Comp. Biochem. Physiol.Vol. 95B, No. 1, pp. 165-169, 1990 Printed in Great Britain

0305-0491/90 $3.00 + 0.00 © 1989 PergamonPress plc

A HEAT-STABLE ALKALINE PHOSPHATASE FROM P E N A E U S J A P O N I C U S BATE (CRUSTACEA: DECAPODA): A PHOSPHATIDYLINOSITOL-GLYCAN ANCHORED MEMBRANE PROTEIN NIN-NIN CHUANG Division of Biochemistry and Molecular Science, Institute of Zoology, Academia Sinica, Nankang, Taipei, Taiwan, Republic of China (Received 1 June 1989)

A heat-stable alkaline phosphatase was purified from Penaeusjaponicus, with a final specific activity of 21,280 U/mg of protein. 2. In polyacrylamide-gel electrophoresis under non-denaturing conditions, the purified shrimp alkaline phosphatase was found to have an identical molecular size and surface charge as the human placental enzyme. 3. By using SDS-PAGE, the monomers of shrimp alkaline phosphatase were discovered to have a Mr 55,000 but those of human placental enzyme with a Mr 70,000. Deglycosylation decreases the M r values of the subunits to 33,000 for shrimp alkaline phosphatase. 4. The purified alkaline phosphatase from shrimp was recovered with both the attachment sites for sialic acids and phosphatidylinositol. 5. The shrimp alkaline phosphatase has an isoelectric point (pI) of 7.6 and the human placental enzyme has a pl of 4.8.

Abstract--1.

INTRODUCTION The alkaline phosphatases (EC 3.1.3.1) are intrinsic plasma membrane enzymes found on the membranes of almost all animal cells. In higher primates three distinct groups of isoenzymes: placental, intestinal (fetal and adult) and hepatic/renal/skeletal are recognized (McComb et al., 1980; Seargeant and Stinson, 1979; Moss, 1982). Recently, the studies with peptide 'mapping' and N-terminal-sequence analysis indicate that these isoenzymes are the products of the same gene locus (Knoll et aL, 1988; Hua et aL, 1986; Garattini et al., 1986). The evolution of this gene family has presumably involved the duplication of a primordial hepatic/renal/skeletal alkaline phosphatase gene to create the intestinal alkaline phosphatase gene, followed by additional duplication to create the placental alkaline phosphatase gene. That is, the placental alkaline phosphatase is the latest mutated form. Despite the observed highly conserved homology at the amino termini of alkaline phosphatases (Hua et al., 1986; Besman and Coleman, 1985; Garattini et al., 1986), the isoenzymes can still be qualitatively distinguished by either their different electrophoretic mobility on charge-separation gels or by their different stability to denaturation with heat. The placental alkaline phosphatase is found to be the sole isoenzyme being resistant to heating at 65°C, at which all the other non-placental phosphatases are rapidly inactivated (Neale et al., 1965). The difference in thermostability between placental and non-placental enzymes is probably due to tissue specific posttranslational modifications such as glycosylation at the carboxy termini of the enzyme (Moss and King,

1962; Stinson and Seargeant, 1981). Recently, the C-terminal is disclosed to be anchored on the membrane by covalent binding with phosphatidylinositol (Takami et al., 1988; Low and Zilversmit, 1980). The aim of the present study is to prepare the purified alkaline phosphatase from Penaeusjaponicus and use its heat-stability in investigating the nature and extent of differences between two evolutional particular forms of enzyme, the modern placental form and the primitive shrimp form. In addition, it is hoped that the purified shrimp alkaline phosphatase could be used in obtaining the N-terminal amino acid sequence, which may provide structural information to trace the relationships with the human enzymes and their possible evolutionary development. MATERIALS AND METHODS

Materials All reagents used were of the highest grade available commercially. Sialidase (Clostridium perfringens) was obtained from calbiochem, UK. Endoglycosidase F (Flavobacterium meningosepticum ) and phospholipase C (Bacillus cereus) were supplied by Boehringer, FRG. Reagents for isoelectric focusing were from Pharrnacia and for polyacrylamide-gel electrophoresis were as previously described (Chuang, 1987). Purification of alkaline phosphatase Alkaline phosphatase was purified with essentially the same procedures as described by Meyer et al. (1982) but with slight modifications. Fresh shrimps (Penaeusjaponicus) were washed once with cold distilled water. The hepato-pancrease was chopped into small fragments and homogenized in 300ml of buffer A (10 mM Tris-HC1, pH 7.5, containing

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NIN-NIN CHUANG Table 1. Purification of alkaline phosphatase from Penaeusjaponicus Specific Activity Protein activity Yield Purification Purification step (U) (rag) (U/rag) (%) factor Butanol extract 12,723 3360 3.8 100 I Ethanol precipitate 9,668 17l 56 76 14 DE 52 2,623 41 64 21 16 Con A-Sepharose 1,251 4.3 291 9.8 75 AcA 34 1,049 0.76 1380 8.2 354 Gel electrophoresis 532 0.025 21 280 4.1 5455

1 mM MgC12 and 0.1% Triton X-100) for 2min with a Polytron unit and the homogenate was then stirred at 4"C for 30 rain. Following this, 250 ml of butanol was added, and the mixture stirred overnight at 4°C. After centrifugation at 6000g for 20 min, the butanol layer was discarded, the aqueous phase was adjusted to pH 5.0 by adding of 1% acetic acid and, after heating at 65°C for 10 rain, centrifuged at 6000g for 20 rain. The supernatant was adjusted to pH 7 with solid N%CO 3 and ethanol (-20'~C) was added with stirring to bring the concentration of acetone to 25% (v/v). After centrifugation at 6000g for 20 rain, the precipitated pellet was dissolved in 0.9% NaC1 and dialysed overnight against 10 mM NaCI. The recovered solution was applied to DE 52 (Whatman DEAE cellulose) equilibrated in buffer A and was developed with the same buffer superimposed on a gradient of NaC1. Alkaline phosphatase activity was eluted with 0.5 M NaC1. Those fractions with the highest specific activity were pooled and bound to concanavalin A-Sepharose (Pharmacia). After elution with Ct,D-methylmannoside, the enzyme was concentrated by ultrafiltration on an Amicon PM 10 membrane. The concentrated solution (1.5 ml) was then subjected to gel filtration on the column AcA 34 in buffer A. Finally the fraction with high specific activity was pooled and concentrated using Amicon. The concentrated enzyme preparation was further subjected to preparative polyacrylamide-gel electrophoresis under nondenaturing conditions (Chuang, 1987). A single peak of alkaline phosphatase activity was obtained and was sliced for electroelution. Polyacrylamide-gel electrophoresis Polyacrylamide-gel electrophoresis under non-denaturing conditions was carried out at 4°C with alkaline phosphatase, using buffer system 4229 of Jovin et al. (1980). Alkaline phosphatase activity was determined in 2 mm gel slices and recovery of the enzyme after electrophoresis exceeded 90% unless otherwise stated. Sodium dodecyl sulphate~olyacrylamide:gel electrophoresis (SDS-PAGE) was conducted on slab gels containing 10% (w/v) acrylamide with 0.27% (w/v) NN'methylenebis-acrylamide (LaemmlL 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 Moss and Edwards (1984) on purified placental alkaline phosphatase (approximately 50mU of activity) using ampholytes with a pH range of 3.0 10.0. For some experiments, restricted range ampholytes (pH range 5.0-7.0, 7.0-9.0) were employed for better resolution of isoelectric forms. Focusing was performed at a constant voltage (200V) for 18--21 hr. Assay of alkaline phosphatase Alkaline phosphatase activity was measured at 37°C with the use of 10raM 4-nitrophenyl phosphate in 0.75M 2-amino-2-methyl-l-propanol-HC1 buffer, pH 10.3. One unit of enzyme activity = 1 #mol substrate hydrolyzed/min. Enzyme activity after polyacrylamide gel electrophoresis was determined by incubating the gels at 37°C in 2 mg of -naphthylic acid phosphate (Sigma F-7375) per ml, and 1 mg

of Fast Blue BB (Sigma N-0250) per ml in 60 mM borate buffer/pH 9.7 for 30 min to stain the activity bands. 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 scheme used for shrimp alkaline phosphatase, summarized in Table 1, is similar to the one t h a t was successfully applied to the isolation of h u m a n placental alkaline p h o s p h a t a s e (Chuang, 1987). After the gel filtration step, p r e p a r a t i o n activity gel electrophoresis removed m a j o r c o n t a m i n a n t s (Fig. 1). The final specific activity of purified alkaline p h o s p h a t a s e was 21,280 U / r a g of protein, and the overall purification was a b o u t 5500-fold. The existence of a m e m b r a n e binding d o m a i n in alkaline p h o s p h a t a s e may be d e m o n s t r a t e d by the application of phospholipase C. It has been reported t h a t p h o s p h a t i d y l inositol-specific phospholipase C can release alkaline p h o s p h a t a s e from cell membranes (Shukla et al., 1980) a n d phosphatidylinositol

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Fig. 1. Non-denaturing polyacrylamide-gel electrophoresis of alkaline phosphatase in Penaeus japonicus. Partially purified alkaline phosphatase preparation from AcA 34 was electrophoresed into 10% (w/v) polyacrylamide gels under non-denaturing condition in the presence of 0.1% Triton X-100. The gel was stained for alkaline phosphatase activity. ~t" Marks the moving position of enzyme.

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Penaeus alkaline phosphatase

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B

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Fig. 3. Ferguson plot analysis of alkaline phosphatase from Penaeus japonicus. Purified alkaline phosphatase preparation from Penaeus japonicus was electrophoresed on

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4=Front Fig. 2. Conversion by phosphatidyl inositol-specific phospholipase C of the purified alkaline phosphatase from Penaeus japonicus. Purified enzymes (10 mU of each) were incubated at 37°C for 60 rain in the presence of phospholipase C purified from Bacillus cereus (4000 U/ml) (track B) or without (track A), followed by electrophoresis in 0.1% Triton X-100 on a 10% T (w/v total monomer concentration) polyacrylamide gel. The gel was stained for alkaline phosphatase activity. Alkaline phosphatase was incubated with phospholipase C (1 unit) in 50mM Tris-maleate (pH 5.5) and 1 mM CaCI2 at 37°C for 1 hr. provides the enzyme's hydrophobic membrane anchor (Low and Zilversmit, 1980; Low et al., 1986). After the treatment with phospholipase C, the shrimp alkaline phosphatase did show decreased mobility in native activity gel (Fig. 2), indicating that the enzyme retained a phosphatidylinositol-glycan membrane binding domain. In previous studies, an endogenous cellular phospholipase C which can be activated by acidic butanol has been proposed to be responsible both for the acidic butanol-solubilized form of alkaline phosphatase and the soluble serum form (Ogata et al., 1988; Hawrylak and Stinson, 1988; Bailyes et al., 1987; Miki et al., 1985). Our data are consistent with this, since butanol extraction had no effect on the detergent-purified enzyme which does not contain phospholipase C (Bailyes et al., 1987) and alkaline butanol extraction yielded enzymes with phosphatidylinositol attachment (Hawrylak and Stinson, 1988). In the present study, the shrimp alkaline phosphatase was first solubilized by detergent, Triton X-100, then extracted with alkaline butanol. When the purified alkaline phosphatase from Penaeus j a p o n i c u s was electrophoresed under nondenaturing conditions in the p r e s e n c e of Triton

non-denaturing tube gels over different concentrations in the presence of Triton X-100. For each gel concentration, duplicate gels were measured, and the plot of Rf (log scale) vs gel concentration (%) was constructed from the results for three samples using least-square linear regression. X-100, a non-ionic detergent, the enzyme exhibited one homogenous form. Ferguson plot comparative analysis of the shrimp alkaline phosphatase and the enzyme from human placenta reveals that both shared the same size and charge, having a Y0 of 3.0_+ 0.2 and a K r equivalent to a geometric mean radius of 3 . 7 n m with an apparent M r 160,000 (Fig. 3). Despite the somewhat surprising similarities in surface charge and molecular size between the shrimp and human placental enzymes, the physical relationship between them is not indicative of their identity. Using isoelectric focusing and S D S - P A G E , the shrimp alkaline phosphatase was found to have an isoelectric point (pI) of 7.6 + 0.1 (Fig. 4A), an A

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Fig. 4. Isoelectric points of alkaline phosphatase from Penaeus japonicus. Purified alkaline phosphatase from Penaeusjaponicus were untreated (A) or treated (B) with the

bacterial sialidase and subsequently subjected to isoelectric focusing. For comparison, the purified alkaline phosphatase from human placenta with (C) or without (D) the treatment of bacterial sialidase was included. For each sample, triplicate gels were measured. Alkaline phosphatase (10 mU) was incubated with sialidase (0.5 U) in 50 mM sodium acetate (pH 4.5) and 0.1% (w/v) Triton X-100, and the reaction was stopped by immediate freezing.

168

NIN-N1N CHUANG

A

C

B

94k 71k

67k 5sk

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33k 3ok

Fig. 5. SDS- PAGE of the purified alkaline phosphatase from Penaeus japonicus. The purified alkaline phosphatase (I pg) either non-treated (A) or treated (B) with endoglycosidase F was subjected to electrophoresis on a SDS-polyacrylamide gel (10%), then with silver staining. Alkaline phosphatase was incubated with endoglycosidase F (10U) in 100mM sodium phosphate (pH 6.1), 50mM EDTA, 1% Nonidet P-40, 0.1% SDS and 1% 2-mercaptoethanol. After incubation at 37~'C for 14 hr, the reaction was stopped. For comparison, human placental alkaline phosphatase was included (C).

apparent subunit mol. wt (Mr) of 55,000 (Fig. 5A) and the placental enzyme having a pI of 4.8 _+ 0.1 (Fig. 4C), a M r of 70,000 (Fig. 5C). Figures 4B and D demonstrate the effect of sialidase digestion on the two enzymes. The placental enzyme is shifted to a more basic pI, whereas the shrimp enzyme is slightly affected by this treatment. The desialyated shrimp alkaline phosphatase has a pl of 7.8 + 0.1. In other words, the heat-stable alkaline phosphatase in shrimps is a sialyated glycoprotein. As a third approach to the physical relationship between these two enzymes, we used deglycosylation of purified enzymes and analysis by SDS PAGE. Since alkaline phosphatase is known to be highly glycosylated and the shrimp alkaline phosphatase appeared to have subunits with tool. wt very close to that of the deglycosylated form of human liver alkaline phosphatase (M r 50,000; Garattini et al., 1986), the purified shrimp alkaline phosphatase was treated with endoglycosidase F, an enzyme that removes asparagine-linked oligosaccharides from glycoproteins. Figure 5B shows that deglycosylation shifted the M r values of the subunits from 55,000 to 33,000 for shrimp alkaline phosphatase. The shrimp enzyme apparently contains six-fold as much Nlinked carbohydrate (33%) as does the placental enzyme (5%; Takami et al., 1988). In the present study, we provide the first evidence that a unique complement of enzymic machinery for post-translational glycosylation of alkaline phosphatase does exist in shrimps. Further studies on comparing primary structure of alkaline phosphatase from shrimp and human placenta are being undertaken.

Acknowledgements--I thank Professor Chung-Chia Huang for his careful reading of the manuscript. I am grateful to Su-Ling Shih for her technical assistance. This work was supported by National Science Council, Republic of China.

REFERENCES

Berger J., Howard A. D., Brink L., Gerber L., Hauber J., Cullen B. R. and Udenfriend S. (1988) COOH-terminal requirements for the correct processing of a phosphatidylinositol-glycan anchored membrane protein. J. biol. Chem. 263, 10,016 10,021. Chuang N.-N. (1987) Alkaline phosphatase in human milk: a new heat-stable enzyme. Clinica chim. Acta 169, 165-174. Endo T., Ohbayashi H., Hayashi Y., Ikehara Y., Kochibe N. and Kobata A. (1988) Structural study on the carbohydrate moiety of human placental alkaline phosphatase. J. Biochem. 103, 182 187. Garattini E., Hua J.-C., Pan Y.-E. and Udenfriend S. (1986) Human liver alkaline phosphatase, purification and partial sequencing: homology with the purified isozyme. Archs Biochem. Biophys. 245, 331 337. Hawrylak K. and Stinson R. A. (1988) The solubilization of tetrameric alkaline phosphatase from human liver and its conversion into various forms by phosphatidylinositol phospholipase C or proteolysis. J. bioL Chem. 263, 14,368 14,373. Hua L-C., Berger J., Pan Y. E., Hulmes J. D. and Udenfriend S. (1986) Partial sequencing of human adult, human fetal and bovine intestinal alkaline phosphatases: comparison with the human placental and liver enzyme. Proc. natn. Acad. Sci. USA 83, 2368 2372. Jovin T. M., Dante M. L. and Chrambach A. (1980) Multiphasic buffer system output, National Technical Information Service, Springfield, VA. 22151, PB numbers 196090, 259309 259312.

Penaeus alkaline phosphatase

Knoll B. J., Rothblum K. N. and Longley M. (1988) Nucleotide sequence of the human placental alkaline phosphatase gene. J. biol. Chem. 263, 12,020-12,027. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, Lond. 227, 680-685. Lane L. C. (1978) A simple method for stabilizing protein-sulfhydryl groups during SDS~el electrophoresis. Analyt. Biochem. 86, 655-664. Low M. G., Ferguson M. A. J., Futerman A. H. and Silman I. (1986) Covalently attached phosphatidylinositol as a hydrophobic anchor for membrane proteins. TIBS 11, 212--215. Low M. G. and Zilversmit D. B. (1980) Role of phosphatidylinositol in attachment of alkaline phosphatase to membranes. Biochemistry 19, 3913 3918. Lowry O. 1t., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the folin phenol reagent. J. biol. Chem. 193, 265-275. McComb R. B., Bowers G. N. and Posen S. (1980) Alkaline Phosphatase, pp. 373 524. Plenum Press, New York. McKenna M. J., Hamilton T. A. and Sussman H. H. (1979) Comparison of human alkaline phosphatase isoenzymes. Biochem. J. 181, 67 73. 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 211, 1437 1438. Meyer L. J., Lafferty M. A., Raducha M. G., Foster C. J., Gogolin K. J. and Harris H. (1982) Production of a monoclonal antibody to human liver alkaline phosphatase. Clinica chim. Acta 126, 109 117. Miki A., Kominami T. and Ikehara Y. (1985) pH-dependent conversion of liver-membranous alkaline phosphatase to

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a serum-soluble form by n-butanol extraction. Biochem. biophys. Res. Commun. 126, 89-95. Moss D. W. (1982) Alkaline phosphatase isoenzymes. Clin. Chem. 28, 2007-2016. Moss D. W. and Edwards R. K. (1984) Improved electrophoretic resolution of bone and liver alkaline phosphatases resulting from partial digestion with neuraminidase. Clinica chim. Acta 143, 177 182. Moss D. W. and King E. J. (1962) Properties of alkaline phosphatase fractions separated by starch-gel electrophoresis. Biochem. J. 84, 192-195. Neale F. C., Clubb J. S., Hotchkis D. and Posen S. (1965) Heat-stability of human placental alkaline phosphatase. J. clin. Pathol. 18, 359-363. Ogata S., Hayashi Y., Takami N. and Ikehara Y. (1988) Chemical characterization of the membrane-anchoring domain of human placental alkaline phosphatase. J. biol. Chem. 263, 10,489 10,494. Seargeant L. E. and Stinson R. A. (1979) Affinity elution from a phosphoric acid-Sepharose derivative in the purification of human liver alkaline phosphatase. Nature 281, 152 154. Shukla S. D., Coleman R., Finean J. B. and Michell R. H. (1980) Selective release of plasma-membrane enzymes from rat hepatocytes by a phosphatidylinositol-specific phospholipase C. Biochem. J. 187, 277 280. Stinson R. A. and Seargeant L. E. (1981) Comparative studies of pure alkaline phosphatases from five human tissues. Clinica chim. Acta 110, 261 272. Takami N., Ogata S., Oda K., Misumi Y. and Ikehara Y. (1988) Biosynthesis of placental alkaline phosphatase and its post-translational modification by glycophospholipid for membrane anchoring. J. biol. Chem 263, 3016-3021.