19 Citations (Scopus)

Abstract

Human BChE (butyrylcholinesterase) protects against the toxicity of organophosphorus nerve agents and pesticides. BChE purified from human plasma is limited and pathogen carry-over is a concern. Unlike the native BChE tetrameric complex with a residence time of days, rBChE (recombinant BChE) is produced predominantly as dimers andmonomers that are cleared from the circulation within minutes. Assembly into tetramers requires incorporation of proline-rich peptides, a process that was thought to occur intracellularly. Our goal was to determine whether polyproline added to rBChE under cell-free conditions would promote tetramerization. Secreted rBChE was purified by procainamide affinity chromatography, and synthetic polyprolines (8-mer to 300-mer) were tested to determine their effect on tetramer assembly. These studies demonstrated that 90-98% of purified rBChE (65 μM) could be assembled into tetramers when incubated with synthetic 17-mer or 50-mer polyproline peptides (100 μM) for 1.5 h at 25°C. However, rBChE tetramerizationwas inefficient with smaller 8-mer polyproline peptides and larger 300-mer polyproline proteins. Collectively, these studies demonstrated that the eukaryotic cellularmachinery is not required for assembly of active BChE into tetramers and that this process can occur in vitro with purified rBChE in the presence of peptides containing 15-50 consecutive proline residues.

Original languageEnglish (US)
Pages (from-to)329-335
Number of pages7
JournalBiochemical Journal
Volume462
Issue number2
DOIs
StatePublished - Sep 1 2014

Fingerprint

Butyrylcholinesterase
Peptides
Proline
Plasma (human)
Affinity chromatography
Procainamide
Pathogens
Affinity Chromatography
Pesticides
Dimers
Toxicity
polyproline
Proteins

Keywords

  • Enzyme stability
  • Human butyrylcholinesterase
  • Polyproline peptide
  • Recombinant expression
  • Tetrameric bioscavenger

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

@article{5ec744f507dc473482ff9e90503a0a75,
title = "Polyproline promotes tetramerization of recombinant human butyrylcholinesterase",
abstract = "Human BChE (butyrylcholinesterase) protects against the toxicity of organophosphorus nerve agents and pesticides. BChE purified from human plasma is limited and pathogen carry-over is a concern. Unlike the native BChE tetrameric complex with a residence time of days, rBChE (recombinant BChE) is produced predominantly as dimers andmonomers that are cleared from the circulation within minutes. Assembly into tetramers requires incorporation of proline-rich peptides, a process that was thought to occur intracellularly. Our goal was to determine whether polyproline added to rBChE under cell-free conditions would promote tetramerization. Secreted rBChE was purified by procainamide affinity chromatography, and synthetic polyprolines (8-mer to 300-mer) were tested to determine their effect on tetramer assembly. These studies demonstrated that 90-98{\%} of purified rBChE (65 μM) could be assembled into tetramers when incubated with synthetic 17-mer or 50-mer polyproline peptides (100 μM) for 1.5 h at 25°C. However, rBChE tetramerizationwas inefficient with smaller 8-mer polyproline peptides and larger 300-mer polyproline proteins. Collectively, these studies demonstrated that the eukaryotic cellularmachinery is not required for assembly of active BChE into tetramers and that this process can occur in vitro with purified rBChE in the presence of peptides containing 15-50 consecutive proline residues.",
keywords = "Enzyme stability, Human butyrylcholinesterase, Polyproline peptide, Recombinant expression, Tetrameric bioscavenger",
author = "Larson, {Marilynn A} and Oksana Lockridge and Hinrichs, {Steven Heye}",
year = "2014",
month = "9",
day = "1",
doi = "10.1042/BJ20140421",
language = "English (US)",
volume = "462",
pages = "329--335",
journal = "Biochemical Journal",
issn = "0264-6021",
publisher = "Portland Press Ltd.",
number = "2",

}

TY - JOUR

T1 - Polyproline promotes tetramerization of recombinant human butyrylcholinesterase

AU - Larson, Marilynn A

AU - Lockridge, Oksana

AU - Hinrichs, Steven Heye

PY - 2014/9/1

Y1 - 2014/9/1

N2 - Human BChE (butyrylcholinesterase) protects against the toxicity of organophosphorus nerve agents and pesticides. BChE purified from human plasma is limited and pathogen carry-over is a concern. Unlike the native BChE tetrameric complex with a residence time of days, rBChE (recombinant BChE) is produced predominantly as dimers andmonomers that are cleared from the circulation within minutes. Assembly into tetramers requires incorporation of proline-rich peptides, a process that was thought to occur intracellularly. Our goal was to determine whether polyproline added to rBChE under cell-free conditions would promote tetramerization. Secreted rBChE was purified by procainamide affinity chromatography, and synthetic polyprolines (8-mer to 300-mer) were tested to determine their effect on tetramer assembly. These studies demonstrated that 90-98% of purified rBChE (65 μM) could be assembled into tetramers when incubated with synthetic 17-mer or 50-mer polyproline peptides (100 μM) for 1.5 h at 25°C. However, rBChE tetramerizationwas inefficient with smaller 8-mer polyproline peptides and larger 300-mer polyproline proteins. Collectively, these studies demonstrated that the eukaryotic cellularmachinery is not required for assembly of active BChE into tetramers and that this process can occur in vitro with purified rBChE in the presence of peptides containing 15-50 consecutive proline residues.

AB - Human BChE (butyrylcholinesterase) protects against the toxicity of organophosphorus nerve agents and pesticides. BChE purified from human plasma is limited and pathogen carry-over is a concern. Unlike the native BChE tetrameric complex with a residence time of days, rBChE (recombinant BChE) is produced predominantly as dimers andmonomers that are cleared from the circulation within minutes. Assembly into tetramers requires incorporation of proline-rich peptides, a process that was thought to occur intracellularly. Our goal was to determine whether polyproline added to rBChE under cell-free conditions would promote tetramerization. Secreted rBChE was purified by procainamide affinity chromatography, and synthetic polyprolines (8-mer to 300-mer) were tested to determine their effect on tetramer assembly. These studies demonstrated that 90-98% of purified rBChE (65 μM) could be assembled into tetramers when incubated with synthetic 17-mer or 50-mer polyproline peptides (100 μM) for 1.5 h at 25°C. However, rBChE tetramerizationwas inefficient with smaller 8-mer polyproline peptides and larger 300-mer polyproline proteins. Collectively, these studies demonstrated that the eukaryotic cellularmachinery is not required for assembly of active BChE into tetramers and that this process can occur in vitro with purified rBChE in the presence of peptides containing 15-50 consecutive proline residues.

KW - Enzyme stability

KW - Human butyrylcholinesterase

KW - Polyproline peptide

KW - Recombinant expression

KW - Tetrameric bioscavenger

UR - http://www.scopus.com/inward/record.url?scp=84905910734&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84905910734&partnerID=8YFLogxK

U2 - 10.1042/BJ20140421

DO - 10.1042/BJ20140421

M3 - Article

C2 - 24916051

AN - SCOPUS:84905910734

VL - 462

SP - 329

EP - 335

JO - Biochemical Journal

JF - Biochemical Journal

SN - 0264-6021

IS - 2

ER -