Jan 13, 1992 - Yael Loewenstein, Shlomo Seidman,. Gal Ehrlich and Hermona Soreq1 ..... analogues/inhibitors (Hasan et al., 1980, 1981; Cohen et al.,. 1645 ...
The EMBO Journal vol. 1 1 no.4 pp. 1 641 - 1649, 1992
Intramolecular relationships in cholinesterases revealed by oocyte expression of site-directed and natural variants of human BCHE Lewis F.Neville, Averell Gnatt, Yael Loewenstein, Shlomo Seidman, Gal Ehrlich and Hermona Soreq1 Department of Biological Chemistry, The Life Sciences Institute, The Hebrew University of Jerusalem, Jerusalem 91904, Israel 'Corresponding author Communicated by A.R.Fersht
Structure-function relationships of cholinesterases (CHEs) were studied by expressing site-directed and naturally occurring mutants of human butyrylcholinesterase (BCHE) in microinjected Xenopus oocytes. Sitedirected mutagenesis of the conserved electronegative Glu441,1le442,Glu443 domain to Gly441,1le442,Gln443 drastically reduced the rate of butyrylthiocholine (BTCh) hydrolysis and caused pronounced resistance to dibucaine binding. These findings inplicate the charged Glu441, Ile442,Glu443 domain as necessary for a functional CHE catalytic triad as well as for binding quinoline derivatives. Asp7O to Gly substitution characteristic of 'atypical' BCHE, failed to alter its Km towards BTCh or dibucaine binding but reduced hydrolytic activity to 25% of control. Normal hydrolytic activity was restored to Gly7O BCHE by additional His114 or Tyr561 mutations, both of which co-appear with Gly7O in natural BCHE variants, which implies a likely selection advantage for these double BCHE mutants over the single Gly7O BCHE variant. Gly7O BCHE variants also displayed lower binding as compared with Asp7O BCHE to cholinergic dru, certain choline esters and solanidine. These effects were ameliorated in part by additional mutations or in binding solanidine complexed with sugar residues. These observations indicate that structural interactions exist between N' and C' terminal domains in CHEs which contribute to substrate and inhibitor binding and suggest a crucial involvement of both electrostatic and hydrophobic domains in the build-up of the CHE active center. Key words: anionic sites/butyrylcholinesterase alleles/ cholinergic drugs
Introduction In man, the existence of the acetylcholine hydrolyzing enzyme butyrylcholinesterase (BCHE, EC 3.1.1.8) was established over 50 years ago based upon the distinct substrate and inhibitor specificities (Alles and Hawes, 1940), which distinguish it from the closely related enzyme acetylcholinesterase (ACHE, EC 3.1.1.7). Apart from its likely role in supporting ACHE in terminating cholinergic neurotransmission (Massoulie and Toutant, 1988), the intensive expression of BCHE in fetal tissues (Zakut et al., (© Oxford University Press
1985; Layer and Sporns, 1987; Zakut et al., 1991) and bone marrow stem cells
(Patinkin et al., 1990), together with its gene amplification in germ cells (Prody et al., 1989) as well as in blood cells (Lapidot-Lifson et al., 1989) and ovarian carcinomas (Zakut et al., 1990) indicate a probable growth related role for BCHE (Soreq and Zakut, 1990). Serum BCHE exerts a key clinical role in the degradation of drugs such as succinylcholine (SucCh; Hodgkin et al., 1965), heroin (Valentino et al., 1981), physostigmine and ecothiophate (Silver, 1974) and activates pro-drugs such as bambuterol (Olsson and Svensson, 1984). Ecologically, BCHE serves in the scavenging and subsequent detoxification of both naturally occurring (e.g. solanum related alkaloids or quinoline compounds) and synthetic (e.g. carbamate or organophosphorous) CHE inhibitors (Silver, 1974; Whittaker, 1986). Characterized mutations in the anionic subsite of BCHE result in altered binding properties of various substrates and inhibitors (Neville et al., 1990a). An interesting but yet unexplained phenomenon regarding the human BCHE gene is that it exhibits a high rate of non-lethal mutation. At present, 10 different allelic forms each possessing phenotypically distinct properties (LaDu et al., 1991; Soreq et al., 1991) have been described. The availability of these compatible-with-life variants makes BCHE an appropriate model for structure -function relationship studies in CHEs. This has been achieved in the present study by engineering and expressing plasmid vectors containing various human derived allelic coding regions for BCHE or combined fragments from such alleles. Once modified domains within BCHE were correlated with distinctly altered enzyme properties, rational site-directed mutagenesis (Russell and Fersht, 1987) was devised to further modify key residues in the enzyme. The resultant series of naturally occurring and site-directed mutants was employed to pursue the roles of particular loops included in the BCHE polypeptide in order to correlate individual amino acids or domains within the molecule with the binding of specific substrates and inhibitors and to shed new light on intramolecular interactions in CHEs.
Results Construction of recombinant BCHE vectors including natural and site-directed mutations Recombinant and site-directed engineering of four naturally occurring allelic BCHE cDNA clones (I-IV) were employed to create 10 distinct BCHE cDNA constructs
(Numbers 2-11) carrying different combinations of single point mutations, all of which result in amino acid substitutions (Figure lA; see Materials and methods). Substrate hydrolytic activities of native and recombinant oocyte produced enzymes At a substrate concentration of 10 mM, all mutations examined had reduced the ability of these oocyte produced
1641
L.F.Neville et al.
enzymes to hydrolyze butyrylthiocholine (BTCh), with the exception of the Gly7O, HisI 14 double mutant (Figure 2A). Both this mutant (construct 9, Figure IA) and the usual recombinant BCHE (construct 1) routinely hydrolyzed BTCh at the rate of 9.0 nmol/oocyte/h over values exhibited by control oocytes that had been injected with Barth's medium. The least effective mutants regarding hydrolytic activity were those carrying the site-directed Gly441,Gln443 mutations (Figure 2A, constructs 10 and 11). Even under optimized conditions (following plasmid linearization and a modified microinjection protocol; see Materials and methods), when
the usual BCHE hydrolyzed BTCh at 161.56 nmol/oocyte/h, Gly441,Gln443 BCHE displayed a net rate of only 5.54 nmol/oocyte/h. The addition of Gly70 further reduced its activity to 1.13 nmol/oocyte/h. Thus, the exceedingly low activity of the triple mutant prevented us from further characterizing its enzyme product. To examine whether the dramatic loss in catalytic activities which occurred in the Gly441,Gln443 and Gly7O,Gly441,Gln443 BCHE mutants reflected changes in protein quantity or altered catalytic properties, an immunoblot analysis was performed on detergent extracts of oocytes
11.1
..
.--U;. . ,"t"',
1642
-1. '.
I
':
Mutagenesis and oocyte expression of BCHE variants
expressing these enzymes. The intensity of the relevant immunoreactive protein band produced from the mutated constructs appeared in this test to be indistinguishable from that obtained with the usual BCHE (Figure 2A, inset). This implied that both the natural Gly7O mutation and the sitedirected Gly441,Gln443 mutations impair BCHE activity without affecting its rate of synthesis and/or general stability in the oocytes. The single Tyr561 and Pro425 mutations decreased hydrolytic activities, but much less than the single Gly7O or double Gly7O,Pro425 mutant. In contrast, the Tyr561 substitution partially reversed the depressant effects of the Gly7O mutation, while the introduction of His1 14 into Gly7O BCHE totally restored its activity (Figure 2A). These separate potentiating effects of Hisi 14 and Tyr561 on substrate hydrolysis were not observed, however, when both these substitutions appeared together. Thus the triple mutant Gly7O,Hisl 14,Tyr561 hydrolyzed the substrate at a slower rate than even Gly7O alone. Nevertheless, the inclusion of Hisi 14 together with Gly7O and Pro425 improved activity over Gly7O,Pro425 BCHE. Figure 2B represents schematically the pairwise interactions between these single amino acid substitutions. Substrate specifcity altered in recombinant BCHE variants To determine if reduced catalytic activities observed for the various BCHE variants reflected changes in the microenvironment of their active sites, their affinities to various substrate analogues were examined (Figure 3A and B). The choline esters studied included BTCh, benzoyl- (BenzCh), acetyl- (ACh), succinyl- (SucCh) and propionylcholine (PropCh), all of which interact with BCHE through its presumed anionic site. Normal Michaelis - Menten constants (Km1) with BTCh and associative binding constants (Ki) for BenzCh were observed for all mutants, varying slightly within a range of 2-fold (Figure 3A). Tyr561 BCHE exhibited similarly normal binding to the quarternary charged choline ester SucCh, with a Ki of 1.68 mM. However, all the other mutants displayed markedly higher (> 15-fold) Ki values for SucCh, indicative of major reductions in the enzyme's affinity towards this choline ester. The triple
mutant Gly70,Hisl 14,Tyr561 displayed the highest affinity whereas the double mutants Gly7O,Tyr561 and Gly7O,His114 exhibited weakened binding capacities toward SucCh. However, the addition of Pro425 to Gly7O,Hisl 14 resulted in total resistance of SucCh binding with no IC50 value obtainable even at 200 mM SucCh, similar to previous observations with the double Gly7O,Pro425 BCHE variant (Neville et al., 1990a). All Gly7O containing mutants displayed -4- to 5-fold reductions in binding ACh as compared with the usual and Tyr561 enzymes, showing a profile similar to that observed with SucCh. In contrast, PropCh binding was unchanged in the Tyr561 and Gly7O,Tyr561 mutants (Figure 3B). Addition of His1 14 to Gly7O, with or without Tyr561, resulted in a 4-fold reduction in PropCh binding and addition of Pro425 to Gly7O,Hisl 14 BCHE further decreased binding. Different BCHE variants display resistance to inhibition by solanum and quinoline related poisons Stable mutations in metabolically important proteins are frequently advantageous to organisms in which they occur, explaining the evolutionary survival of such mutations (Whittaker, 1986). To reveal whether any of the tested variants may belong to this category, we examined their sensitivity to inhibition by naturally occurring solanum related toxins (aglycones and their glycoalkaloids) and the cocaine derivative dibucaine. Dose-response curves revealed normal binding of dibucaine to Tyr561 BCHE (IC50 = 30 uM). Gly7O mutants carrying either Hisi 14 or Hisi 14,Pro425 displayed marked resistant to dibucaine, but were susceptible to inhibition at a concentration of 1 mM. In contrast, the triple Gly7O,Hisl 14,Tyr561 mutant showed a decreased and an interestingly biphasic dibucaine binding profile; at 100,^M dibucaine, this mutant was significantly inhibited, but at 1 mM dibucaine its inhibition was not further modified (Figure 4A). In order to compare the effects of the site-directed mutations Gly441,Gln443 on dibucaine with those on SucCh binding, enzymes were incubated with two concentrations of dibucaine (0.1 mM and 1 mM) and SucCh (10 mM and 50 mM) and their remaining BTCh hydrolyzing activities determined. Whilst the ability of Gly441,Gln443 BCHE to
Fig. 1. Construction of native and site-directed BCHE mutants. (A) Positions of mutations examined and the respective amino acid substitutions. BCHE cDNA clones I, II and III representing the normal BCHE (Soreq et al., 1989), the unusual BCHE species present in human neuroblastomas and glioblastomas (Gnatt et al., 1990) and a novel XgtlO BCHE cDNA clone (Gnatt, 1990), respectively, were used to engineer constructs 1-9 by enzymatic restriction with PstI and BamHI and religation of the resultant a-g fragments in the noted combinations. Amino acid substitutions in each of these BCHE variants are marked in the single letter code. Site-directed mutagenesis of the Gly441 -Gln443 domain into the normal BCHE coding region created Clone IV, from which constructs 10 and 11 were generated in a similar manner, containing the Gly441,Gln443 domain with or without the Gly7O mutation. Respective positions of each of these substitutions along the BCHE polypeptide chain are noted below. (B) Substitution of the Glu441 -Glu443 domain by site-directed mutagenesis. DNA from the usual BCHE Clone I was restricted with BamHI and HindIII and the resultant 800 bp fragment containing the Glu441 -Glu443 (EIE) domain was sub-cloned into identically restricted double-stranded M13mpl8RF vector (Boehringer, Mannheim). Single-stranded uracil enriched DNA including this fragment was obtained (see Materials and methods) and doublemutated M 24mer oligonucleotide, containing two mis-matches at the EIE region was hybridized to the extracted phage single-stranded DNA. Second strand DNA synthesis was subsequently catalyzed by DNA polymerase and ligase in the presence of dNTPs. Following transformation into competent E. coli MV 1190 cells, selective degradation of the uracil enriched strand occurred and the resultant DNA containing the corresponding two mismatches was restricted with BamHI and HindII and religated back into the identically restricted pSP64-41 plasmid containing the human BCHE cDNA. (C) Mutagenesis of Glu441-Glu443 to Gly441 -Gln443 introduces an informative EcoRI site in BCHE cDNA. Nucleotides 1471-1497 in BCHE cDNA (Prody et al., 1987, upper sequence) encode the amino acid residues 438-446 in the mature BCHE protein, including the electronegative domain of interest (top peptide sequence). By utilizing the minus strand 24mer M oligonucleotide (lower nucleotide sequence) which contains two mis-matches directed at the EIE domain 5'-TTQAATTCC-3', a mutated protein which is encoded by the complementary mutant strand 5'-GGAATTCAA-3' (noted below) results in the sequence Gly441 -Isoleu442 -Gln443 (GIQ). This sequence introduces a third and novel EcoRI site in BCHE cDNA which has been used to isolate GIQ encoding mutants. Samples 1, 3, 5 and 7 extracted from constructs 1, 5, 10 and 11, respectively, were each subjected to enzymatic restriction with BamHI and Hindll. Parallel restrictions with EcoRP are shown in lanes 2, 4, 6 and 8. Note that the BCHE insert of the mutated plasmids 5 and 7 produced two fragments with EcoRI which were 1.2 kb and 1.0 kb, as compared with a single 2.2 kb insert fragment for samples 1 and 3.
1643
L.F.Neville et al.
"7.
E
*0110
R
't -
i"
if
yd
CH,
0 ButyryleMn
cm
,-Nl-
0 cm- cm-
-O-I-CH
-CH-
C,
CH3 CH,
E B.nzoylholln
CH
-
0
c -Cc II-C
CH a
Fig. 2. Recombinant Xenopus oocyte produced BCHE variants hydrolyze BTCh with different efficiencies. (A) Substrate hydrolysis and immunoreactive intensities. Hydrolytic activities of recombinant BCHE variants produced from constructs 1 -11 (Figure IA), measured using 10 mM BTCh, are presented as a percentage activity of 'usual' recombinant BCHE (None; construct 1). Barth's injected oocyte activities of 6.61 nmol/oocyte/h (of which 5.09 nmoles/oocyte/h was due to spontaneous hydrolysis of substrate) have been subtracted. Immunoreactive 70 kDa BCHE produced in injected oocytes was detected by chemiluminescent protein blot analysis following SDS-PAGE of the respective, solubilized oocyte extracts. Extracts of control oocytes injected with Barth's medium and a 0.05 I1 sample of human serum served as controls. Equivalent amounts of immunoreactive protein products were observed in oocytes injected with mRNA from three different constructs, with no detectable immunoreactive protein product at this size range of 70 kDa in the Barth's medium injected oocytes. (B) Mutational co-operativity on BTCh hydrolysis in constructed BCHE variants. Dominant and inert effects of combined mutations within BCHE on substrate hydrolysis are shown in relation to cysteine loops A, B and C. Dominance by one amino acid over another is indicated by an arrow connecting these mutations and its either potentiatory or depressant effects on general hydrolytic activities are indicated by + and - signs, respectively. Note the dominant, suppressive effect of the Gly70 mutation over other naturally occurring mutations (the single Pro425 and Tyr561 mutations, the double HisI 14,Pro425 and Hisl 14,Tyr561 mutations). Dominance of the single Hisl 14 and double Gly441,Gln443 mutations over Gly70 is reflected by dramatic changes in hydrolytic activities (Figure 2A). Position of each mutation along the BCHE protein is noted above, together with the active site Ser200 residue.
bind SucCh was virtually unaffected, its ability to bind dibucaine was markedly reduced (Table I) demonstrating a clear involvement of this specific peptide domain in the binding of quinoline compounds to cholinesterases. The steroidal alkaloid solanidine, which selectively interacts with BCHE but not ACHE (Roddick, 1989), effectively inhibited usual and Tyr561 BCHEs with identical IC5Os and 55 izM. In marked contrast, the hydrolytic activities of all the Gly7O containing BCHE mutants, 1644
Z
0
b
b b
z c,
SuccinyIchoUne
CH
.-C4 CHa-N'-C-a-CHa-O-C-CHi-CHi-C-0-CHi-CH.a..a.--aa,-. -. 0
CH.
*ActtWchol@e
b
CHa
CH.,- N'-C a,-aC a-O-C-CH, CHa
CH,
O
-aC-CA- CHa []Proplony1cholln CHa.- N- a.CH-
CHS
Fig. 3. Alterations in binding affinities of recombinant BCHE variants to choline esters. (A) Limited influence on binding to BTCh and BenzCh substrates. Binding of BTCh and BenzCh to recombinant BCHE mutants was measured as described in Materials and methods and is presented in apparent K,. and Ki values (columns). Variations in the K,,, of BTCh ranged between 1.64 mM for the single Tyr561 containing mutant and 4.25 mM for the triple Gly7O,Hisl 14,Pro425 variant. Also, the site-directed Gly441,Gln443 BCHE mutant displayed apparently normal binding (K,,, = 2.64 mM) towards BTCh and the binding affinities of all BCHE variants towards BenzCh were - 10-fold higher than affinities to BTCh. Note that BenzCh binding to Gly441,Gln443 was not evaluated in this study. (B) Decreased affinity to various substrate analogs induced by Gly7O and modulated by other mutations. Ki determinations (mM) for three choline esters (SucCh, PropCh and ACh) were derived from the equation Kj = IC50/1 +SIK,,, and are presented in columns. Note that the triple BCHE mutant Gly7O,Hisl 14,Pro425 and, to a lower extent, other Gly7O variants present Ki values which increased as much as 100-fold for SucCh. 6.5-fold for PropCh and 5-fold for ACh as compared with the usual recombinant enzyme. The corresponding structures are displayed for each of these choline esters.
irrespective of the presence of other mutations, were totally unaffected by solanidine even at a concentration of 200 uM. A similar trend in inhibition was observed for the steroidal glycoalkaloid alpha-solanine, which potently inhibited the usual recombinant and Tyr561 BCHEs with IC5Os of 5.2
Mutagenesis and oocyte expression of BCHE variants
_0 C
10
100
1000
10000
(B) Alpha-Solanine
100i so
N
SOLAPIDIE
20
1000I
10
Xso ,
