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CLIN. CHEM. 38/12, 2493-2500 (1992) Allelic Amino Acid Substitutions Germ-Cell Alkaline Phosphatase Marc F. Hoylaerts,”2 Thomas Affect the Conformation Phenotypes Manes,’ and Jos#{233} Luis gene (PLAP) gene encoding placental alkaline phosphatase displays a well-documented allelic polymorphism. Likewise, different phenotypes exist for the PLAP-related germ-cell alkaline phosphatase (GCAP). We investigated the extent to which various allelic GCAP positions are critical in determining the enzymatic, structural, and immunological properties of GCAP phenotypes. Three homozygous GCAP phenotypes [JEG3, BeWo, and wildtype (wt) GCAP] were analyzed and compared with a “core” GCAP mutant that contains the seven amino acid substitutions that are consistently different between PLAP and GCAP but are common to the three known allelic GCAP genotypes. Although some substitutions could in influence the electrophoretic behavior PLAP. The selective of the phenotypes, the kinetic properties the immunoreactivity detected with a panel antibodies (MAbs) to immunoreactivity of the PLAP/ GCAP-discnminating MAb C2 was critically dependent on the nature of the allelic residues 133 and 361 in GCAP. Residue 133 was also importantfor the general stability of the molecule because BeWo and wt GCAP, which have Asn1 and Val1, respectively, instead of Met1, showed a consistently reduced heat stability compared to core GCAP and JEG3. Because the core GCAP mutant consistently shows the characteristics of wt GCAP, its use as an antigen should allow the generation of monoclonal antibodies to GCAP that will not cross-react with and whose immunoreactivity will only marginally enced by allelic GCAP variation. Additional . Keyphrases: monoclonal allelic polymorphism antibodies . PLAP be influ- genetic variation isoenzymes Human alkaline phosphatases (ALPs) are encoded by a gene family composed offour loci [for review, see (1)]. Whereas the three tissue-specific ALP (TSAP) genes, placental (PLAP), germ-cell (GCAP), and intestinal ALPs, are composed ofli exons and occupy less than 5.0 kb of DNA, the single tissue-nonspecific ALP (TNAP) 1 La Jolla Cancer Research Foundation, Cancer Research Center, 10901 North Torrey Pines Road, La Jolla, CA 92037. 2 Department of Nephrology-Hypertension, University of Ant- werp, Antwerp, Belgium. Author for correspondence. 4 Nonstandard abbreviations: TSAP, tissue-specific alkaline 3 ALP, phosphatase; alkaline TNAP, phosphatase; tissue-nonspe- cific alkaline phosphatase; PLAP, placental alkaline phosphatase; GCAP, germ-cell alkaline phosphatase; wt, wild type; MAb, monoclonal antibody; DEA diethanolamine; and pNPP, p-nitrophenyl phosphate. Received May 13, 1992; accepted August 3, 1992. of Milan”3 The the allelic differences did not affect of GCAP. However, they did affect and conformation of the variants as of 1 8 epitope-mapped monoclonal and Immunoreactivity contains an additional, differentially spliced exon 5’ region and significantly larger introns that 40-50 kb of DNA. The TSAP genes are colocalized in the long arm of chromosome 2, but the TNAP gene resides at the end of the short arm of chromosome 1. The PLAP gene is subject to a high degree of polymorphism. Three common alleles (Plo, pF’, and P1’) account the occupy for over 90% of the PLAP phenotypes (2). However, more than 20 rare allozymes have been described, often only in heterozygous combinations because of their low allelic frequency (3). The two most common PLAP phenotypes, S (slow) and F (fast), differ only in an Arg#{176} to p2o9 substitution (4), but additional amino acid replacements have been identified for the I (intermediate) variant (5). Although GCAP is encoded by a different gene from PLAP, the primary structure of GCAP shows 98% sequence identity with PLAP (6, 7). However, when various GCAP samples were analyzed with respect to their immunoreactivity with a number of monoclonal antibodies raised against PLAP, some of the antibodies reacted with high, intermediate, or low affinity (8). From these findings, the existence of a GCAP polymorphism with up to nine allelic GCAP variants was anticipated (8-10). Determination of the sequence of GCAP, derived from JEG3 choriocarcinoma cells (11) and BeWo cells (12), confirmed the existence of allelic variation in the GCAP gene. Using a series of site-directed PLAP mutants, we recently showed that individual amino acid substitutions in the PLAP isoenzyme had a considerable effect on the immunoreactivity and conformation ofthe resulting mutants, as detected by a panel of 18 monoclonal antibodies (MAbs) to PLAP (4). These results emphasized the importance of characterizing in great detail the reactivity of those MAbs used clinically for the serological evaluation of ALP isoenzymes. PLAP and GCAP are useful tumor markers in the management of patients with adenocarcinoma of the ovary and seminoma of the testis (13-1 7). The existence of allelic GCAP differences can influence the accuracy of the immunochemical detection of GCAP phenotypes, their electrophoretic identification, and the molecular stability of the GCAP allotypes. To evaluate these variables, we used site-directed mutagenesis to construct a series of PLAP and GCAP mutants. We then compared these mutants with those GCAP phenotypes for which the sequence is known. Although the presence of Gly at position 429 in GCAP is the key element determining the enzymatic properties ofGCAP (18), our results show that different amino acids at certain allelic positions can CLINICAL CHEMISTRY, Vol. 38, No. 12, 1992 2493 influence significantly Materials PLAP the and Mutants conformation affect antibody of the GCAP recognition. molecule and with tively, low a rabbit Genotypes The study PLAP (F phenotype) and GCAP used in this have been described previously (18) and are referred to as wild-type (wt) PLAP and wt GCAP. A 2.0-kb Eco RI-Kpn I fragment of the PLAP cDNA (6) was used as the source of template DNA to generate a series of PLAP mutants. Site-directed mutagenesis experiments were performed according to Kunkel (19) by using the mutagene M13 in vitro mutagenesis kit (Bio-Rad Laboratories, Richmond, CA). The generation of the single amino acid mutants, [Gln’5]PLAP, [Thr67]PLAP, [PhessIPLAP, [Ser84]PLAP, [His’]PLAP, [Leu]PLAP, and [G1y429JPLAP, was described previously (4). These cDNAs were used as a source of fragments to reassemble the more complex mutants. [HisZl, Leu, G1y429]PLAP ([HLG]PLAP) was constructed by ligating a 568-bp BamHI-SacI fragment containing the [His’1 mutation (BamHI-[His’]-SacI), the 154-bp Sac I-[Leu254]-SacII, and the 937-bp SacII-[Gly4]-KpnI fragments into pSVT7-PLAP digested with BamHI and KpnI. A 1414-bp BstEll-KpnI fragment from the [HLG] PLAP construct was then isolated and ligated with a 276-bp BamHI-[SerJ-BstEII fragment into either cut with BamHI and KpnI to create Leu254, Gly429]PLAP ([SHLG]PLAP) or digested with BamHI and KpnI to SerM, His241, Leu2M, G1y429]PLAP ([QSHLG]PLAP). Finally, a 276-bp Barn HI-[Ser, r67, PhessJBstEll fragment was ligated with the 1414-bp BstEII-[His1, Leu, Gly4}-KpnI fragment into pSVT7-[Gln’5IPLAP to generate core GCAP. The sequence of the mutagenesis primer used to generate [Leu361]PLAP was as follows: 5’-GGA GAA GAj GTG GGA GTG GTC-3’ (the underlined base indicates the change). The wt and mutagenized PLAP cDNAs were antiserum concentrations tants, core GCAP, the insolubilized Methods and GCAP coated and GCAP MAbs. Upon fraction was measured total enzyme concentration surements were carried age concentration divided by the fraction the binding step increasing that expressed in the in triplicates (<20%) an affinity values residual for index the binding. calculated by to the B/F ratio relative Mea- the aver- bound enzyme (B) was of the unbound (free) between 5% and 10%, for those phenotypes (%) were to the wells. and were low mu- added to the bound relative deposited to obtain ConsecuPLAP were recorded affinities B/F ratio and out IgG. PLAP, The to 15-20% showed ative wt phenotypes equilibration, (±SD) of concentration (F), in order binding. to mouse of the (B/F) SD occasionally and mutants Finally, expressing recorded for of the relthe for wt PLAP. Gel Electrophoresis Starch described PLAP lated gel electrophoresis (23). Prior to mutants, by treatment at pH 8.6 was performed as electrophoresis, the wt PLAP, and GCAP phenotypes with neuraminidase 24 h at 37 #{176}C to eliminate any linked to differences in the degree different cell were desialy(0.33 U/mL) for charge heterogeneity ofsialylation between types. pSVT7-PLAP [SerM, His241, pSVT7-[Gln’5IPLAP create [Gln15, subcloned into the vector pSVT’7 downstream from the SV4O early promoter (20) and transfected by the calcium phosphate procedure (21) into Chinese hamster ovary (CHO) cells. Transfected cells were selected as described (18) and, at confluency, washed with 20 mmol/L Tris-HC1 buffer, pH 7.5, containing 140 mmolJL NaC1. The cells were extracted (30 mm) with a 1:1 mixture of n-butanol and 50 mmolfL acetate buffer, pH 5.5, containing 100 mmol/L NaC1, 20 j.unol/L ZnCl2, 1 mmoLfL MgCl2, and 0.5 g/L thimerosal (Sigma Chemical Co., St. Louis, MO). JEG3 cells grown in the presence of 2 mmolfL sodium butyrate to maximally stimulate the production of GCAP (22) and BeWo cells grown to confluency were extracted in the same way. Upon titrating the pH to 7.5 with a 1.0 molfL Tris solution, these extracts were aliquotted and stored at -80 #{176}C. Relative Affinity Measurements Immunoreactivities were measured Briefly, 16 MAbs to PLAP and intestinal ALP were incubated 2494 CLINICAL CHEMISTRY, as (4). MAbs to plates pre- described 2 cross-reacting in microtiter Vol. 38, No. 12, 1992 Heat Inactivation The wt PLAP, PLAP mutants, and were diluted in 1 molfL diethanolamine pH 9.8, supplemented alone or 20 mol/L with either ZnCl2 and 0.5 GCAP 0.5 mmol/L phenotypes (DEA) buffer, mmol/L MgCl2, MgCl2 and incubated in a water bath at 56 #{176}C or 65 #{176}C. At fixed time intervals, 50-pL samples were removed and pipetted into each well of a microtiter plate kept on ice. Residual activities were then measured in duplicate upon the simultaneous addition to the wells of200 L of 10 mmol/L p-nitrophenyl DEA buffer, pH 9.8, MgCl2. These activities activity of the unheated Enzyme Activation phosphate (pNPP) in 1 mol/L supplemented with 0.5 mmoLTL were expressed relative to the enzyme. and Inhibition Kinetics Michaelis-Menten kinetics of the core GCAP and different GCAP phenotypes were performed as recently described, with Km and Vm being derived from Lineweaver-Burk plots (24). Catalytic rate constants (k) were calculated from Vm upon determination of the total enzyme concentration, [El#{176}, in an enzyme antigen immunoassay based on the PLAP MAbs C2 and H7 (8). Residual activities released during the inhibition of the wt PLAP, PLAP mutants, and GCAP phenotypes by increasing concentrations of L-Leu (0.05-10 mmol/L) were measured in microtiter plates by adding 10 mmol/L pNPP in 1 mol/L DEA buffer, pH 9.8, supplemented with 0.5 mmolJL MgC12. These activities were expressed relative to the enzyme activity in the absence of inhibitor. Position Amino wt PLAP [G]PLAP [HLGJPLAP acid at the residue tSHLGIPIAP 15 Glu Glu Glu G)u 38’ II. II. II. U#{149} b II. U. 67 II. II. Pro Pro 68 84 133’ 241 254 297’ 361 429 Pro Pro Pro Mn Asn Met Met Met Met Met: Arg Arg Met Met Arg Arg Arg Arg Arg Vat Vat Vel Vat Vat Pro Pro Pro Glu Pro Giy rs position GCAP JEG3 OCAP BeWo OCAP II. II. Arg Arg Vat wi GCAP lie Asn 479* indicated (OSHLG)PLAP- Met Vat Leu Pro Arg $IYL GJy Gly Pro Cly . Pro Pro Fig. 1 . Amino acid sequence differences that define wt PLAP, PLAP mutants, and GCAP genotypes The shaded residues indicate those substitutions consistently different between wt PLAP and the JEG3, BeWo, and wt GCAP genotypes. An asterisk identities those positions known to be polymorphic by sequence analysis. Boxed residues indicate actual allelic substitutions A Results and PLAP Mutants and GCAP 479, The comparisons genotypes and from wt GCAP wt GCAP and the the most (at positions Enzyme Electrophoresis at positions 38, 133, and 297. JEG3 GCAP phenotypes differ 38, 133, 297, 361, and 479). on Starch Gel agreement with the charge difference between the F PLAP phenotype (Arg for Pro at position 209), gel electrophoresis separates the desialylated FS heterozygous PLAP phenotype into three bands, correspending to the FF, FS, and SS dimers, respectively (Figure 2A). The single substitution in PLAP of Glu429 by Gly429 ([GIPLAP) causes an important electrophoretic retardation of the resulting mutant, compared with the S PLAP phenotype (Figure 2B). This effect is primarily conformational, because the S PLAP phenotype ([2O9, Glu429]PLAP) and [G]PLAP ([Pro2#{176}, GIy429JPLAP) have an identical charge density. This result corroborates our finding that the substitution of residue 429 in PLAP is associated with an important conformational change in the molecule (4). The electroIn S and starch C Genotypes (Figure 1) ofthe three different sequenced to date indicate the existence of at least five allelic amino acid positions (residues 38, 133, 297, 361, and 479). These three GCAP phenotypes differ from the wt PLAP sequence at 12 amino acid positions. Seven of these differences are invariant and common to all three GCAP alleles. We have constructed, through site-directed mutagenesis, PLAP mutants containing an increasing number (from one to seven) of substitutions designed to progressively confer more GCAP character to the resulting mutants (Figure 1). The most complex mutant, [QTFSHLGIPLAP, contains the core of seven amino acid differences that distinguish GCAP from PLAP. This core GCAP mutant represents a GCAP genotype containing the minimal number of consistent differences compared with wt PLAP but displaying a full GCAP enzymatic character (see below) common to all known GCAP phenotypes. This core GCAP differs from BeWo GCAP at position 133, from JEG3 GCAP at positions 361 and Sequence GCAP B Discussion I 2 3 1 2 3 4 5 1 2 3 4 Fig. 2. Starch gel electrophoresis of PLAP and GCAP phenotypes (A) Common PLAP phenotypes: F phenotype (lane 1), FS phenotype (lane 2), and S phenotype (lane 3); (B) increasingly more complex PLAP mutants: reference PLAP S phenotype (lane 1), [GIPLAP (lane 2), [HLG)PLAP (lane 3), [SHLGJPLAP (lane 4), and [QSHLGJPLAP (lane 5); (C) diflerent GCAP phenotypes: core GCAP (lane 1), JEG3 GCAP (lane 2), wt GCAP (lane 3), and BeWo OCAP (lane 4) phoretic behavior of the mutant enzymes is further modulated in increasingly more complex PLAP mutants in which three, four, or five amino acids are substituted for the corresponding GCAP residues (Figure 1). Comparison ofthe electrophoretic mobility ofcore GCAP and the other GCAP phenotypes confirms that GCAP does not behave as an electrophoretically unique entity. The somewhat higher anodic mobility ofwt GCAP than that of the other GCAP molecules (Figure 2C) can be explained by the substitution in wt GCAP of Arg97 for Leu297. These findings show that the electrophoretic microheterogeneity of GCAP in seminoma does not depend only on the carbohydrate heterogeneity and differences in hydrophobicity, as was suggested (25), because allelic amino acid substitutions involving residues also contribute to the electrophoretic heterogeneity, analogous to the situation with the electrophoretic PLAP polymorphism (26). charged Immunoreactivity of PLAP Mutants We recently used a panel of 18 epitope-mapped conformationally dependent MAbs against PLAP to define conformational changes induced by each ofthe 10 amino acid differences between PLAP and GCAP (4). Measurements of the relative affinities of the same panel of antibodies for the increasingly complex mutants EGIPLAP, [HLG}PLAP, [SHLG]PLAP, and [QSHLG]PLAP indicate that the resulting immunoreactivity is a composite function of the contributions of each single CLINICAL CHEMISTRY, Vol. 38, No. 12, 1992 2495 (G]PLAP lx (OSHLG] 1 PLAP wt GCAP 120 so i.ii..IihiL :BIIdIllhIIhLso F$,s QJ8U IiLhIiiIIIIIk :[I111.1itttii1 : [JJ]C[JJJ #{174}jJjjjP[J g&’ 4c;9 Fig. 3. ImmunoreactiVity 18 epitope-mapped Values represent of a series of complex PLAP 81p4’g mutants, the GCAP relative affinities with respect to wt PLAP. Note differences amino acid substitution (Figure 3). The conformational effect of combining different amino acid substitutions in a single mutant is not necessarily equal to the sum of the conformational contributions for the individual substitutions. For example, when His’, Leu, and Gly4 are individually substituted in PLAP, the affinity of antibodies D10, Fil, B2, and GlO is greatly reduced from that of wt PLAP (4). Yet when these three substitutions are combined into a single mutant ([HLG]PLAP), the affinity for D10 and Fli is largely reconstitoted (Figure 3). Furthermore, the immunoreactivity of the triple mutant [HLG]PLAP for most ofthe antibodies was different from that of a triple mutant studied previously, [SerM, Leu254, Gly429]PLAP. The epitope for MAb 17E3 is almost entirely constituted by 241 (4) The predominant contribution of the His24’ substitution in [HLG]PLAP, as well as in [SHLG]PLAP and [QSHLGJPLAP, is apparent from the lack of reactivity of these mutants with 17E3 and from the relatively high reactivity with H5. The additional inclusion of SerM ([SHLG}PLAP) and of GIn’5 ([QSHLG]PLAP) in the mutant causes further modulation ofthe immunoreactivities toward lower affinities for most of the antibodies in the panel. Immunoreactivity of GCAP Comparisons MAb panel of the with the Phenotypes pattern immunoreactivity core GCAP mutant and the of the three GCAP phenotypes of known sequence revealed major differences in affinity between the different GCAP allelic variants. First, in comparison with [QSHLG]PLAP, the two additional substitutions in core GCAP ([QTFSHLG]PLAP) further affected the reactivity of 8 out of 18 MAbs by reducing their affinities fivefold compared with those of PLAP (Figure 3). Together, the seven amino acid substitutions that distinguish core GCAP from PLAP shape a GCAP phenotype that is structur2496 CLINICAL phenotypes, and the single [Leu1JPLAP mutant with the panel of MAbs to PLAP CHEMISTRY, Vol. 38, No. 12, 1992 in the scales of the yaxes ally different from PLAP. This conformational difference is detected by MAbs B2, Gb, and E6, which recognize the F/S allelic PLAP difference, as well as by MAbs A3, E5, F6, 327, and 7E8, which bind to the central antigenic domain of the molecule. The BeWo GCAP phenotype can be regarded as resulting from the additional substitution in core GCAP of a single amino acid, Met’33 for Asn’” (Figure 1). This additional substitution is important for two reasons: first, the domain recognized by the F/S discriminating antibodies B2, Gb, and E6 is reexposed, and second, the reactivity with C2 drops by a factor of 10 (Figure 3). The C2 antibody has been described before as a reagent reacting with PLAP but not with GCAP (8). We recently concluded that this selectivity is largely conformationally determined (4). It is somewhat surprising that the substitution of seven GCAP amino acids in PLAP (core GCAP) only leads to a twofold decrease in the immune reactivity for C2, whereas the additional substitution of the allelic Met’ generates the expected C2 selectivity. Moreover, this substitution is operational only in a context, because our previous analysis (4) could not define any structural role for Met’33 when we analyzed the phenotype in a PLAP context by measuring the immune reactivity pattern of[Val’]PLAP. The wt GCAP showed a more modest decrease in immunereactivity than the BeWo GCAP, but the drop was more general, in that all antibodies tested reacted to a lower extent with wt GCAP than with PLAP. Although in wt GCAP Met’33 is substituted for Val’33 (and not for Asn’33), the same low reactivity with C2 is observed, indicating conformational similarities between the BeWo GCAP and wt GCAP. The slightly higher affinities ofwt GCAP with some antibodies are a consequence ofthe Arg297 to Leu7 substitution, known to positively GCAP influence immune The JEG3 GCAP recognition shows (4). a distinctive immunoreactiv- ity pattern compared with the other GCAP phenotypes. The affinity for the group of antibodies binding to the central antigenic domain of the molecule (A3, E5, F6, 7E8) is entirely reconstituted in JEG3 GCAP compared with wt GCAP. On the contrary, the reactivity measured with C2 is even five- to tenfold lower for JEG3 GCAP than for wt and BeWo GCAP. Likewise, lower reactivities are found with MAb 17E3 for the JEG3 GCAP than for the other GCAPs. The low reactivity with C2 cannot be accounted for, in this case, by residue 133 since this position is not substituted in JEG3 GCAP. Moreover, the larger reduction in affinity for this phenotype in comparison with the others suggests the involvement of the other two residues that are substituted in the JEG3 GCAP: Val36’ (for Leu361) and Prom (for Arg’79). The construction of a PLAP mutant carrying the single Va136’ to Leu36’ substitution generated a mutant with a reactivity pattern that largely corresponded to that of JEG3 GCAP itself (Figure 3), suggesting that residue 361 played an important conformational role in JEG3 GCAP. If one considers all the individual mutations investigated previously (4), apart from the essential G1u429 to Gly4 substitution, the Va138’ to Leu36’ substitution is the only single amino acid replacement that could reduce the immunoreactivity ofC2 by 50%. The low reactivity ofJEG3 GCAP can be explained by this mutation, which, when analyzed in the GCAP context ofthe JEG3 GCAP phenotype, is fully operational. These data clearly show that the low reacof C2 with different GCAP phenotypes is very dependent on conformation, and that several combinations of substitutions also influence reactivity, which explains why different GCAP phenotypes have varying residual reactivities with C2. On the contrary, the low reactivity of GCAP with 17E3 largely depends on the Arg24’ to His24’ substitution (4), which is common to all tivity four GCAP genotypes. Reactivities of GCAP Phenotypes Cloned GCAP Alleles Deduced from the The immunoreactivity patterns of the three GCAP phenotypes investigated here are far from identical and provide a rational explanation for the differences in immunoreactivity observed previously during the screening ofrandom GCAP-positive tumor extracts and serum samples (8, 10, 15). GCAP, like PLAP, is a dimeric enzyme that results from the random association oftwo (either identical or allelic) GCAP monomers. The genotypes we studied could give rise to three heterozygous GCAP phenotypes: wt GCAP-JEG3 GCAP, wt GCAP-BeWo GCAP, and JEG3-BeWo GCAP. These heterozygous GCAP phenotypes would have immunoreactivities intermediate between those depicted count for the homozygotes for the existence ofsix and different could, therefore, ac- GCAP phenotypes distinguishable immunologically with MAbs from the panel. Although additional GCAP genotypes are likely to be discovered in the future, on the basis ofour current knowledge of the binding of epitope-mapped MAbs, we are able to predict the likely substitutions. We recently reported that the epitopes for the MAbs F11 and 17E3 were almost entirely constituted by Arg#{176} and Arg41, respectively (4). Previously, a type H GCAP phenotype (8) was characterized as being fully reactive with F11 compared with PLAP (S phenotype). This indicates that the type II GCAP probably has a Pro#{176} to Arg#{176} substitution. Similarly, type VU and VIII GCAP were defined as highly reactive with MAb 17E3 (10). This points to the existence of GCAP phenotypes that have a His241 to Arg24’ substitution and identifies residue 241 as another allelic position. The core GCAP mutant constructed in this study contains the minimal number of substitutions that confer a GCAP character to the molecule. The MAb reactivity ofcore GCAP, that is, low reactivity with 7E8, 17E3, 327, and E6, is compatible with the definition oftype IX GCAP (10) and thus may well represent a naturally existing genotype. Finally, the reactivity pattern of those same antibodies indicate that JEG3 GCAP is compatible with a type V GCAP phenotype, whereas the BeWo and wt GCAP currently cannot be correlated with any of the previously defined phenotypes. Heat Stability At ing of PLAP Mutants physiological temperatures and GCAP pH, PLAP is extremely of 65 #{176}C for 60 mm. Phenotypes stable, resistTo facilitate an of the general stability of PLAP mutants phenotypes, we conducted heat stability tests at a higher pH (1 mollL DEA buffer, pH 9.8). Heating enzyme samples at 65 #{176}C for 6 mm in the presence of 0.5 mmol/L MgCl2 caused a moderate (20%) loss of enzymatic activity for wt PLAP, whereas the same treatment caused a considerable reduction (>60%) in the activity of the PLAP mutants and the GCAP phenotypes (Figure 4A). These results show that the conformational change that occurs in PLAP when Glu429 is substituted for Gly4 has a major impact on the general stability of the isoenzyme. They also show that further substitutions can partially correct for this decreased stability. However, because all PLAP mutants and GCAP phenotypes were not inactivated to the same degree, we also measured residual activities after mactivation at a less critical temperature (56 #{176}C). Exposure to 56 #{176}C for 30 mm (Figure siB) caused a comparable destabilization for wt PLAP (80% residual activity), but differences within the group of PLAP mutants and GCAP phenotypes were also evident. The stability ofthe PLAP mutants and GCAP phenotypes, expressed relative to that of wt PLAP, is greater at 56 #{176}C than at 65 #{176}C, even when the enzymes are exposed for longer time intervals. The addition of ZnCl2 during incubation at elevated temperatures could partially protect some GCAP phenotypes from denaturation (not shown). Therefore, to describe the kinetics of heat inactivation in more detail, we analyzed the residual activity of wt PLAP time-dependently in 1 molJL DEA buffer, pH 9.8, containing 20 moI/L ZnCl2 and 0.5 mmol/L MgCl2, both at 56 #{176}C and at 65 #{176}C (Figure 5A). At 65 #{176}C, the wt PLAP inactivation showed a biphasic process, with the first phase lasting approximately 10 assessment and GCAP CLINICAL CHEMISTRY, Vol. 38, No. 12, 1992 2497 100 A 100 65C A 80 50 60 20 wt PLAP 56 C : 0 wtPLAP 65C:I I ) 100 I 10 I 20 I I 30 40 50 60 30 40 50 60 40 50 60 56C B .80 B 100 >1 u6O a 4O 50 20 0 0 Q Q9 20 C, [GJPLAP:o [HLG] PLAP : [SHLG] PLAP : o b 0 Fig. 4. Residual enzyme activity of the wt PLAP, PLAP mutants, and GCAP phenotypes after heat inactivation in 1 mol/L DEA buffer, pH 9.8, containing 0.5 mmol/L MgCI2 at (A) 65 #{176}C for 6 mm and at (B) 10 [QSHLG]PLAP:u Cl) 10 I 20 56 #{176}C for 30 mm Values expressed mm; tion relative to the activity of the unheated samples 100 at 56 #{176}C, a slower process was heat inactivation PLAP mutants turation but almost monophasic inactivaapparent. Likewise, kinetic analysis of of [GIPLAP and the more complex at 56 #{176}C (Figure 5B) showed that dena- occurred by way of a monophasic mechanism. The introduction of additional mutations in [GIPLAP increased the stability of the more complex mutants. The heat inactivation behavior ofthe GCAP phenotypes at 56 #{176}C (Figure 5C) could also be described as a monophasic process. Core GCAP and JEG3 GCAP had a general stability comparable with that of the multiply substituted PLAP mutants. However, wt GCAP and BeWo GCAP consistently showed lower stability. This difference must be related to the single amino acid substitution of Met’ (core GCAP) for Asn’ (BeWo Met’33 for Val’33 (wt GCAP) substitution effect on the stability of wt GCAP. This already described as being conformationally critical, causing a 10-fold loss in immunoreactivity with MAb C2. Therefore, from the antibody affinity studies and the heat inactivation analysis, we can attribute a structural role to the allelic amino acid residue at position 133. The double Val36’ (for Leu361) and Pro (for Arg479) substitution in core GCAP is silent in terms of heat inactivation behavior. Yet, from the pronounced effect of the Val36’ to Leu36’ substitution in wt PLAP on the immunoreactivity of C2, we can attribute a structural role to this amino acid position that is evident only when it is combined with additional amino acid substitutions that confer the general GCAP structure. GCAP). The had a similar residue was Active Site Properties of GCAP Allelic Variants We showed recently that the Glu429 for Gly429 substitution in wt PLAP is accompanied by a small decrease 2498 CLINICAL CHEMISTRY, 50 Vol. 38, No. 12, 1992 20 CoreGCAP: 0 JEG3 GCAP : wtGCAP: 0 10 IBeW0GCAP: a io 20 Time Fig. 5. Kinetics containing ofthe heat inactivation 30 (mm) in 1 mol/L DEA buffer, pH 9.8, 20 moVL ZnCI2 and 0.5 mmol/L MgCI2 of (A) wt PLAP at 56 #{176}C and 65 #{176}C, (B) the different PLAP mutants at 56 #{176}C, and (C) the different GCAP phenotypes at 56 #{176}C Michaelis constant (Km) from 0.35 mmol/L to 0.1 when measured in 1 mol/L DEA containing 0.5 mmol/L MgCl2 (18, 24). This substitution only slightly affected the turnover number (k) from 460 s’ (wt PLAP) to’344 s’ ([Gly429]PLAP). Additional substitutions in wt PLAP did not further affect these kinetic parameters. We have now confirmed that this result also holds for th core GCAP mutant and the three GCAP phenotypes studied; they have very similar Km (0.1 mmolJL) and (280-300 1) values. The substitution in wt PLAP of Glu429 for Gly429 accounts for the differential inhibition ofGCAP by L-Leu (18), a phenomenon explained by steric hindrance exerted by the Glu4 side chain in PLAP, but absent in GCAP, during positioning of the inhibitor in the active site of the enzyme (24). Our present comparison ofthe inhibition of the different GCAP phenotypes by increasing concentrations of L-Leu (Figure 6A) confirms that all GCAPs in mniol/L #{149} A 100 These mutant we phenotype displaying the consistent characteristics of GCAP, including immunoreactivity, molecular stability, and inhibition properties. The use ofcore GCAP as an antigen for the production of monoclonal antibodies is likely to allow the generation ofreagents that will show very low cross-reactivity with PLAP and will enable us to produce antibodies for which immunoreactivity is only constructed 80 40 >1 20 0 0.05 0 0.1 1 10 studies show behaves that as the core a prominent GCAP GCAP marginally determined by allelic amino in GCAP. These monoclonal antibodies valuable in the specific determination serum of patients. acid variations would prove of GCAP in the 100 801- Supported by grant CA42595 from the National Health and by the Veremging voor Kankerbestrijding, We thank Elisabeth Bossi for help with the transfections 60 DNAS. 0) 40 References (GI PLAP: [HLG] PIAP: 201- 1. Harris H. The human and what we don’t know. 2. Beckman G, Beckman PLAP: PLAP: I. 0L 0.05 0.1 1 10 [L-Leu] (mM) Fig. 6. Inhibition of the enzymatic activity of (A) the different GCAP phenotypes and (B) the different PLAP mutants by increasing L-Leu concentrations (0.05-i 0 mmol/L) in comparison with wt PLAP investigated efficiencies are than Significance of Core inhibited by L-Leu with 10-fold higher wt PLAP. The [Gly41PLAP and the triple [His’, Leu4, Gly429]PLAP mutant are inhibited with even slightly higher affinities (Figure 6B). We have shown previously that Ser in GCAP plays a modulating role on the L-Leu inhibition. As soon as this mutation is superimposed on top of the other PLAP mutations, L-Leu inhibition profiles are identical to those obtained for GCAP. All the raised immunological MAbs GCAP and 130) used in this study Therefore, it is not surprising that GCAP was generally recognized with lower affinities than PLAP. However, it is clear from the present study that amino acids in allelic GCAP positions can strongly affect the conformation and immunoreactivity ofthe enzyme. Only two MAbs (17E3 and C2) consistently showed a low reactivity with the different GCAP phenotypes, a property that has been exploited in the clinical evaluation and quantitation of PLAP and GCAP in serum (9, 10). A third MAb (H317), with properties comparable with those of 17E3 and C2, has also been described (27); however, to date there are no were (except against reagents Institutes of Belgium. of mutant 151 PLAP. that allow the specific mea- surement of GCAP in the presence of PLAP. Yet, a correct assessment ofthe GCAP concentration would be clinically valuable because in seminoma (9, 10, 13-15, 25), other testicular tumors (14, 15), and ovarian cancer (16, 1 7) GCAP concentrations increase in serum and fluids. alkaline phosphatases: what we know Clin Chim Acta 1990;186:133-50. L. The placental alkaline phosphatase polymorphism. Hum Hered 1969;19:524-9. 3. Donald U, Robson EB. Rare variants of placental alkaline phosphatase. Ann Hum Genet Lond 1974;37:303-13. 4. Hoylaerts MF, Mill#{225}nJL. Site-directed mutagenesis and epitope-mapped monoclonal antibodies define a catalytically important conformational difference between human placental and germ cell alkaline phosphatase. Eur J Biochem 1991;202:605-16. 5. Henthorn PS, Knoll BJ, Raducha M, et a!. Products of two common alleles at the locus for human placental alkaline phosphatase differ by 7 amino acids. Proc Natl Acad Sd USA 1986;83: 5597-601. 6. Millan JL Molecular cloning and sequence analysis of human placental alkaline phosphatase. J Biol Chem 1986;261:3112-5; J Biol Chem [Letter] 1991;266:4023. 7. Millan JL, Manes T. Seminoma-derived Nagao isozyme is encoded by a germ-cell alkaline phosphatase gene. Proc Nat! Acad Sci USA 1988;85:3024-8. 8. Millan JL, Stigbrand T. Antigenic determinants of human placenta! and testicular placental-like alkaline phosphatases as mapped by monoclonal antibodies. Eur J Biochem 1983;136:1-7. 9. Wahren B, HinkulaJ, Stigbrand T, et a!. Phenotypes of placental-type alkaline phosphatase in seminoma sara as defIned by monoclonal antibodies. hit J Cancer 1986;37:595-600. 10. Hendrix PG, Hoylaerts MF, Nouwen EJ, De Bros ME. Enzyme immunoassay ofhuman placental and germ-cell alkaline phosphatase in serum. Cim Chem 1990;36:1793-9. 11. Watanabe 5, Watanabe T, Li WB, Soong BW, Choy JY. Expression of the germ cell alkaline phosphatase gene in human choriocarcinoma cells. J Biol Chem 1989;264:12611-9. 12. Lowe ME, Strauss AW. Expression ofa Nagao-type, phosphatidylinositol-glycan anchored alkaline phosphatase in human choriocarcinoma. Cancer Res 1990;50:3956-62. 13. Lange PH, Mill#{225}nJL, Stigbrand T, Vesse!la RI, Ruoslahti E, Fishman WH. Placental alkaline phosphatase as a tumor marker for seminoma. Cancer Rae 1982;42:3244-7. 14. PaivaJ, Damjanov I, Lange PH, Harris ical localization of placental-like alkaline H. Immunohistochem- phosphatase in testis and germ cell tumors using monoclonal antibodies. Am J Pathol 1983;111:156-65. 15. Jeppsson A, Wahren B, Brehmer-Andersson E, Si!fverswArd C, Stigbrand T, Millan JL. Eutopic expression of placental-like alkaline phosphatase in testicular tumors. mt J Cancer 1984;34: 757-61. 16. Vergote I, Onarud M, Nustad K. Placental alkaline phosphatase as a tumor marker in ovarian cancer. Obstet Gynecol 1987; 69:228-32. 17. De Bros ME, Pollet DE. Multicenter evaluation of human placental alkaline phosphatase as a possible tumor-associated antigen in serum. Clin Chem 1988;34:1995-9. CLINICAL CHEMISTRY, Vol. 38, No. 12, 1992 2499 18. Hummer uncompetitive tase. Biochem C, Mill#{225}n JL. Gly4 is the major determinant of inhibition of human germ cell alkaline phosphaJ 1991;274:91-5. 19. KunkelTA. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Nat! Acad Sci USA 1985;82:488-92. 20. Bird P, Gething M-J, Sambrook J. Translocation in yeast and mammalian cells: not all signal sequences are functionally equivalent. J Cell Biol 1987;105:2905-14. 21. German CM, Moffat LF, Howard BH. Recombinant genomes which express chloramphethcol acetyltransferase in mammalian cells. Mo! Cell Biol 1982;2:1044-51. 22. Ito F, Chou JY. Induction of placental alkaline phosphatase biosynthesis by sodium butyrate. J Biol Chem 1984;259:2526-30. 23. Poulik MD. Starch gel electrophoresis in a discontinuous system and buffers. Nature (London) 1957;180:1477-9. 24. Hoylaerts MF, Manes T, Mill#{225}n JL. Molecular mechanism of uncompetitive inhibition of human placental and germ cell alkaline 25. phosphatase. Biochem K, Stigbrand J 1992; 286:23-30. T, Hisazumi H, Wahren B. Electrophoretic heterogeneity ofalkaline phosphatase isozymes in seminoma and normal tissue. Tumour Biol 1989;10:181-9. 26. Harris H. The principles of human biochemical genetics, 3rd ed. Amsterdam: ElseviertNorth Holland, 1980. 27. McLaughlin PJ, Johnson PM. A search for human placentaltype Koshida alkaline phosphatases using monoclonal antibodies. Prog Clin Biol Res 1984;166:67-75. Appendix The technical issue discussed here was raised when this manuscript was being reviewed and merits consideration. Reviewer’s question: The reactivities of antibodies C2 and H7 with the different isoenzymes and mutants are not uniform, as shown in Figure 2 of this paper and in previous reports [Mill#{225}n JL, Stigbrand T. Eur J Biochem 1983;136:1-7; Hoylaerts MF, Mill#{225}nJL. Eur J Biochem 1991;202:605-16]. The concentration of antigen [El#{176} was determined by use of these monoclonal antibodies. Why can you get a true value of [E]#{176}? Authors’ response: Initially we used an ELISA procedure [Hoylaerts MF, Manes T, Mill#{225}nJL. Biochem J 1992;286:23-30] in which plates were coated with a polyclonal antiserum to PLAP. After deposition of the 2500 CLINICAL CHEMISTRY, Vol. 38, No. 12, 1992 samples, we used 500 gfL ofH 7/C2 for the detection of bound PLAP/GCAP mutants, using H7 preferentially for the GCAP-related enzymes (or mutants). After revealing the bound monoclonal antibody with biotinylated rabbit antiserum to mouse IgG and Vectastain ABC reagent, the absorbance was read on calibration curves constructed with purified PLAP. Concentrations thus determined yield only estimates of [El#{176}. Because, in principle, the enzyme concentrations could be estimated from their catalytic activity, we have determined Km and for each enzyme mutant. Km determinations are straightforward, but to circumvent the problems encountered in the ELISA during the determination of [El#{176}, we adapted our immunoassay as follows: A limited amount of H7 (10 ngfL) was bound onto rabbit antiserum to mouse Ig-coated plates and, during the incubation amount of antibody was increasing concentrations ofPLAP, GCAP, or mutant enzymes. The activity of the bound enzymes was measured at 405 nm. Plots of 1/A (405 nm) vs the dilution factor, in the range of saturating concentrations, are then linear. The A (405 nm) at infinite concentration (intersection with y-axis) represents the activity of fully saturated H7 monoclonal antibody. At saturation, independent of the mutant studied or of its affinity, the absolute amount of the enzyme is constant. Therefore, the differences in the A (405 nm) values at the intersection reflect differences in When relating these A (405 nm) values to that found for the reference PLAP (for which can be calculated easily), it is possible to calculate for the progressively different step, this saturated enzyme mutants. low with Thus, we found that values are only marginally influenced by the different substitutions investigated. This, we believe,justifies the choice of enzyme concentrations based on activity measurements, as was done during the measurements of relative affinities.