Modifications in a Flexible Surface Loop Modulate the Isozyme ...

Vol. 268, No. 34, Issue of December 5, pp. 25409-25416,1993 Printed in U.S. A.

OF BIOLOGICAL CHEMISTRY THEJOURNAL

Inc. ~ 0 1993 by The American Society for Biochemistry and M o l d a Biology,

Modifications in aFlexible Surface Loop Modulate the Isozymespecific Propertiesof Mammalian Alkaline Phosphatases* (Received for publication, February 26, 1993,and in revised form, July 26, 1993)

Massimo BossiSP, MarcF. Hoylaertsn, andJo& Luis MillinSII From the $LaJolla Cancer Research Foundation, La Jolla, California 92037 and the (Centerfor Molecular and Vascular Biology, Uniuersity of Leuuen, B-3000 Leuuen, Belgium

We have analyzed to what extent the surface loop variety of organs like liver, bone, kidney, etc. and threetissuedomain of alkaline phosphatases (APs) is responsible specific AP (TSAP) genes coding for the intestinal AP (IAP), forisozyme-specificfunctionalproperties.Unique placentalAP (PLAP), and germcell AP (GCAP). TNAP Aut11and RsrII restriction sites were introduced by shows approximately 50% sequence similarity with the TSAP site-directed mutagenesis at identical positions in mu- isozymes, while IAP is 90% identical to PLAP/GCAP, these rine tissue-nonspecific AP (TNAP) and human placen- last two isozymes differing by only 12 of their 513 amino acid tal AP (PLAP) cDNAs to allow thehomologous ex- residues (98% identity). The human APs, as well as other change of the loop domain of the TNAP (T domain) mammalian and APs, show 25-35% sequence similarity with the PLAP (P domain) isozymes and the generation of the E. coli enzyme in those regions adopting a-helix and B-strand reciprocally chimeric molecules PLAP-T and TNAP- conformations that are critical for catalysis. The catalytic P. The introduction of the T loop into PLAP reduced residues, i.e. Aspg1,Serg2,Arg’66, and ligands coordinating the the heat stability of PLAP-T to almost that of TNAP. metal ions zinc and magnesium are all conserved (Millhn, The domain substitution was accompanied by a confor1988) suggesting that the mammalian APs may perform hymational change that resulted in the loss of immune reactivity with four of 17 epitope-mapped anti-PLAP drolase/transphosphorylase reactions via a similar mechanism as their bacterial counterpart. However, contrary to the monoclonalantibodies.TheTandPloopsprovided stabilization to the side chainspecific of uncompetitive bacterial enzyme that is known to be involved in phosphate AP inhibitors. The introduction of the T domainalso transport (Horiuchi et al., 1959), the biological role and subconferred collagen-binding propertiesto PLAP-T ac- strates of the mammalian APs await elucidation. Yet, three counting for halfof the binding affinity of TNAP for properties of mammalian AP not shared by their bacterial collagen, while not affecting PLAP binding to IgG. ancestor may provide clues to their function. Our data indicate that the surface loop determines First, while the bacterial AP is released after synthesis to overall enzyme stability, differs conformationally in the periplasmic space, mammalian APs are anchored to the the various isozymes, and modulates catalytic param- exterior of the cytoplasmic membrane via a phosphatidylinoeters in the presence of protein ligands, thus, account-sitol glycan moiety (Low and Saltiel, 1988). In the case of ing in part for isozyme-specific protein interactions. human PLAP, this structure is covalently attached to Aspm, following proteolytic processing of a hydrophobic stretch of amino acids at the carboxyl-terminal end of the de nouo Alkaline phosphatases (EC 3.1.3.1) (AP)’ are highly ubiq- synthesized molecule (Micanovic et al., 1988; Takami et al., uitous enzymes found in almost all species from bacteria to 1988). Second, mammalian APs, but not the bacterial enman (McComb et al., 1979). The Escherichia coli A P was the zymes, are susceptible to inhibition by stereo-specific amino first AP whose amino acid sequence was determined (Brad- acids and peptides, particularly L-homoarginine, L-Phe, Lshaw et al., 1981). The crystallographic structures of the E. Leu, and the related peptides L-Phe-Gly-Gly and L-Leu-Glycoli AP and of two phosphoryl intermediates have been re- Gly (Fishman and Sie, 1971; Doellgast and Fishman, 1976; solved at the 2.0-A resolution (Kimand Wyckoff, 1991). Mulivor et al., 1978). While PLAP and GCAP only differ in Recently, several mammalian APs, including human APs, 12 of 513 residues ( M i l l h and Manes, 1988), GCAP displays have been cloned and sequenced (for review see Millan, 1988; a 17-fold higher sensitivity to L-Leu inhibition than PLAP Harris, 1989). The human genome contains four AP loci, one (Doellgast andFishman, 1976). Recently, this differential coding for the tissue-nonspecific A P (TNAP) expressed in a inhibition was found to reside on a single Gly for Glu substitution at position 429 in the GCAP molecule (Hummer and * This work was supported by Grant CA 42595 from the National Millhn, 1991; Watanabe et al., 1991; Hoylaerts and Millhn, Institutes of Health. The costs of publication of this article were 1991). The surface loop of PLAP/GCAP that harbors residue defrayed in part by the payment of page charges. This article must 429 plays an important role in determining the accessibility therefore be hereby marked “advertisement” in accordance with 18 and stability of the inhibitor in the active site duringuncomU.S.C. Section 1734 solely to indicate this fact. 5 Supported by a fellowship from the Swiss National Fond and petitive inhibition(Hoylaerts et al., 1992a). Third,TNAP interacts with collagen type I, 11, and X, and this interaction Zurcher Krebsliga. 11 To whom correspondence should be addressed La Jolla Cancer was suggested to contribute to the mineralization process of Research Foundation, Cancer Research Center, 10901 N. Torrey osteoid to bone (Vittur et al., 1984; Wu et al., 1991, 1992). Fax: 619-455-0181. Using sequence comparisons we were able to identify a putaPines Rd., La Jolla, CA 92037.Tel.: 619-455-6480; The abbreviations used are: AP, alkaline phosphatase; TNAP, domain in APs based on tissue-nonspecific AP; TSAP, tissue-specific AP; IAP, intestinal AP; tive protein-proteininteraction PLAP, placental AP; GCAP, germ cell AP; kb, kilobase; wt, wild- sequence homology with Von Willebrand factor, complement type; TES, N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic factor B, cartilage matrix protein, Mac-1, p150, all molecules acld. with protein binding capabilities, and we suggested that this

25409

25410

Structural-Functional Domain

sequence may represent a putative functionaldomain of APs (Tsonis et al., 1988). The region of identified homology contains the loop found to be of critical importance for the stabilization and specificity of the uncompetitive inhibitors of APs. The recent findings that the Fc portion of human IgGs interacts with PLAP (Makiya and Stigbrand, 1992a) and thehypothesis that PLAPmight function as theplacental Fc receptor (Makiya and Stigbrand, 1992b), further support a role for AP-protein interactions. Since this surface loop of the mammalian APs shows the lowest degreeof sequence similarity between the different AP isozymes, this region potentially represents a domain conferring isozyme-specific properties to this family of homologous molecules. In thispaper we provide evidence that thesequence and conformation of this loop plays an important role in determining the stability of the isozymes toward thermal denaturation. Also, this loop is in part responsible for the isozyme-specific binding of TNAP to collagen as well as for the stabilization of the side chains of peptide inhibitors of APs. The loop is notinvolved in thebinding of IgG byPLAP.

of Alkaline Phosphatases

method (Gorman et al., 1982). The wild-type PLAP andGCAP stable transfectants were previously described (Hummer and Millh, 1991) as were[Ala’lIPLAP and [Asnsl]PLAP mutants (Hoylaerts et al., 1992a). TWOdays after plasmid transfection, APs were extracted as described (Hoylaerts et al., 1992a) and the extracts aliquoted and stored at -70 “C until used. Kinetic Measurements-Experimental conditions, mathematical calculations, and graphical representations were done as previously reported (Hoylaerts and Millin, 1991; Hoylaerts et al., 1992a). AP activity was measured using 10 mM p-nitrophenylphosphate as substrate in 1.0 M diethanolamine buffer, pH = 9.8, containing 0.5 mM M&1, and 20 p M ZnClz. During measurements of K,,,, uncompetitive inhibition, and antibody recognition the final assay volume was 200 pl maintaining the substrate concentration at 10 mM. Changes in absorbance were measured at 405 nm. For K,,, determinations in the presence of proteins, the stock solutions of human IgG (10 mg/ml) and bovine or chicken collagen I1 (3 mg/ml in 1mM HCl) were diluted to a final concentration of 0.25 p M in 1 M diethanolamine, pH 9.8, 0.5 mM MgCl,, and 20 pM ZnCl,. Kinetic measurements in the presence of proteins were also carried out in 1M Tris, pH 7.5,0.5 mM MgCl,, and 20 p~ ZnCIZ. Prior to heat inactivation kinetics, AP samples were diluted in 10 mM Tris-buffered saline, pH 7.5, containing 10% saturated casein, 20 p M ZnClz, 1 mM MgCl, and incubated in a water bath at 56 “C. At fixed time intervals 50-pl samples wereremoved and kept on ice. MATERIALS ANDMETHODS Residual activities were then measured in duplicates upon simultaConstruction of Chimeric A P cDNAs-A 2.0-kb EcoRI-KpnI frag- neous addition to thewells of 200 pl of substrate solution. ment of the human PLAP cDNA (Millin, 1986) and a 1.1-kb S m I The inhibitors L-homoarginine, L-Phe, L-Leu, L-Phe-Gly-Gly, and EcoRI fragment of the mouse TNAP cDNA (Hahnel and Schultz, L-Leu-Gly-Gly were purchased from Sigma. The tripeptide L-Phe1989) were subcloned into the M13mp18 phage vector. In vitro site- hydroxyPro-Gly and the two undecapeptides, i.e. Val-Thr-Glu-Serdirected mutagenesis (Muta-Gene@M13 in vitro mutagenesis kit, Bio- Glu-Ser-Gly-Ser-Pro-Glu-Tyr and Val-Ser-Met-Val-Asp-Tyr-AlaRad) was used to introduce an RsrII restriction site at position 1290 His-Asn-Asn-Tyr, were synthesized in-house using an Applied Bioin the PLAP cDNA and 1434 for mouse TNAP and anAatII site at systems 430A peptide synthesizer. The carboxyl-terminal modificapositions 1414 and 1558of the respective cDNAs. The following bases tion of L-Phe-Gly-Gly was done manually by first protecting the were replaced 1295 ( A - G ) and 1415 (G-C) in PLAP and 1436 amino group with t-Boc followed by activation of the protected (G+T), 1439 (T-G), and 1559 ( k c ) in TNAP. These substitutions tripeptide to an active ester and subsequent addition of 2-amino-4did not change the codon specificity but allowed the excision of nitro-phenol and deprotection of the amino group to produce L-Pheequivalent 124-base pair RsrII-AatII fragments derived from the Gly-Gly-pNP. Acetylation of the tripeptides L-Phe-Gly-Gly and LPLAP cDNA (P fragment) and TNAP cDNA (T fragment), respec- Leu-Gly-Gly was done following procedures described elsewhere tively. (Bodanszky and Bodanszky, 1981). A 316-base pair SmaI fragment of the PLAP cDNA (extending Measurements of Monoclonal Antibody Reactivity-Immunoreacfrom position 1251 to 1567) and a 1.4-kb SmaI-EcoRI fragment of tivities were measured as described (Hoylaerts and Millin, 1991; the TNAP cDNA were subcloned into Bluescript KS+ (Stratagene, Hoylaerts el at., 1992b).Briefly, 15 anti-PLAPmonoclonal antibodies San Diego, CA) and used as templates for the exchange of the P and and two cross-reacting anti-intestinal AP antibodies were incubated T fragments, respectively, by digestion with RsrII and AatII and in microtiter plates, precoated with a rabbit anti-mouse IgG antiligation with the exchanged fragments. A complete PLAP-T cDNA serum. Consecutively, low concentrations of the PLAP, PLAP-T, and was obtained by ligating the hybrid SmaI fragment into a SacII-KpnI PLAP-t chimeric enzymes (0.5-1.0 units/liter) were added to the PLAP-Bluescript plasmid previously digested with SrnaI, and finally insolubilized monoclonal antibodies. Upon equilibration, the bound the SacII-KpnI fragment was ligated into the SacII-KpnI-digested fraction was measured and expressed relative to the total enzyme expression vector PLAP-pSVT7 (Hummer and Millin, 1991). The concentration deposited in the wells. Measurements were carried out TNAP-P cDNA was obtained in a one step two-way ligation assem- in triplicates and theaverage concentration (+S.D.) of bound enzyme bling a 1.4-kb HindIII-XmnI fragment (the HindIII site derived from (B) was divided by the concentration of the totalfraction ( O ) , in order the polylinker region of pcDNA I) containing the 5’ half of the TNAP to obtain an affinity index (B/O) for the binding. PLAP B/O was cDNA with the hybrid 1.0-kb XrnnI-EcoRI fragment into theexpres- given a value of 1, and all others are expressed as a percentage of sion vector pcDNA I (Invitrogen, San Diego, CA)previously digested PLAP binding. During peptide competition experiments, each of the with HindIII and EcoRI. The integrity of the exchanged domains and undecapeptides, in concentrations ranging from 10 nM to 100 mM, ligation junctions were confirmed by sequencing using the Sequenase was added simultaneously with wild-type PLAP. Bound PLAP was expressed as a percentage of PLAP bound in the absence of peptides. 2.0 kit (United States Biochemical Corp. Cleveland, Oh). Wild-type Affinity Chromatography on Collagen-Sephurose and IgG-SephaPLAP and TNAP cDNAs containing the point mutations, but no rose-Forty mg of collagen type I1 were isolated from 12 g of adult domain exchange, were also rebuilt to be used as wild-type controls. The third hybrid, PLAP-t, was built following the same strategy chicken sternal cartilage by the method of Miller (Miller and Rhodes, depicted above except that thecorresponding doubled-stranded AatII- 1982) and bound to Sepharose CL-4B activated with CNBr following RsrII fragment was synthesized on a Applied Biosystems Automated the method of Cuatrecasas (1970). Twenty mgof either chicken or 380 B-DNA synthesizer. The forward strand of the 124-base pair bovine (Seikagaku America Inc., St. Petersburg, FL) type I1 collagen domain was synthesized as two oligonucleotides with the following were coupled to 5 ml of activated Sepharose (4 mg/ml wet gel, 67% sequence: 5”GT CCG GGC TAT GTG CTC AAG GAC GGC GCC yield). Binding and elution of the APs to the collagen column was CGGCCGGATGTTTCCATGGTAGATTACGCTCAC-3’and done following the conditions described in Wu et al. (1991). Sixty mg 5”AAC AAC TAT CGG CAG CAG TCA GCA GTG CCC CTG GAC of human IgG were purified from 10 ml of human control serum by GAAGAGACCCACGCA GGC GAG GAC GT-3’ containing the protein A-Sepharose affinity chromatography and 30 mg were bound correct endings RsrII at the 5’ end and the AatII at the 3’ end. to CNBr-activated Sepharose CL-4B (3 mg/ml, 95% yield) as deSimilarly, thereverse set of primers had sequences: 5°C CTC GCC scribed above. The AP preparations were loaded onto the column in T G C G T G G G T C T C T T C G T C C A G G G G C A C T G C T G A C T G 50 mM Tris-HC1, 100 mM NaCl, pH 7.4. After washing with 20 bed CTG CCG ATA GTT GTT GTG-3’ and 5’-AGC GTA ATC TAC volumes of the same buffer, any AP activity retained on the column C A T G G A A A C A T C C G G C C G G G C G C C G T C C T T G A G C A C was eluted with 0.1 M sodium citrate, pH 4.0, as described in Makiya and Stigbrand (1992a). The elution profiles were obtained adding 50 ATA GCC CG-3’. Tissue Culture-Chinese hamster ovary cells were cultivated in 10- p1 of each eluted fraction to 200 pl of substrate solution containing mm Petri dishes until 50% confluence and then transfected with 10 mM p-nitrophenyl phosphate in 1 M diethanolamine buffer, pH PLAP-T, PLAP-t, TNAP-P, or TNAP by the calcium phosphate 9.8, 0.5 mM MgCl,, and 20 p M ZnCl,.

73

Structural-Functional Domain of Alkaline Phosphatases

2541 1

ters, monoclonal antibody recognition, and collagen or IgG binding ability. Heat Stability Studies-TNAP loses its enzymatic activity very rapidly a t 56 "C (Whitby and Moss, 1975),whereas PLAP is stable for several hours even at 65 "C(Fishman, 1974). Fig. 2 shows the progressive inactivation with time of PLAP, PLAP-t, PLAP-T, TNAP,and TNAP-P at 56 "C. Remarkably, the introduction of the T loop into PLAP reduced the heat stability properties of the chimeric molecule to a value similar tothat of TNAP.This destabilization was even achieved by introduction of the smaller t domain as seen in the behavior of PLAP-t (Fig. 2). Reciprocally, as compared to TNAP, TNAP-P gained some stability. These results point out the significant role played by this surface loop in determining the heat stability properties of the molecules. No experimental evidence has previously been provided to explain the heat stability of PLAP or GCAP. In the case of the E. coli AP, which does not have a surface loop equivalent to the mammalian AP loop, mutations of the active site residue Asp'" were shown to decrease the stability of the enzyme (Chaidaroglou and Kantrowitz, 1989). However, mutagenesis of the homologous Aspg' residue in PLAP for Asn or Ala influenced the turnover number of the mutantenzymes (Hoylaerts et al., 1992a) but had no major effect on heat stability as shown in Fig. 2. Our data indicate that in the mammalian APs changes in the active site are less destabilizing than in the bacterial enzyme and that, in this case, it is the surface loop that plays a critical role in determining enzyme stability. Reactivity of PLAP Chimeric Mutants with a Panelof Morw-

RESULTS AND DISCUSSION

Strategy of Surface Loop Swapping-Previous studies (Tsonis et al., 1988; Hoylaerts et al., 1992a) have suggested the possible importance of a surface loop of approximately 40 amino acids in determining or influencing unique properties of the mammalian APs such as collagen binding and uncompetitive inhibition properties. In order to study the function of this surface loop, we focused on two isozymes with considerably different biochemical properties; i.e. on one hand murine TNAP which is very heat labile, binds to collagen type I, 11, and X and is inhibited uncompetitively by L-homoarginine and on the other hand human PLAP,which is extremely heat stable, interacts with the Fc region of IgG and is inhibited uncompetitively by t-Phe, L-Leu, and related peptides. Experimentally, unique restriction sites were introduced, by site-directed mutagenesis, into thePLAP and TNAP cDNAs to allow a homologous swapping of the loop domain to generate reciprocal chimeric molecules, i.e. PLAP-T and TNAP-P as depicted in Fig. 1. The P and T domains correspond to thepreviously identified region of homology (Tsonis et al., 1988) between APs and Mac-1, p150, Von Willebrand factor, complement factor B, and cartilage matrixprotein which are proteins known to interact with collagen. Because in the center of this loop domain a stretch of seven amino acids is entirely different between PLAP and TNAP,we have also substitutedthis small TNAP surface loop domain (t domain) for the corresponding sequence in the wt PLAP, generating a PLAP-t mutant (Fig. 1). These chimeric molecules were compared tothe wild-type TNAPandPLAP molecules for changes in their heat stability, kinetic parame-

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mTNAP cDNA in pcDNAl FIG. 1. Schematic representation of the PLAP and TNAP cDNAs showing the restriction sites involved in the domain swapping strategy. The actual amino acid sequence contained between the experimentally introduced RsrII and AatII sites is presented for the PLAP, PLAP-t, PLAP-T, TNAP-P, and TNAPwell as as for the corresponding region in the E. coli AP which largely lacks this loop region. The TNAP residues in w t TNAP and chimeric mutants are shaded for easy recognition. Glu429 residue (E) is boxed and shaded and represents the only difference between PLAP and GCAP in this area of the molecules.

Structural-Functional Domain

25412

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Incubation time (min.) FIG.2. Percentage residual activity of PLAP, TNAP, and mutants after incubation at 56 “C as a function of time. The symbols represent: PLAP (O), PLAP-t (A), PLAP-T (O), TNAP-P (W), TNAP (O),[Alasl]PLAP (0),[Asngl]PLAP (e). clonal Antibodies-Recently, using site-directed mutants of PLAP and GCAP, we have been able to map the epitopes of a panel of 18 monoclonal antibodies raised against PLAP (Hoylaerts and Millin, 1991). These antibodies proved to be sensitive conformational probes detecting even point mutations between PLAP and GCAP allelic variants (Hoylaerts and Millan, 1991; Hoylaerts et al., 1992b). These antibodies also revealed that the substitution of Glu4” for Gly4” which confers L-Leu sensitivity to the [Gly4”]PLAP molecule was accompanied by a major conformational change of the surface loop harboring this residue. Since the P and T domains swapped in the present investigation contain residue 429, we used this panel of antibodies to monitor the conformational effect of this homologous replacement. As depicted in Fig. 3a, the substitution in PLAP of the t domain caused a loss of affinity for the monoclonal antibodies E5, F6, and C4. This drop in affinity was extended to antibody A3 when the larger T domain was introduced in PLAP (Fig. 3b). During our mapping studies, the epitopes for antibodies E5, F6, C4, and A3 were placed in a centrally located region of the molecule (Hoylaerts and Mill&, 1991). Recently, we have attempted, in an enzyme-linked immunosorbent assay configuration, to compete out binding of E5, F6, C4, and A3 to microtiter well-bound PLAP using the synthetic undecapeptides VTESESGSPUY (derived from the P domain) and VSMVDYAHNNY (derived from the T domain). Neither of these peptides had any effect on antibody recognition further indicating that the loop itself was not part of the epitope recognized by these antibodies. Therefore, the present data strongly indicate that replacement in PLAP of the P domain by the T domain, and even the t domain, affects the conformation and position of the top surface loop in the resulting PLAP-T and PLAP-t mutants. Furthermore, antibodies F11 and 130, whose epitopes were previously placed close to the entrance to the active site and adjacent to the surface loop, reacted more strongly with PLAP-T and PLAP-t than with PLAP. These results suggest that upon substitution of the P domain for the T or t domains, the new PLAP surface loop leans toward the central area of the AP molecule, thus sterically interfering with the recognition by E5, F6, C4, and A3,

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Monoclonal antibody FIG.3. Reactivity of a panel of 17 epitope-mapped monoclonal antibodies to PLAP with thechimeric swapped PLAPt (a)and PLAP-T ( b ) molecules. The ratio B/O obtained with each single antibody on thePLAP-tandPLAP-Tmutants was normalized with the equivalent ratio obtained for w t PLAP with the same antibodywhich is taken as 100%. but increasing exposure of the more peripheral F11 and 130 epitopes. Protein-binding Properties of APs-In the past several years, evidence has accumulated that suggests that APs may have a protein-protein interactiondomain. Vittur et al. (1984) and Wu et al. (1991) have reported that chicken TNAP binds to collagen type I, 11, and X, while Makiya and Stigbrand (1992a) have reported that human PLAP bindsto IgG. Based on sequence analysis and homology comparisons we identified the surface loop of APs as a candidate region that may provide the structuralbasis for the protein-protein interactiondomain of APs (Tsonis et al. 1988). The availability of AP mutants in which this surface loop has been swapped presently enabled us to test experimentally whether, in thecontext of an intact AP molecule, the loop wasinvolved in binding to collagen and IgG. Affinity chromatography of TNAP and PLAP on type I1 collagen resulted in partial retention of AP activity in both cases (Fig. 4, a and b). However, comparing the fractional binding to thecollagen column and calculating relative affinity indexes for this binding (Table I) it is clear that TNAP binds about 30-fold better to collagen than PLAP. The introduction of the t domain and, more importantly, of the T domain in PLAP confers binding abilities progressively enhanced and approximating those of TNAP. These data indicate that the T domain can account for almost half of the binding affinity of TNAP for collagen compared to PLAP. However, the T loop cannot entirely account for the full binding properties because theTNAP-P molecule (which

25413

Structural-Functional Domain of Alkaline Phosphatases a

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Fraction number FIG. 4. Affinity chromatography of TNAP (a)and PLAP ( b ) on collagen 11-Sepharose CL 4B column. AP activity bound to collagen was eluted with a NaCl gradient from 0 to 1 M in 10 mM TES buffer, pH 7.5, containing 0.1% Triton X-100, 1 mM MgC12,and 20 pM ZnC12. TABLEI Relative affinities of P L A P , P U P - t , P U P - T , T N A P - P and , TNAP for chicken type 11 collagen and human IgG during affinity chromatography Relative affinities were calculated as (% bound at 0 mM NaCl)/ (%PLAP,or TNAP, bound at 0 mM NaCl). Relative affinity of AP for Enzyme

Chicken Collagen 11"

Human I&

PLAP 1.0 3.1 PLAP-t 4.4 3.7 PLAP-T 12.4 3.3 0.0 17.0 TNAP-P 29.0 1.0 TNAP 'Bound enzymeactivity was 70% for TNAP and 5% for PLAP.

lacks the T domain) can still bindwith approximately half of the affinity of TNAP, indicating the presence in TNAP of at least one additional independent binding site for collagen. Other AP isozymes tested for collagen binding ability, i.e. human GCAP, bovine IAP, and E. coli A P only bound with affinities 1.3-, 1.8-,and 3.0-fold better than PLAPindicating that a generalized low-specificitycollagen binding property is shared by all these AP isozymes. Affinity chromatography of TNAP, PLAP, and the chimeric APs on human IgG-Sepharose on the contrary revealed that PLAPbound to thecolumn with a 3.1-fold higher affinity than TNAP (Table I), in agreement with the postulated Fc receptor function of PLAP in placenta (Makiya and Stigbrand, 199213). Introduction of the t domain, or of the T domain, in PLAPhad no effect on the affinity of the resulting chimeric molecules for human IgG, and the introduction of the P domain in TNAP (TNAP-P) similarly had no effect (Table I). The binding of bovine IAP and E. coli AP to the column was negligible. Therefore, although the relative affinities calculated for binding to human IgGs fluctuate less than those calculated for collagen binding, it is clear from these experiments that collagen binding and immunoglobulin binding occur on different sites on the molecule. In agreement with this conclusion, the anti-PLAP antibody D10, whose epitope we have recently mapped to be close to residue 209

(Hoylaerts and Millan, 1991), is capable of competing with the Fc-mediated binding of human IgGs to PLAP (Makiya and Stigbrand, 1992a), indicating a more peripheral location for this binding site. Catalytic Properties of the Chimeric Molecules-Recently, we have reported on the critical role played by residue 429 in determining the specificity of uncompetitive inhibition of APs (Hummer and Millh, 1991). A simple transition of G~u''~ (present in PLAP) to Gly4" (present in GCAP) rendered the PLAP molecule L-Leu sensitive while maintaining the L-Phe inhibition property sharedby both isozymes. Even mutations to Ser4" (present in intestinal AP) or His4" (present in TNAP) caused the same L-Leu sensitivity (Hoylaertsand Millh, 1991). These transitionswere associated with a major conformational change of the loop harboring residue 429, and we concluded that the structure of this loop determines accessibility of the inhibitor to the active site residues and stabilizes the side chain during inhibition (Hoylaerts et al., 1992). In view of these facts, it was clearly of interest to analyze the consequences of T and P domain replacements in TNAP and PLAP,respectively, from a kinetic viewpoint. Fig. 5 shows the inhibition of AP activity as afunction of the concentration of L-Phe, L-Leu, and L-homoarginine for the different chimeric enzymes as well as for PLAP,TNAP,and GCAP controls. In the case of L-Phe (Fig. 5a), a poorly discriminatory inhibitor between highly homologous isozymes and mutants, the only notable effect is the displacement to slightly higher inhibitorconcentrations of the TNAP curve upon substitution of the P for the T domain (TNAP-P). During inhibition by L-Leu (Fig. 5b), remarkably the PLAP-t molecule behaves identically to wild-type PLAP, whereas PLAPT responded to L-Leu exactly as GCAP. Alsowith this inhibitor, the TNAP-P was displaced approximately 3-fold from the TNAPcontrol curve. Both these results can be interpreted readily based on our previous knowledge of the critical importance of residue 429 in the inhibition. The net results of introducing the T domain (but not thet domain) in PLAP is to introduce a His residue at position 429, thus increasing the accessibility of the inhibitor to the active site. Indeed, we

25414

Structural-Functional Domainof Alkaline Phosphatases b

120

C

T

120

T

- .

0.0001

0.01

1

100

0.0001

0.01

1

100

o.wo1

0.01

1

100

Inhibitor [mM] FIG. 5. Inhibition response of PLAP, TNAP, GCAP, and the chimeric swapped mutants toward the uncompetitive inhibitors L-Phe (a),L-Leu ( b ) ,and L-homoarginine ( c ) . Symbols represent: PLAP (O), PLAP-t (A), PLAP-T (O), TNAP-P (W), T N A P (O),and GCAP (A).

have previously described a comparable inhibition by L-Leu of [ H ~ S ~ ~ ~ I Pand L AGCAP P (Hoylaerts and Millan, 1991). The opposite effect is true for the substitution of the P domain in TNAP which introduces a Glu4” residue, consequently restricting the accessibility of the inhibitor. Clearly, introduction of the t domain in the PLAP molecule does not affect the nature of residue 429 and has no effect on any of the inhibitions, in spite of the pronounced effects this substitution provoked on the heatstability of the resulting chimeric PLAP-t mutant. Similarly in the L-homoarginine panel (inhibitor specifically used to discriminate TNAP from the other isozymes), we observed a 4-fold shift to higher inhibitor concentrations upon introduction of the P domain in TNAPP (Fig. 5c). While PLAP, GCAP, and PLAP-t are all poorly inhibited by L-homoarginine, substitution of the T domain in PLAP seems capable of slightly facilitating the inhibition. These findings indicate that also during inhibition of TNAP by L-homoarginine the inhibitor is being stabilized by the surface loop. In order to ascertain possible additional interaction points within the T or P domains, we have studied the inhibition using peptide analogs previously known to discriminate between AP isozymes, Le. L-Phe-Gly-Gly and L-Leu-Gly-Gly. The COOH-terminal extension of L-Phe with Gly-Gly increases the selectivity of inhibition of the resulting peptide as can be observed in Fig. 6a. While L-Phe does not discriminate between PLAP, PLAP-T, PLAP-t, and GCAP, L-Phe-GlyGly now clearly distinguishes GCAP and PLAP-T from the other molecules. Moreover, PLAP and PLAP-t are inhibited about 5-fold better by L-Phe-Gly-Gly than by L-Phe. This pattern is analogous, although amirror image, of the selectivity displayed by L-Leu. Interestingly the addition of Gly-Gly onto L-Leu (Fig. 6b) eliminates the ability to distinguish between PLAP, PLAP-T, and PLAP-t while still discriminating between these molecules and theclosely related GCAP isozyme. Recently, we have clarified the detailed mechanism of uncompetitive inhibition using the simpler amino acids L-Phe and L-Leu (Hoylaerts et al. 1992a). While the carboxylic group of L-Phe and L-Leu attack Arg’% during the catalytic process,

the amino group of the inhibitor is stabilized by interaction with zinc 1in the active site, and the R group is stabilized by the surface loop harboring residue 429. Clearly this mechanism cannot readily apply to the peptide analogs since the carboxylic group is involved in a peptide bond to a Gly-Gly side chain. This prompted us to further investigate the AP inhibition by different peptides in an attempt toclarify those elements essential for the inhibition mechanism by peptides. Experiments employing Gly-Gly, hydroxyPro-Gly, or GlyhydroxyPro indicated that these compounds were not inhibitory up to concentrations of 100 mM (not shown). Blocking the carboxyl group of L-Phe-Gly-Gly yielded curves entirely superimposable to those of L-Phe-Gly-Gly (Fig. 7a), while acetylation of the amino group of L-Phe-Gly-Gly and L-LeuGly-Gly resulted ina 20-fold reduction in affinity of the modified inhibitors for the different enzymes and mutants (Fig. 7a) in agreement with earlier observations (Fishman and Sie, 1971). Substituting the internal Gly residue by a hydroxy-Pro (Fig. 7b) or modifying the COOH terminus with p-nitrophenol (Fig. 7c) yieldedcurves entirely superimposable to those of L-Phe-Gly-Gly. Therefore, it is clear that during inhibition by peptide analogs, it is the amino function of the NHz-terminal amino acid that is responsible for the uncompetitive inhibition mechanism while the rest of the peptide chain, not being able to obtain stabilization by interaction with Arg“ in the active site, must be stabilized by additional interactions involving the neighboring loop and nearby amino acids. We have shown before that the substitution in PLAP of Glu4” for Gly429indeed resulted in areduction of the affinity for L-Phe-Gly-Gly (Hummer and Millan, 1991), a phenomenon that we can now explain by the loss of an interaction between Glu4*’ and a peptide bond in L-Phe-Gly-Gly. The stronger inhibition of PLAP and PLAP-t by L-Phe-Gly-Gly and L-Leu-Gly-Gly as compared to L-Phe and L-Leu further points to the existence of secondary stabilizing interactions. Yet, the replacement during inhibition of L-Phe-Gly-Gly by L-Phe-hydroxyPro-Gly, where the COOH-terminal part has a different orientation, still results in a similar inhibition pattern, confirming that most of the tripeptide inhibitor’s binding energy resides in its firstamino acid.

25415

Structural-Functional Domain of Alkaline Phosphatases b

a

120

T

FIG. 6. Inhibition response of PLAP, TNAP, GCAP, and the chimeric swapped mutants toward the uncompetitive inhibitors L-PheGly-Gly (a)and L-Leu-Gly-Gly ( b ) . Symbols are identical to those in the legend to Fig. 5.

0.OWl

1

0.01

100

o.ooo1

0.01

1

1Do

Inhibitor [mM] b

a

C

1

120

IZ0

rmi

-0 0.wal

0.01

1

..ml

100

o.Oo01

0.01

1

100

I

n - 4. . o.wa1

.

. . ...2

"

0.01

1

Inhibitor [mM] FIG. 7. Inhibition response of PLAP, TNAP, GCAP, and the chimeric swapped mutants toward the L-Phe-Gly-Gly derivatives N-acetyl-Phe-Gly-Gly (a),L-Phe-HydroxyPro-Gly ( b ) , and L-Phe-Gly-Gly-pNP (c). Symbols are identical to those in the legend to Fig. 5.

Finally, it is interesting to observe that, in agreement with the collagen interaction site provided by the T domain, the K,,, values of TNAP and PLAP-T measured in the presence ofcollagen, both at PH 9.8 and 7.5, are lower than in its absence (Table 11). This decrease in K,,, values indicates that a conformational change occurs in the loop upon binding to collagen that facilitates access of the substrate to the catalytic site. On the other hand, human IgGs interacting with PLAP and PLAP mutants at a site more distant fromihe entrance to the active site than collagen have a much smaller impact (Table 11). on K,,, Conclusions-Our results clearly demonstrate the important structural role played by the flexible surface loop of the mammalian alkaline phosphatase isozymes. A change of only nine amino acids in the middle of the loop structure was sufficient to reduce the heat stability properties of PLAP at

TABLEI1 K m values of pup, pup-t, pup-T, TNAp-p, and TNAp measured in buffered substrate, at p H 9.8, alone or in the presenceof 0.25 pM type I I colhgen or 0.25 p M IgG Comparable relative data are obtained by performing kinetic determinations at pH 7.5. K,,, (mM)values in the presence of Enzyme

Collagen Buffer

PLAP PLAP-t PLAP-T TNAP-P TNAP

0.38 f 0.11 0.38 f 0.18 0.43 & 0.12 0.67 f 0.16 0.86 f 0.13

I1 0.35 f 0.09 0.40 f 0.09 0.16 f 0.07 0.31 f 0.09 0.30 f 0.13

I& 0.47 f 0.06 0.55 f 0.06 0.27 f 0.10 0.63 f 0.10 0.51 k 0.04

25416

Structural-FunctionaZDomain of Alkaline Phosphatases

56 "C from a half-life of days to 8 min. The substitution of the t or T loop in PLAP also had a clear impact on the structure of the PLAP molecule as detected by the loss of immune recognition by 3 and 4 out of a panel of 17 epitopemapped antibodies, respectively. The T loop constitutes one of at least two independent domains in TNAP that interacts with collagen I. However, the corresponding P loop in PLAP is not responsible for the binding to theFc region of IgG. Acknowledgments-We thank Dr. Marie-Claude Hofmann for her help with the Chinese hamster ovary cell transfectants and Thomas Manes for advice regarding site-directed mutagenesis. REFERENCES Bodanszk ,M , and Bodanszky, A. (19&) in The Practice of Peptide Synthesis, p. 231,Jpringer Verla Berlin Bradshaw, R. A,, Cance&a, F., Ericsson, L. H., Neumann, P. A,, Piccoli, S. P., Schlesinger, M. J., Shriefer, K., and Walsh, K. A. (1981)Proc. Natl. Acud. Sci. U. S. A. 78,3473-3477 Chaidaroglou, A., and Kantrowitz, E. R. (1989)Protein Erg. 3,127-132 Cuatrecasas, P. (1970)Nuture 228,1327-1328 Doellgast, G. J., and Fishman, W. H. (1976)Nature 259, 49-51 Doellgast, G. J., and Fishman, W. H. (1977)Clin. Chim. Acta 75, 449-454 Fishman, W. H., and Sie, H.-G. (1971)Enzymologio 41, 141-167 Gorman, C.M., Moffat, L. F., and Howard, B. H. (1982)Mol. Cell. Bwl. 2, 1044-1051 Hahnel, A. C., and Schultz, G. A,, (1989)Clin. Chim. Acta 186, 171-174 Harris, H. (1989)Clin. Chim. Acta 186, 133-150

Horiuchi, T., Horiuchi, S., and Mizuno, D.(1959)Nature 183,1529-1531 Hoylaerts, M. F., and Millan, J. L.(1991)Eur. J. Biochem. 202, 605-616 Hoylaerts, M. F., Manes, T., and Millin,J. L. (1992a)Biochern. J. 286,23-30 Hoylaerts, M. F., Manes, T., and Millin, J. L. (1992b)Clin. Chem. 38,24932500 Hummer, C., and Millin, J. L. (1991)Biochem. J. 274,91-95 Kim, E. E., and Wyckoff, H. W. (1991)J. Mol. BWL 218,449-464 Low, M. G., and Saltiel, A. R. (1988)Science 239,268-270 Makiya, R., and Stigbrand,T. (1992a)Eur. J. Biochem. 205,341-345 Makiya, R., and Stigbrand, T. (1992b)Clin. Chem. 38,2543-2545 McComh. R. B., Bowers. G. N.. and Posen.. S. (1979) . . Alkaline PhosDhatases. Plenum Press; New York Micanovic, R., Bailey, C. A,, Brink, L., Gerber, L., Pan, Y.-C. E., Hulmes, J. D., and Udenfriend, S. (1988)Proc. Nutl. Acud. Sci. U. S. A. 86,1398-1402 Millin, J. L. (1986)J. BWl. Chem. 261,3112-3115 Millin, J. L.(1988)Anticancer Res. 8,995-1004 Millin, J. L. (1990)Prog. Clin. BWl. Res. 344,453-475 Millin, J. L., and Manes, T. (1988)Proc. Nutl. Acud. Sci. U. S. A. 85, 30243028 Miller, E. J., and Rhodes, R. K. (1982)Methods Enzymol. 82,33-64 Mulivor, R. A., Plotkin, L. I., and Harris, H. (1978)Ann. Hum.Genet. 42, 113 Takami. N.. Oeata. S.. Oda. K.. Misumi.. Y.., and Ikehara Y. (1988) . . J. BWl. Chem: 263,3016-3021 ' ' Tsonis, A., Argraves, W. S., and Millan, J. L. (1988)Biochem. J. 264,623-624 Vittur, F., Stagni, F., Moro, L., and De Bernard, B. (1984)Experknth (Basel) 4n. - - ,826-827 -- - - - . Watanabe, T., Wada, N., Kim, E. E., Wyckoff, H. W., and Chou, J. Y. (1991) J.BWL Chem. 266,21178-21178 Whitby, L.G., and Moss, D.W. (1975)Clin. Chim. Acta 59, 361-367 Wu, L. N. Y.,Genge,B.R., Lloyd, G. C., and Wuthier, R. E. (1991)J. BWl. Chem. 266,1195-1203 Wu, L. N. Y., Genge, B. R., and Wuthier, R. E. (1992)Bone Mineral 17,247252 '