Studies on a heterologous complex formed by human butyrylcholinesterase

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Biochemical Genetics [bigi] pp772-bigi-461674 April 2, 2003 19:1 Style file version Nov 9th, 2002

Biochemical Genetics, Vol. 41, Nos. 5/6, June 2003 (C© 2003)

Studies on a Heterologous Complex Formedby Human Butyrylcholinesterase

R. L. R. Souza,1 L. Furtado,1 A. C. P. Diniz,1 A. C. D. Silva,1 J. Kaiss,1

M. L. Petzl-Erler, 1 and E. A. Chautard-Freire-Maia1,2

Received 31 January 2002—Final 7 August 2002

An electrophoretic band with butyrylcholinesterase activity was detected in 71CHE2 C5+ and 378 CHE2 C5− individuals and was named C4/5 in view of itssimilar mobility to either C4 or C5, depending on the pH of the agar gel used. Thepresent data suggest that C4/5 is a heterologous complex of butyrylcholinesterase.Although the C4/5 band may have the same mobility as C5, depending on theconditions of electrophoresis, our hypothesis is that these two bands result fromthe association of BChE with different molecules.

KEY WORDS: serum cholinesterase;BCHE locus;CHE2 locus; C4/5 and C5 bands.

INTRODUCTION

Human butyrylcholinesterase (BChE, EC 3.1.1.8) is determined by theBCHElocus (3q26.1-q26.2). The first study of electrophoretic variations of BChE inhuman serum showed four molecular forms (C1–C4) normally present in all in-dividuals and a fifth band named C5 (Harris et al., 1962) that showed geneticvariation (Harriset al., 1963), being present in about 10% of Caucasians. TheseBChE molecules appear as a monomer (C1), a monomer linked to albumin (C2),a dimer (C3), a trimer, a tetramer (C4), and a tetramer linked to an unknownmolecule (C5). The C5 complex is determined by the interaction of theBCHEandCHE2 (2q33-q35) genes; theCHE2∗C5+ allele determines its presence (CHE2C5+ phenotype) that follows a dominant pattern of inheritance, while theCHE2∗

1 Department of Genetics, Federal University of Paraná, P.O. Box 19071, 81531-990 Curitiba, PR,Brazil.

2 To whom correspondence should be addressed.

141

0006-2928/03/0600-0141/0C© 2003 Plenum Publishing Corporation

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142 Souza, Furtado, Diniz, Silva, Kaiss, Petzl-Erler, and Chautard-Freire-Maia

C5− allele is responsible for its absence and brings about the homozygous CHE2C5− phenotype. Data reported by Juul (1968) on the detection of 12 forms ofBChE suggest the existence of other heterologous forms in serum.

Van Ros and Vervoort (1973), using acid agar gel electrophoresis, achievedgood resolution for the detection of theCHE2locus phenotypes with the C5 com-plex moving to the positive pole, separated from all the other BChE forms whichconstitute only one band (COF – other forms) that moves to the negative pole inthese conditions. After modifications of this method, a band that moves to thenegative or to the positive pole (C4/5) depending on the pH of the agar gel wasdetected in our laboratory. Our initial objective was to verify whether C4/5 could bedetermined by theCHE2∗C5− allele which in this case would not be responsiblefor a null phenotype (CHE2 C5−). This interest was reinforced by the fact thatthe activity of the C5 complex was negatively associated with weight (Chautard-Freire-Maiaet al., 1991) and body mass index (Alcântaraet al., 2001), whereasthe activity of the C4/5 band was found to be positively correlated with these twoanthropometric variables (Alcântara, 2000).

MATERIAL AND METHODS

The serum or plasma samples were stored at−20◦C. The C4/5 band was examinedin 378 CHE2 C5− individuals from two populations: 311 individuals from thegeneral population of Curitiba (284 blood donors and 27 members of the FederalUniversity of Paraná) and 67 Brazilian Guarani Indians, subtribe Guarani-M’bya,from the Rio das Cobras Reserve, county of Laranjeiras do Sul (52◦35′W; 25◦20′S).This band was also studied in 71 CHE2 C5+ Guarani-M’bya Indians.

TheCHE2 locus was phenotyped by acid agar gel electrophoresis accordingto the following modifications of the original methodology (Van Ros and Vervoort,1973). Agar gel (50 mL of Difco Bactoagar 1.4% in a 50 mM citric acid – sodiumcitrate buffer of pH 6.7) was poured onto a 230× 160× 3 mm glass plate, and thesame buffer at pH 6.7 was used for the bridge. Serum or plasma (2.5 or 3.0µLdiluted 1:1 in saline solution or in distilled water) was loaded directly half way intothe gel with the aid of a metal sheet. An electric current of 35 mA was applied for3 h (approximately 75 V/plate) in a run at 4◦C. The zones of enzyme activity weredeveloped with a solution of 0.1% Fast Red TR Salt (Sigma F2768) in 200 mMsodium phosphate buffer, pH 7.1, containing 2.5% of the 30 mMα-naphthyl acetatesubstrate dissolved in acetone and made up to volume with distilled water (1:1).The same technique was used for the detection of the C4/5 band, changing onlythe pH of the agar gel which was tested from 4.0 to 6.7.

Two kinds of assays were used to test the activity of the C4/5 band afteragar gel electrophoresis at pH 5.3: (1) plasma samples with EDTA (0.01 M finalconcentration) and control samples in distilled water (1:1) were applied to agargel, (2) one hour incubation (37◦C) of half of the agar gel with the BChE inhibitor

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Studies on a Heterologous Complex Formed by Human Butyrylcholinesterase 143

(Ro2-0683; 2.5× 10−4M in phosphate buffer, pH 7.1) after electrophoresis andbefore the staining procedure. The other half of the agar gel was a control on whichthe phosphate buffer was used instead of the solution containing inhibitor.

CHE2 C5− and CHE2 C5+ plasma samples (5µL) were added to 5µL ofa trypsin (bovine type III) solution (1000µg/mL) in pH 7.4 buffer (0.15M Tris-HCL) for 60 min in a water bath (37◦C), and control plasmas were diluted in 5µLof the same buffer. After incubation, 2µL of each sample was submitted to agargel electrophoresis at pH 5.3 and pH 6.7 for CHE2 C5− and CHE2 C5+ samples,respectively.

The bands from CHE2 C5− and CHE2 C5+ individuals were examined intwo kinds of two-phase electrophoresis. In the first, agar gel at pH 5.3 was thesupport medium for the first 3 h run. After this run, the gel was cut into four strips:one containing the C4/5 and C5 bands that move to the positive pole on agar gel atpH 5.3, another containing the COF bands that move to the negative pole, and thetwo extreme strips which were discarded. The part containing the COF bands wastaken from the glass plate and stained, whereas the strip containing the C4/5 andC5 bands was left on the glass plate which was then filled with agar gel at pH 6.7,and a second run was carried out for 3 h. After this run, the gel was stained. Thesecond kind of two-phase electrophoresis was done with the first run in agar gelat pH 6.7 and the second run in agar gel at pH 5.3. This also differed from thefirst kind of two-phase electrophoresis by the fact that, after the first run, the stripcontaining the COF bands was transferred to another glass plate that was then filledwith agar gel (pH 5.3) and the second run was also carried out for 3 h. After theseruns, both agar gels were submitted to the staining procedure.

Plasma from two individuals (one of them CHE2 C5− and the other CHE2C5+) was applied 140 times each to the agar gel for band isolation after elec-trophoresis. After the staining procedure, the bands were cut from the gel: theC4/5 bands were cut from agar gel of pH 5.3 and the C5 bands from agar gel ofpHs 5.3 and 6.7. The bands separated in these three groups were kept frozen for1 day. After thawing, the liquid released from the agar gel was concentrated usinga saturated gum arabic solution for dialysis at room temperature.

Starch gel electrophoresis at pH 8.6 (Poulik, 1957; with modifications fromPetzl and Primo-Parmo, 1979) was used to isolate the C1 to C3 bands from theCHE2 C5− samples. In this case, only one column of the starch gel was stained toindicate the position of these bands which were cut out and submitted to the samefreezing and thawing procedure, with dialysis carried out at 4◦C.

The effect of a saturated 3 M saline solution on the C4/5 and C5 bands wastested after electrophoresis in agar gel at pH 5.3 and pH 6.7 for the CHE2 C5−and CHE2 C5+ phenotypes, respectively. In these experiments, after a 3-h run, thepart of the agar gel corresponding to the COF bands was taken from the glass plateand submitted to the staining procedure. The glass plate was filled with agar gelof the same pH and 2.5µL of a 3 M saline solution was inserted into each of three

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grooves in the region corresponding to the positions of the C4/5 or C5 bands; inone control, 2.5µL of a 0.9% saline solution were inserted into each of the threecorresponding grooves and no treatment was used in another control. Another 3 hrun followed these procedures.

RESULTS AND DISCUSSION

A total of 70 electrophoretic runs were performed with CHE2 C5− samples, using8 different pHs of the agar gel: 4.0, 4.5, 4.7, 5.0, 5.3, 5.6, 6.2, and 6.7. Almostno migration occurs when the pH of the agar gel is lower than 4.7 because of thecloseness of the isoelectric points of the BChE forms (Masson, 1979). The C4/5

band may appear from pH 4.7 to pH 6.2, occupying a position similar to C5 in thedirection of the positive pole. However, only at a pH of around 5.3 was the C4/5

band clearly detected as a separate band (Fig. 1(B-1)). Under these conditions, theother BChE molecular forms appear as only one band (COF) moving to the negativepole. When the pH of the agar gel is higher than 6.2, all the BChE molecules formonly one band moving to the negative pole, as shown in Fig. 1(A-1) for the CHE2C5− samples. So, a pH higher than 6.2 is recommended for the agar gel in order todiscriminate the CHE2 C5+ from the CHE2 C5− phenotype (Fig. 1(A-2)), sincein this condition the C4/5 band does not appear at the position of C5.

Fig. 1. The same CHE2 C5− (1) and CHE2 C5+ (2) samples were applied in A (6.7 pH agar gel) andB (5.3 pH agar gel), following the same sample order.

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Fig. 2. Results for the CHE2 C5− (1) and CHE2 C5+ (2) samples, showing the C5 and C4/5 bandsafter the second run in agar gel at pH 6.7 in two-phase electrophoresis; the first run took place inagar gel of pH 5.3. The part of the gel that contained the COF bands in the first run is not shown.

Two-phase gel electrophoresis was used to test whether this dual behaviorof the C4/5 band seen in the CHE2 C5− phenotype was due to its differentialmobility depending on the pH of the agar gel. This same kind of electrophoresiswas used to test whether CHE2 C5+ samples contained both the C4/5 and C5

bands in the same position when the run was done on a gel at pH 5.3. Figure 2shows that during the second run (pH 6.7) the C4/5 band from the CHE2 C5−samples migrated to the negative pole, with separation of the C5 and C4/5 bandswhich constituted only one band in the CHE2 C5+ samples when the first run wasdone on agar gel at pH 5.3 (Fig. 1(B-2)). This indicates that the mobility of theC4/5 band in pH 6.7 agar gel is toward the negative pole and that the CHE2 C5+individuals examined also have the C4/5 band. This conclusion is supported by theresults illustrated in Fig. 3 which show that C4/5 migrates together with COF atpH 6.7, while it separates from it when the pH of the gel is 5.3 in the second run(Fig. 3(B)).

The C4/5 band was not inhibited by EDTA, a fact excluding the possibilitythat this is the human serum paraoxonase that could also hydrolyze theα-naphthylacetate substrate used (Erdöset al., 1960).

The C4/5 band was completely or partially inhibited by the BChE inhibitorRo2-0683 (Kalow and Davies, 1958) in samples from the usual (BCHE U) oratypical (BCHE A) phenotypes, respectively. The C4/5 behavior was similar tothat of the COF band composed by all the other BChE molecular forms. Theseresults show that the C4/5 band has BChE activity.

Samples submitted to trypsin digestion showed that the C4/5 bands werenot affected, presenting the same behavior as the control samples. However, theC5 bands were affected by trypsin and did not appear after electrophoresis, aswas expected from previously reported data (Masson, 1991). These data seem toexclude the possibility of C4/5 being C2 (a BChE monomer linked to albumin) orthe complex formed by BChE andα-2-macroglobulin, since these two complexesare affected by the action of trypsin (Masson, 1978).

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Fig. 3. Results for the CHE2 C5− (1) and CHE2 C5+ (2) samples from two-phase elec-trophoresis in agar gel at pH 6.7 and pH 5.3 in the first and second run, respectively. A – C5bands; B – Respective COF and C4/5 bands.

The C1, C2, and C3 bands isolated after starch gel electrophoresis and appliedto acid agar gel electrophoresis (pH 5.3) migrated to the negative pole (similar tothe COF band), showing that none of these three bands is C4/5, thus confirming thedata obtained with the use of trypsin that indicated that C4/5 and C2 are differentBChE forms.

The C4/5 band isolated from agar gel at pH 5.3 and the C5 band isolatedfrom agar gel at pH 5.3 and pH 6.7 when applied to another electrophoretic(pH 5.3) run showed not only the expected band moving to the positive polebut also another band moving to the negative pole, similar to COF (Fig. 4). Thebands which moved to the negative pole are assumed to represent BChE ho-mopolymers disconnected from the BChE heterologous complexes in the isola-tion procedures, as was already shown by Massonet al. (1990; Masson, 1991)and Primo-Parmoet al. (1990) for C5. A comparable behavior was observed inthe experiments with the saturated saline solution (3 M) that affected both theC4/5 (Fig. 5) and C5 bands, provoking an altered migration towards the negativepole. The results obtained in these two types of experiments suggest that C4/5 isa heteropolymer like C5, and is affected by treatments which separate the BChEmolecules from the substance they were linked to. These data also suggest thatBChE and this other substance are predominantly linked by ionic forces in the C4/5

complex.

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Fig. 4. Segment of the agar gel plate (pH 5.3) showing control plasmas (1 – CHE2 C5+; 2 – CHE2C5−) and the previously isolated bands: C5 (3 – from agar gel of pH 5.3; 4 – from agar gel of pH6.7) and C4/5 (5 – from agar gel of pH 5.3).

A CHE2 C5+phenotype population subsample was selected from the GuaraniAmerindian tribe, subtribe Guarani-M’bya, that has a very high frequency of thisphenotype (45.9%; Alcântaraet al., 1995) in order to test the hypothesis that theC4/5 band could be determined by an allele at theCHE2 locus. The expected fre-quency of homozygotes for theCHE2∗C5+ allele was 7.0% in this tribe, leading toan expected number of approximately 11 (15.24%) of these homozygotes amongthe 71 CHE2 C5+ phenotypes examined. The C4/5 band was detected in all these

Fig. 5. Effect of 3 M saline solution on CHE2 C5− samples (2, 4) as compared to a control submittedto 0.9% saline solution (1) and to a control without any treatment (3) in a second electrophoreticrun from which the COF bands had been excluded. Both runs were in agar gel of pH 5.3.

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71 individuals when examined by two-phase electrophoresis (agar gel at pH 5.3 andpH 6.7 in the first and second runs, respectively). The absence of homozygosity—that would be revealed by the lack of C4/5—is significantly different from whatwould be expected on the basis of the allele hypothesis (χ2

(1) = 12.77; p < 0.001).The nonrejection of the allele hypothesis would imply a very complex systemassuming lethal homozygotes for theCHE2∗C5+ allele and a heterozygous ad-vantage in order to maintain the polymorphic frequency of this allele in nonisolatedpopulations.

All the analyzed individuals showed the C4/5 band: 378 CHE2 C5− (agargel electrophoresis, pH 5.3) and 71 CHE2 C5+ individuals (two-phase agar gelelectrophoresis with agar gels at pH 5.3 and pH 6.7 in the first and second runs,respectively). Since no variant of the C4/5 band was detected by these methods,one can consider this phenotype as a monomorphism on the basis of the data fromthe Curitiba sample (N = 311).

It is likely that the C4/5 band has already been detected in other studies.Although the C4/5 activity increases with storage (Alcântara, 2000) it is also seenin fresh blood, a fact that may exclude it from being one of the bands (S1 and S2)detected by Harriset al. (1962) after storage at temperatures of 4◦C and−7◦C.Many studies have reported nonstandard electrophoretic bands of BChE (Ashtonand Simpson, 1966; Delbrück and Henkel, 1979; Gallango and Arends, 1969; VanRos and Druet, 1966; Yamamotoet al., 1986, 1987; Yoshida and Motulsky, 1969)whose possibility of being C4/5 can be excluded since these previously reportedbands are not monomorphic. The studies by La Mottaet al.(1968) and Juul (1968)referred to the occurrence of 7 and 12 bands of BChE, respectively. Although theseauthors did not study the C4/5 band, it is possible that it appeared among thosedetected by them.

The data obtained in the present study indicate that theCHE2 locus does notinteract with theBCHE locus in the determination of the C4/5 molecular form ofbutyrylcholinesterase, excluding the possibility of C5 and C4/5 being conditionedby alleles.

The isolation of the C4/5 band from the other BChE bands allows that thisBChE complex may be examined for further studies. Considering that the physio-logical role of BChE is not yet established and that several molecular forms of thisenzyme occur in blood, it is of interest to search for possible different functionsof these molecules.

ACKNOWLEDGMENTS

We are grateful to Conselho Nacional de Desenvolvimento Cient´ıfico eTecnologico (CNPq) for research grants and fellowships and to Funda¸cao daUniversidade Federal do Paraná (FUNPAR) for grants. We thank the Centro deHematologia e Hemoterapia do Paraná (HEMEPAR) for donating blood samples

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and Hoffmann-La Roche & Co. (Basel, Switzerland) for furnishing the inhibitorRo2-0683.

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Studies on a heterologous complex formed by human butyrylcholinesterase

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