery (mg) units (U/nag) (%) Homogenate 1044 3000 2.87 100 Adjustment with acid 312 2828 9.06 94 Heating at 65°C for 30 min 19.6 266 13.6 8.9 DE 52 0.02 80 4000 2.7 SuperoserMl2 0.0004 12.5 31,250 0.4
was centrifuged at 6000 g for 20 min, and the precipitated pellet was dissolved in 50 mM sodium citrate (pH 5.5) and dialyzed against 10 vols of distilled water. The enzyme solution obtained after dialysis was heated at 65°C for 30 rain before centrifugation at 10,000g for 15 min. The recovered supernatant was then applied to a DE 52 column (Whatman DEAE cellulose) (1.5 × 6cm) that had been equilibrated with 50 mM sodium citrate buffer (pH 5.5) and was developed with the same buffer superimposed on a gradient of NaC1. a-L-Fucosidase was unbound to the column and was pooled and concentrated by ultrafiltration on an Amicon PM I0 membrane. The concentrated solution was then subjected to gel filtration on a SuperoseTM12 (Pharmacia, Sweden) developed with 100 mM ammonium acetate buffer (pH 5.5).
Polyacrylamide-gel electrophoresis Sodium dodecyl sulfate-polyacrylamide-gel electrophoresis (SDS-PAGE) was conducted on slab gels containing 10% (w/v) acrylamide with 0.27% (w/v) NN'methylenebis-acrylamide (Laemmli, 1970). Samples were reduced and alkylated (Lane, 1978) before application to the gels. Gels were silver-stained according to the method of Merril et aL (1981). Isoelectric focusing was carried out essentially as described by Chuang (1990) using ampholytes with a pH range
Mr 3 xlO
Purification (fold) 1.0 3.2 4.7 1394 10,889
of 3.0-10.0. Focusing was performed at a constant voltage (200V) for 18-21 hr.
Assay of a-L-fucosidase activity" a-L-Fucosidase activity was measured at 37°C with 0.2 mM 4-methylumbeUiferyl-~-L-fucopyranoside in 50 mM sodium citrate buffer (pH 5.5). The reaction was stopped by adding 0.2 M glycine NaOH buffer (pH 9.0). The 4-methylumbelliferone liberated was determined with a Hitachi Fluorometer 850 (a primary filter transmitting at 367 nm and a secondary filter transmitting at 448 nm). One unit of enzyme was defined as the amount of enzyme which hydrolyzed 1/tmol of 4-methylumbelliferyl-a-L-fucopyranoside per min under the conditions described above. Assay of protein Bovine serum albumin served as the standard in the measurement of proteins. The amount of protein was determined by the Lowry method (1951). RESULTS AND DISCUSSION The purification o f a-L-fucosidase from the hepatopancreas o f Penaeus monodon is summarized in Table 1. Both the ion-exchange c h r o m a t o g r a p h i c step
D
C
B
A
94 67 43
30
20 Fig. I. SDS-Polyacrylamide gel electrophoresis of Ct-L-fucosidase purified from Penaeus monodon. The purified enzyme was subjected to electrophoresis on a SDS-polyacrylamide gel (10%), which was then silver-stained. (B). The enzyme preparation obtained after DE 52 ion exchange column chromatography; (C), the enzyme preparation purified by SuperoserM12 gel filtration column chromatography; (D), the enzyme obtained after inactivation upon freezing-thawing. For comparison, purified a-L-fucosidase from human placenta was included (A).
Ct-L-Fucosidaseof Penaeus monodon 0.5
At
0.4
B
c 0.3
~
,
I
I
I
I
t
l
,
t
n
3
397
4
n, n,
, i
,
i
I
i
I
I
I
5
6
7
8
9
10
,
11
pH 0.2
Fig. 4. Isoelectric points of purified Ct-L-fucosidase from Penaeus monodon. Purified Ct-L-fucosidase from Penaeus monodon was untreated (A), or treated (B) with the bacterial
0.1 I
I
lO
5o
M.W. ( x l O 4 )
Fig. 2. Plot of K~v against log of mol. wt corresponding to the determination of mol. wt of ~t-L-fucosidase by SuperoserM12 column equilibrated with 100mM ammonium acetate buffer, pH 5.5. The protein standards were: (1), ribonuclease A; (2), ovalbumin; (3), aldolase; (4), ferretin. O, ct-L-Fucosidase from Penaeus monodon. Kay = ( V ~ - Vo)/(Vt- Vo), Vt=total bed volume, V0=column void volume, V~= elution volume. on DE 52 and the gel filtration step on SuperoseTM12 (Fig. IB) removed major protein contaminants. The final spec. act. of purified ~t-L-fucosidase was 31,250 U/mg of protein, and the overall purification was about 10,889-fold. The purified enzyme from Penaeus monodon was practically devoid of other glycosidase activities: activities of fl-galactosidase, ~-galactosidase, ~-mannosidase and sialidase were not detected. The purity of the final preparation of enzyme was examined by SDS-PAGE. The relative mass of ~t-Lfucosidase from Penaeus monodon was found to be 63,000 (Fig. 1C), whereas that after denaturation by freezing-thawing was shown to be 31,000 (Fig. 1D). No additional protein, such as Mr 57,000 as evidenced in the study of ~-L-fucosidase from mouse liver by Laury-Kleintop et al. (1987), was detected in the final preparation of shrimp enzyme. The relative mass of ~-L-fucosidase from human placenta was shown to have M, of 46,000 and Mr, of 20,000 (Fig. 1A). The mol. wt of the active Ct-L-fucosidase was estimated by gel filtration on SuperoseTM12. The plot
sialidase, and then subjected to isoelectric focusing. For comparison, purified ~t-L-fucosidasefrom human placenta (C) was included. For each sample, triplicate gels were examined. ~t-L-Fucosidase (10 mU) was incubated at 37°C for l hr with sialidase (0.5U) in 50mM sodium acetate (pH 4.5) and the reaction was stopped by rapid freezing. of Kay (as defined in the figure legend of Fig. 2) versus the mol. wt (logarithmic scale) was constructed by use of least-squared linear regression. The enzyme's relative mass was estimated to be Mr 233,000. From the size of the native enzyme and that of the denatured enzyme on SDS-PAGE, the purified ~t-L-fucosidase from Penaeus monodon appear to be tetrameric. The catalytic activity against 4-methylumbelliferyl~t-L-fucopyranoside of the purified enzyme from shrimp was heat-labile (Fig. 3); 50% of the original activity was lost after preincubation at 65°C for 60 min. Nevertheless, the ~t-L-fucosidase from shrimp was more thermostable than the ~-L-fucosidase from human placenta. Incubation at 65°C for 10 min resulted in 10% reduction in the original activity in the case of the ~-L-fucosidase from shrimp (Fig. 3), whereas the activity of the Ct-L-fucosidase from human placenta disappeared completely. When the two purified enzyme preparations from shrimp hepatopancreas and human placenta were applied to isoelectric focusing analysis, the shrimp's enzyme activity migrated more cathodally than human placental ~t-L-fucosidase (Fig. 4A and C). The pI value of shrimp ct-L-fucosidase was about 8.4-8.6, while that of human enzyme was found to be about 4.0. This difference in pI values between shrimp and human enzyme is not responsible for the thermostability difference between them, since the other study with mouse liver shows that the basic isoelectric form
% 100 ^
l ~
c @
5o
>,, .m
.>_
®
0.1
0
0
i
0.2
C
2~0
n
4~)~
'
~0
Time , m i n ,
Fig. 3. Heat inactivation of Ct-L-fucosidase. The stability of the enzymes was investigated by incubating them for 0-60 min at 65°C in optimal pH buffer prior to assaying with 4-methylumbelliferyl-ct-L-fucopyranoside. O, ~-LFucosidase from Penaeus monodon; C), ~t-L-fucosidase from human placenta.
N C W
3
4
5
6
pH
Fig. 5. Effect of pH on the ~t-L-fucosidase activity. O, ~-L-Fucosidase from Penaeus monodon; O, Ct-L-fucosidase from human placenta.
398
NIN-NXNCHUANGet al.
from mouse liver is considerably more thermolabile than the human enzyme (Laury-Kleintop et al., 1985a,b). This difference in the pI values between shrimp and human enzymes cannot be abolished by treatment with sialidase from Clostridium perfringens. However, the treatment with bacterial sialidase resulted in a shifted migration of the enzyme (Fig. 4B). In other words, the ~-L-fucosidase from shrimp is a sialyated glycoprotein. The apparent K m value of ~-L-fucosidase from shrimp hepatopancreas with 4-methylumbelliferyl-~L-fucopyranoside as substrate was 2 2 . 2 # M . The shrimp enzyme exhibits a relatively sharp p H optimum at 5.5, as determined with 4-methylumbelliferyl-ct-L-fucopyranoside as substrate (Fig. 5). Nevertheless, a broader pH optimum is discovered for the activity of ~t-L-fucosidase from human placenta. The present study provides the first evidence that the Ct-L-fucosidase from the hepatopancreas of shrimp exists as an alkaline isoelectric form. Since no other acidic isoelectric form was discovered in this primitive enzyme, the human acidic Ct-L-fucosidase should be evolved from the alkaline Ct-L-fucosidase in later life. Further studies, comparing the primary structures of the acidic and alkaline isoelectric forms of Ct-L-fucosidases, are underway. Acknowledgement--Financial support for this work was provided by the National Science Council, Republic of China. REFERENCES
Alhadeff J. A. and Janowsky A. J. (1978) Human serum Ct-L-fucosidase. Clinica chim. Acta 82, 133-140. Alhadeff J. A. and O'Brien J. S. (1977) Fucosidosis. In Practical Enzymology o f the Spingolipidoses (Edited by Glew R. H. and Peters S. P.), pp. 247-281. Alan R. Liss, New York. Alhadeff J. A., Miller A. L. and O'Brien J. S. (1974) Purification of human placental ~t-L-fucosidase by affinity chromatography. Analyt. Biochem. 60, 424-430. Chien S.-F. and Dawson G. (1980) Purification and properties of two forms of human ~t-L-fucosidase. Biochem. biophys. Acta 614, 476-488. Chuang N.-N. (1990) A neutral beta-galactosidase from the hepatoapancreas of the shrimp Penaeus monodon (Crustacea: Decapoda): dimeric and sialyated. Comp. Biochem. Physiol. 96, 747-751. DiCioccio R. A., Barlow J. J. and Matta K. L. (1982) Substrate specificity and other properties of ~t-Lfucosidase from human serum. J. biol. Chem. 257, 714-718. DiMatteo G., Orfeo M. A. and Romeo G. (1976) Human ct-fucosidase. Biochim. biophys. Acta 429, 527-545. Fisher K. J. and Aronson N. N. (1989) Isolation and sequence analysis of a cDNA encoding rat liver Ct-Lfucosidase. Biochem. J. 264, 695-701. Grove D. S. and Serif G. S. (1981) Porcine thyroid fucosidase. Biochim. biophys. Acta 662, 246-255.
Hopfer R. L. and Alhadeff J. A. (1985) Solubilization and characterization of pellet-associated human brain Ct-L-fucosidase activity. Biochem. J. 229, 679-685. Hopfer R. L., Johnson S. W., Masserini M., Giuliani A. and Alhadeff J. A. (1990) Hydrolysis of fucosyl-Gml ganglioside by purified pellet-associated human brain and human liver Ct-L-fucosidases without activator proteins or detergents. Biochem. J. 266, 491-496. Jain R. S., Binder R. L., Levy-Benshimol A., Buck C. A. and Warren L. (1977) Purification of alpha-L-fucosidase from various sources by affinity chromatography. J. Chromat. 139, 283-290. Kress B. C., Freeze H. H., Herd J. K., Alhadeff J. A. and Miller A. L. (1980) Purification and characterization of I-cell disease Ct-L-fucosidase. J. biol. Chem. 255, 955-961. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T 4. Nature 277, 680-685. Lane L. C. (1978) A simple method for stabilizing proteinsulfhydryl groups during SDS-gel electrophoresis. Analyt. Biochem. 86, 655-664. Laury-Kleintop L. D., Alhadeff J. A. and Damjanov I. (1985a) Isoelectric forms of a-L-fucosidase in mouse teratocarcinoma derived cell lines. Devl. Biol. 111, 520-524. Laury-Kleintop L. D., Damjanov I. and Alhadeff J. A. (1985b) Characterization of mouse liver Ct-L-fucosidase. Biochem. J. 230, 78-82. Laury-Kleintop L. D., Damjanov I. and Alhadeff J. A. (1987) Antibody-affinity purification of novel Ct-L-fucosidase from mouse liver. Biochem. J. 245, 589-593. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the folin phenol reagent. J. biol. Chem. 193, 265-275. Merril C. R., Goldman D., Sedman S. A. and Ebert M. H. (1981) Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science 21L 1437-1438. Roger L., Bernard M.-A., Percheron F. and Foglietti M.-J. (1989) Ct-L-Fucosidase activity in normal human lymphocytes. Clinica chim. Acta 180, 303-310. Srivastava P. N., Arbtan K., Takei G. H., Huang T. T. F. and Yanagimachi R. (1986) Ct-L-Fucosidase from bull seminal plasma: its purification and acrosome reaction promoting property. Biochem. biophys. Commun. 137, 1061-1068. Turner B. M. (1979) Purification and characterization of alpha-L-fucosidase from human placenta, pH-dependent changes in molecular size. Biochim. biophys. /lcta 578, 325-336. Van Hoof F. (1973) Enzyme replacement in lysosomal diseases. In Lysosomes and Storage Diseases (Edited by Hers H. G. and Van Hoof F.), pp. 277-290. Academic Press, New York. Watkins P. and Alhadeff J. A. (1980) Kinetic and immunochemical characterization of Ct-L-fucosidase from vertebrate livers. Comp. Biochem. Physiol. 68, 517-520. White W. J., Schray K. J., Legler G. and Alhadeff J. A. (1987) Further studies on the catalytic mechanism of human liver Ct-L-fucosidase. Biochim. biophys. Acta 912, 132-138. Wright K., Northcote D. H. and Davey R. M. (1976) Preparation of rat epididymal alpha-L-fucosidase free from other glycosidases: its action on root-cap slime from Zea mays L. Carbohydr. Res. 47, 141-150.
0305-0491/91 $3.00+0.00 © 1991 Pergamon Press plc
THE BASIC ISOELECTRIC FORM OF ~-L-FUCOSIDASE FROM THE HEPATOPANCREAS OF THE SHRIMP P E N A E U S M O N O D O N (CRUSTACEA: DECAPODA) NIN-NIN CHUANG,*CHE-CHUNGYEH and KUNG-SHIH LXN Division of Biochemistry and Molecular Science, Institute of Zoology, Academia Sinica, Nankang, Taipei, Taiwan, Republic of China
(Received 22 November 1990) Abstract--1. ~-L-Fucosidase was purified ca 10,889-fold to homogeneity from Penaeus monodon, with a final spec. act. of 31,250 U/mg of protein. 2. By using SDS-polyacrylamide gel electrophoresis, the monomers of shrimp ~t-L-fucosidase were discovered to have mol. wts of 63,000 and those of human placental enzyme, 46,000 and 20,000. Since the active shrimp ~t-L-fucosidase was found to have a mol. wt of 233,000 by SuperoseTM 12 FPLC, it was concluded that the purified shrimp enzyme was tetrameric. 3. In contrast to the discovery of thermolability with human placental Ct-L-fucosidase, the shrimp enzyme was found to be stable to heating at 65°C for 10 min. 4. The shrimp Ct-L-fucosidase has an isoelectric point (pI) of 8.5, but the human placental enzyme has a pI of 4.0. The shrimp enzyme was sialyated. 5. The shrimp Ct-L-fucosidase has a pH optimum at 5.5 and its K~ was 22.2pM with 4-methylumbelliferyl-a-L-fucopyranoside as substrate. The human enzyme has a broad pH optimum between 5.0 and 6.5.
INTRODUCTION ~t-L-Ftr,..osidase (~-L-fucoside ftr, ohydrolase, EC 3.2.1.51) is an ubiquitous lysosomal enzyme that has been purified and characterized from various mammalian tissues and cells, including placenta (Turner, 1979; Di Matteo et al., 1976; Alhadeff et al., 1974), liver (Fisher and Aronson, 1989; White et al., 1987; Kress et al., 1980), epididymis (Wright et al., 1976; Jain et al., 1977), spleen (Chien and Dawson, 1980), thyroid (Grove and Serif, 1981), brain (Hopfer et al., 1990; Hopfer and Alhadeff, 1985), seminal plasma (Srivastava et al., 1986), serum (Alhadeff and Janowsky, 1978; Di Cioccio et al., 1982) and lymphocytes (Roger et al., 1989). This enzyme is essential for the degradation of fucoglyco-conjugates, and deficiency of its activity results in the rare and fatal neurovisceral storage disease, fucosidosis (Van Hoof, 1973). In most mammalian species, Ct-L-fucosidase is present in four to eight electrophoretic forms with isoelectric points (pls) ranging from 4.5 to 7.5 (Alhadeff and O'Brien, 1977). In addition to these usual acidic and neutral forms of ~-L-fucosidase, mouse tissues (Laury-Kleintop et al., 1987), camel and zebra livers (Watkins and Alhadeff, 1980) contain unusual basic forms of the enzyme. All these isoelectric forms of ~t-L-fucosidase appear to be developmentally regulated, but no phylogenetic trend among them has been found. The aim o f the present study is to prepare the purified Ct-L-fucosidases from a species with distant evolutionary development from mammals, such as *Author to whom correspondence should be addressed.
Penaeus monodon, and use them in investigating the nature and extent of differences from the human enzyme. It is hoped that from this comparison, an index can be born which can be applied to set up the evolutionary relationship among different isoelectric forms of Ct-L-fucosidase. MATERIALS AND METHODS
Materials All reagents used were of the highest grade available commercially. Sialidase (Clostridium perfringens) was obtained from Calbiochem, UK. 4-Methylumbelliferyl-ct-Lfucopyranoside was purchased from Sigma, USA. Reagents for isoelectric focusing were from Pharmacia, Sweden. Experimental animals Shrimps (Panaeus monodon, 1 kg), collected off the coast of Taiwan, were kept at 18°C for less than 3 days in a recirculating sea-water system. Hepatopancreases were dissected out immediately after death. Human placentas were removed from the body not more than 1 hr after delivery and stored at -20°C for up to a week at most before use. Purification of ~-L-fucosidase Hepatopancreases were chopped into small fragments and homogenized in 30 ml of 50 mM sodium citrate (pH 5.5) for 5min with a Polytron unit. After centrifugation at 6000g for 20rain, the precipitate was discarded and the supernatant was adjusted to pH 3.0 by adding 1 M sodium citrate and centrifuged at 6000g for 20 min. The supernatant was adjusted to pH 5.5 with 0.5 M sodium hydroxide and acetone (-20°C) was added with stirring to bring the concentration of acetone to 40% (v/v). After centrifugation at 6000g for 20min, the precipitate was discarded and the acetone (-20°C) was added to the supernatant to a concentration of 70% (v/v). The mixture 395
396
NIN-NIN CHUANGet al. Table 1. Purification of a-L-fucosidasefrom Penaeus monodon Specific Total protein Total activity Recovery (mg) units (U/nag) (%) Homogenate 1044 3000 2.87 100 Adjustment with acid 312 2828 9.06 94 Heating at 65°C for 30 min 19.6 266 13.6 8.9 DE 52 0.02 80 4000 2.7 SuperoserMl2 0.0004 12.5 31,250 0.4
was centrifuged at 6000 g for 20 min, and the precipitated pellet was dissolved in 50 mM sodium citrate (pH 5.5) and dialyzed against 10 vols of distilled water. The enzyme solution obtained after dialysis was heated at 65°C for 30 rain before centrifugation at 10,000g for 15 min. The recovered supernatant was then applied to a DE 52 column (Whatman DEAE cellulose) (1.5 × 6cm) that had been equilibrated with 50 mM sodium citrate buffer (pH 5.5) and was developed with the same buffer superimposed on a gradient of NaC1. a-L-Fucosidase was unbound to the column and was pooled and concentrated by ultrafiltration on an Amicon PM I0 membrane. The concentrated solution was then subjected to gel filtration on a SuperoseTM12 (Pharmacia, Sweden) developed with 100 mM ammonium acetate buffer (pH 5.5).
Polyacrylamide-gel electrophoresis Sodium dodecyl sulfate-polyacrylamide-gel electrophoresis (SDS-PAGE) was conducted on slab gels containing 10% (w/v) acrylamide with 0.27% (w/v) NN'methylenebis-acrylamide (Laemmli, 1970). Samples were reduced and alkylated (Lane, 1978) before application to the gels. Gels were silver-stained according to the method of Merril et aL (1981). Isoelectric focusing was carried out essentially as described by Chuang (1990) using ampholytes with a pH range
Mr 3 xlO
Purification (fold) 1.0 3.2 4.7 1394 10,889
of 3.0-10.0. Focusing was performed at a constant voltage (200V) for 18-21 hr.
Assay of a-L-fucosidase activity" a-L-Fucosidase activity was measured at 37°C with 0.2 mM 4-methylumbeUiferyl-~-L-fucopyranoside in 50 mM sodium citrate buffer (pH 5.5). The reaction was stopped by adding 0.2 M glycine NaOH buffer (pH 9.0). The 4-methylumbelliferone liberated was determined with a Hitachi Fluorometer 850 (a primary filter transmitting at 367 nm and a secondary filter transmitting at 448 nm). One unit of enzyme was defined as the amount of enzyme which hydrolyzed 1/tmol of 4-methylumbelliferyl-a-L-fucopyranoside per min under the conditions described above. Assay of protein Bovine serum albumin served as the standard in the measurement of proteins. The amount of protein was determined by the Lowry method (1951). RESULTS AND DISCUSSION The purification o f a-L-fucosidase from the hepatopancreas o f Penaeus monodon is summarized in Table 1. Both the ion-exchange c h r o m a t o g r a p h i c step
D
C
B
A
94 67 43
30
20 Fig. I. SDS-Polyacrylamide gel electrophoresis of Ct-L-fucosidase purified from Penaeus monodon. The purified enzyme was subjected to electrophoresis on a SDS-polyacrylamide gel (10%), which was then silver-stained. (B). The enzyme preparation obtained after DE 52 ion exchange column chromatography; (C), the enzyme preparation purified by SuperoserM12 gel filtration column chromatography; (D), the enzyme obtained after inactivation upon freezing-thawing. For comparison, purified a-L-fucosidase from human placenta was included (A).
Ct-L-Fucosidaseof Penaeus monodon 0.5
At
0.4
B
c 0.3
~
,
I
I
I
I
t
l
,
t
n
3
397
4
n, n,
, i
,
i
I
i
I
I
I
5
6
7
8
9
10
,
11
pH 0.2
Fig. 4. Isoelectric points of purified Ct-L-fucosidase from Penaeus monodon. Purified Ct-L-fucosidase from Penaeus monodon was untreated (A), or treated (B) with the bacterial
0.1 I
I
lO
5o
M.W. ( x l O 4 )
Fig. 2. Plot of K~v against log of mol. wt corresponding to the determination of mol. wt of ~t-L-fucosidase by SuperoserM12 column equilibrated with 100mM ammonium acetate buffer, pH 5.5. The protein standards were: (1), ribonuclease A; (2), ovalbumin; (3), aldolase; (4), ferretin. O, ct-L-Fucosidase from Penaeus monodon. Kay = ( V ~ - Vo)/(Vt- Vo), Vt=total bed volume, V0=column void volume, V~= elution volume. on DE 52 and the gel filtration step on SuperoseTM12 (Fig. IB) removed major protein contaminants. The final spec. act. of purified ~t-L-fucosidase was 31,250 U/mg of protein, and the overall purification was about 10,889-fold. The purified enzyme from Penaeus monodon was practically devoid of other glycosidase activities: activities of fl-galactosidase, ~-galactosidase, ~-mannosidase and sialidase were not detected. The purity of the final preparation of enzyme was examined by SDS-PAGE. The relative mass of ~t-Lfucosidase from Penaeus monodon was found to be 63,000 (Fig. 1C), whereas that after denaturation by freezing-thawing was shown to be 31,000 (Fig. 1D). No additional protein, such as Mr 57,000 as evidenced in the study of ~-L-fucosidase from mouse liver by Laury-Kleintop et al. (1987), was detected in the final preparation of shrimp enzyme. The relative mass of ~-L-fucosidase from human placenta was shown to have M, of 46,000 and Mr, of 20,000 (Fig. 1A). The mol. wt of the active Ct-L-fucosidase was estimated by gel filtration on SuperoseTM12. The plot
sialidase, and then subjected to isoelectric focusing. For comparison, purified ~t-L-fucosidasefrom human placenta (C) was included. For each sample, triplicate gels were examined. ~t-L-Fucosidase (10 mU) was incubated at 37°C for l hr with sialidase (0.5U) in 50mM sodium acetate (pH 4.5) and the reaction was stopped by rapid freezing. of Kay (as defined in the figure legend of Fig. 2) versus the mol. wt (logarithmic scale) was constructed by use of least-squared linear regression. The enzyme's relative mass was estimated to be Mr 233,000. From the size of the native enzyme and that of the denatured enzyme on SDS-PAGE, the purified ~t-L-fucosidase from Penaeus monodon appear to be tetrameric. The catalytic activity against 4-methylumbelliferyl~t-L-fucopyranoside of the purified enzyme from shrimp was heat-labile (Fig. 3); 50% of the original activity was lost after preincubation at 65°C for 60 min. Nevertheless, the ~t-L-fucosidase from shrimp was more thermostable than the ~-L-fucosidase from human placenta. Incubation at 65°C for 10 min resulted in 10% reduction in the original activity in the case of the ~-L-fucosidase from shrimp (Fig. 3), whereas the activity of the Ct-L-fucosidase from human placenta disappeared completely. When the two purified enzyme preparations from shrimp hepatopancreas and human placenta were applied to isoelectric focusing analysis, the shrimp's enzyme activity migrated more cathodally than human placental ~t-L-fucosidase (Fig. 4A and C). The pI value of shrimp ct-L-fucosidase was about 8.4-8.6, while that of human enzyme was found to be about 4.0. This difference in pI values between shrimp and human enzyme is not responsible for the thermostability difference between them, since the other study with mouse liver shows that the basic isoelectric form
% 100 ^
l ~
c @
5o
>,, .m
.>_
®
0.1
0
0
i
0.2
C
2~0
n
4~)~
'
~0
Time , m i n ,
Fig. 3. Heat inactivation of Ct-L-fucosidase. The stability of the enzymes was investigated by incubating them for 0-60 min at 65°C in optimal pH buffer prior to assaying with 4-methylumbelliferyl-ct-L-fucopyranoside. O, ~-LFucosidase from Penaeus monodon; C), ~t-L-fucosidase from human placenta.
N C W
3
4
5
6
pH
Fig. 5. Effect of pH on the ~t-L-fucosidase activity. O, ~-L-Fucosidase from Penaeus monodon; O, Ct-L-fucosidase from human placenta.
398
NIN-NXNCHUANGet al.
from mouse liver is considerably more thermolabile than the human enzyme (Laury-Kleintop et al., 1985a,b). This difference in the pI values between shrimp and human enzymes cannot be abolished by treatment with sialidase from Clostridium perfringens. However, the treatment with bacterial sialidase resulted in a shifted migration of the enzyme (Fig. 4B). In other words, the ~-L-fucosidase from shrimp is a sialyated glycoprotein. The apparent K m value of ~-L-fucosidase from shrimp hepatopancreas with 4-methylumbelliferyl-~L-fucopyranoside as substrate was 2 2 . 2 # M . The shrimp enzyme exhibits a relatively sharp p H optimum at 5.5, as determined with 4-methylumbelliferyl-ct-L-fucopyranoside as substrate (Fig. 5). Nevertheless, a broader pH optimum is discovered for the activity of ~t-L-fucosidase from human placenta. The present study provides the first evidence that the Ct-L-fucosidase from the hepatopancreas of shrimp exists as an alkaline isoelectric form. Since no other acidic isoelectric form was discovered in this primitive enzyme, the human acidic Ct-L-fucosidase should be evolved from the alkaline Ct-L-fucosidase in later life. Further studies, comparing the primary structures of the acidic and alkaline isoelectric forms of Ct-L-fucosidases, are underway. Acknowledgement--Financial support for this work was provided by the National Science Council, Republic of China. REFERENCES
Alhadeff J. A. and Janowsky A. J. (1978) Human serum Ct-L-fucosidase. Clinica chim. Acta 82, 133-140. Alhadeff J. A. and O'Brien J. S. (1977) Fucosidosis. In Practical Enzymology o f the Spingolipidoses (Edited by Glew R. H. and Peters S. P.), pp. 247-281. Alan R. Liss, New York. Alhadeff J. A., Miller A. L. and O'Brien J. S. (1974) Purification of human placental ~t-L-fucosidase by affinity chromatography. Analyt. Biochem. 60, 424-430. Chien S.-F. and Dawson G. (1980) Purification and properties of two forms of human ~t-L-fucosidase. Biochem. biophys. Acta 614, 476-488. Chuang N.-N. (1990) A neutral beta-galactosidase from the hepatoapancreas of the shrimp Penaeus monodon (Crustacea: Decapoda): dimeric and sialyated. Comp. Biochem. Physiol. 96, 747-751. DiCioccio R. A., Barlow J. J. and Matta K. L. (1982) Substrate specificity and other properties of ~t-Lfucosidase from human serum. J. biol. Chem. 257, 714-718. DiMatteo G., Orfeo M. A. and Romeo G. (1976) Human ct-fucosidase. Biochim. biophys. Acta 429, 527-545. Fisher K. J. and Aronson N. N. (1989) Isolation and sequence analysis of a cDNA encoding rat liver Ct-Lfucosidase. Biochem. J. 264, 695-701. Grove D. S. and Serif G. S. (1981) Porcine thyroid fucosidase. Biochim. biophys. Acta 662, 246-255.
Hopfer R. L. and Alhadeff J. A. (1985) Solubilization and characterization of pellet-associated human brain Ct-L-fucosidase activity. Biochem. J. 229, 679-685. Hopfer R. L., Johnson S. W., Masserini M., Giuliani A. and Alhadeff J. A. (1990) Hydrolysis of fucosyl-Gml ganglioside by purified pellet-associated human brain and human liver Ct-L-fucosidases without activator proteins or detergents. Biochem. J. 266, 491-496. Jain R. S., Binder R. L., Levy-Benshimol A., Buck C. A. and Warren L. (1977) Purification of alpha-L-fucosidase from various sources by affinity chromatography. J. Chromat. 139, 283-290. Kress B. C., Freeze H. H., Herd J. K., Alhadeff J. A. and Miller A. L. (1980) Purification and characterization of I-cell disease Ct-L-fucosidase. J. biol. Chem. 255, 955-961. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T 4. Nature 277, 680-685. Lane L. C. (1978) A simple method for stabilizing proteinsulfhydryl groups during SDS-gel electrophoresis. Analyt. Biochem. 86, 655-664. Laury-Kleintop L. D., Alhadeff J. A. and Damjanov I. (1985a) Isoelectric forms of a-L-fucosidase in mouse teratocarcinoma derived cell lines. Devl. Biol. 111, 520-524. Laury-Kleintop L. D., Damjanov I. and Alhadeff J. A. (1985b) Characterization of mouse liver Ct-L-fucosidase. Biochem. J. 230, 78-82. Laury-Kleintop L. D., Damjanov I. and Alhadeff J. A. (1987) Antibody-affinity purification of novel Ct-L-fucosidase from mouse liver. Biochem. J. 245, 589-593. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the folin phenol reagent. J. biol. Chem. 193, 265-275. Merril C. R., Goldman D., Sedman S. A. and Ebert M. H. (1981) Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science 21L 1437-1438. Roger L., Bernard M.-A., Percheron F. and Foglietti M.-J. (1989) Ct-L-Fucosidase activity in normal human lymphocytes. Clinica chim. Acta 180, 303-310. Srivastava P. N., Arbtan K., Takei G. H., Huang T. T. F. and Yanagimachi R. (1986) Ct-L-Fucosidase from bull seminal plasma: its purification and acrosome reaction promoting property. Biochem. biophys. Commun. 137, 1061-1068. Turner B. M. (1979) Purification and characterization of alpha-L-fucosidase from human placenta, pH-dependent changes in molecular size. Biochim. biophys. /lcta 578, 325-336. Van Hoof F. (1973) Enzyme replacement in lysosomal diseases. In Lysosomes and Storage Diseases (Edited by Hers H. G. and Van Hoof F.), pp. 277-290. Academic Press, New York. Watkins P. and Alhadeff J. A. (1980) Kinetic and immunochemical characterization of Ct-L-fucosidase from vertebrate livers. Comp. Biochem. Physiol. 68, 517-520. White W. J., Schray K. J., Legler G. and Alhadeff J. A. (1987) Further studies on the catalytic mechanism of human liver Ct-L-fucosidase. Biochim. biophys. Acta 912, 132-138. Wright K., Northcote D. H. and Davey R. M. (1976) Preparation of rat epididymal alpha-L-fucosidase free from other glycosidases: its action on root-cap slime from Zea mays L. Carbohydr. Res. 47, 141-150.
