Mutation Report for: D70G/S425P_human-BCHE

Structure-function relationships of cholinesterases (CHEs) were studied by expressing site-directed and naturally occurring mutants of human butyrylcholinesterase (BCHE) in microinjected Xenopus oocytes. Site-directed mutagenesis of the conserved electronegative Glu441,Ile442,Glu443 domain to Gly441,Ile442,Gln443 drastically reduced the rate of butyrylthiocholine (BTCh) hydrolysis and caused pronounced resistance to dibucaine binding. These findings implicate the charged Glu441,Ile442,Glu443 domain as necessary for a functional CHE catalytic triad as well as for binding quinoline derivatives. Asp70 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 Gly70 BCHE by additional His114 or Tyr561 mutations, both of which co-appear with Gly70 in natural BCHE variants, which implies a likely selection advantage for these double BCHE mutants over the single Gly70 BCHE variant. Gly70 BCHE variants also displayed lower binding as compared with Asp70 BCHE to cholinergic drugs, 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.

To study the molecular origin of the altered regulation of butyrylcholinesterase (BCHE) in nervous system tumors, BCHE complementary DNA (cDNA) sequences from human glioblastoma and neuroblastoma cDNA libraries were compared with BCHE cDNAs from normal fetal and adult tissues. A single 2.6-kilobase BCHE cDNA sequence was found in all normal tissues, whereas an additional alternatively terminated BCHE cDNA clone was found in both tumor libraries. The tumor-specific cDNA contained a 3',0.7-kilobase nontranslatable extension, as well as several nucleotide alterations in the normal polyadenylation site. Single-base mutations in the coding region of this unusual BCHE cDNA infer two amino acid substitutions: Asp70----Gly and Ser425----Pro. The Asp70----Gly change has recently been implicated with "atypical" BCHE, which is deficient in its capacity to hydrolyze succinylcholine. The 3.6-kilobase mRNA was less abundant in RNA blot hybridization than the 2.6-kilobase mRNA, which is in agreement with the low ratios between the 3.6- and 2.6-kilobase BCHE cDNA clones in glioblastoma and neuroblastoma libraries. Furthermore, size fractionation and microinjection of glioblastoma polyadenylated RNA, followed by enzyme activity and selective inhibition measurements, demonstrated two peaks of functional BCHE mRNA, the heavier one probably reflecting the longer transcripts. Chromosomal mapping of the 0.7-kilobase 3' fragment by in situ hybridization localized it to a unique 3q26-ter position, where we recently found an inheritably amplified "silent" defective CHE gene in a family exposed to the cholinesterase inhibitor methyl parathion. Our findings confirm previous genetic linkage mapping of the functional CHE gene to the 3q26-ter position and demonstrate that extended functional mRNA transcripts encoding a BCHE form with two modified amino acids are produced from this gene in glioblastoma and neuroblastoma cells.

The "atypical" allelic variant of human butyrylcholinesterase (BCHE) can be characterized by its failure to bind the local anesthetic dibucaine, the muscle relaxant succinylcholine, and the naturally occurring steroidal alkaloid solanidine, all assumed to bind to the charged anionic site component within the normal BCHE enzyme. A single nucleotide substitution conferring a change of aspartate-70 into glycine was recently reported in the CHE gene encoding BCHE from several individuals having the "atypical" BCHE phenotype, whereas in two other DNA samples, this mutation appeared together with a second alteration conferring a change of serine-425 into proline. To separately assess the contribution of each of these mutations toward anionic site interactions in BCHE, three transcription constructs were engineered with each of these substitutions alone or both of them together. Xenopus oocyte microinjection of normal or mutated synthetic BCHEmRNA transcripts was employed in conjunction with biochemical analyzes of the resultant recombinant BCHE variants. The presence of the Gly-70 mutation alone was found to render the enzyme resistant to 100 microM solanidine and 5 mM succinylcholine; concentrations sufficient to inhibit the "normal," Asp-70 containing BCHE by over 50%. Furthermore, when completely inhibited by the organophosphorous poison diisopropylfluorophosphate (DFP), Gly-70 BCHE failed to be reactivated by 10 mM of the cholinesterase-specific oxime pyridine 2-aldoxime methiodide (2-PAM); a concentration restoring about 50% of activity in the "normal" Asp-70 recombinant enzyme. The Pro-425 mutation alone had no apparent influence on BCHE interactions with any of these ligands. However, it conferred synergistic effects on some of the anionic site changes induced by the Gly-70 mutation.