Polyproline tetramer organizing peptides in fetal bovine serum acetylcholinesterase

Abstract

Acetylcholinesterase (AChE) in the serum of fetal cow is a tetramer. The related enzyme, butyrylcholinesterase (BChE), in the sera of humans and horse requires polyproline peptides for assembly into tetramers. Our goal was to determine whether soluble tetrameric AChE includes tetramer organizing peptides in its structure. Fetal bovine serum AChE was denatured by boiling to release non-covalently bound peptides. Bulk protein was separated from peptides by filtration and by high performance liquid chromatography. Peptide mass and amino acid sequence of the released peptides were determined by MALDI-TOF-TOF and LTQ-Orbitrap mass spectrometry. Twenty polyproline peptides, divided into 5 families, were identified. The longest peptide contained 25 consecutive prolines and no other amino acid. Other polyproline peptides included one non-proline amino acid, for example serine at the C-terminus of 20 prolines. A search of the mammalian proteome database suggested that this assortment of polyproline peptides originated from at least 5 different precursor proteins, none of which were the ColQ or PRiMA of membrane-anchored AChE. To date, AChE and BChE are the only proteins known that include polyproline tetramer organizing peptides in their tetrameric structure.

Figure 1

MALDI mass spectrum of the peptides released from FBS AChE by boiling. Peptides were separated from bulk protein by filtration through a YM10 membrane (10,000 MW cutoff). The right-hand y-axis indicates the signal intensity in counts-per-second.

Figure 2

MALDI MSMS spectrum of the 1885.0 Da, 19-proline peptide (PPP)PP PPPPP PPPPP PPPP Na⁺ that was isolated from boiled FBS AChE by HPLC. Amino acid sequences are marked to show the y-ion series and a-ion series. The left-hand y-axis shows the signal intensity as a percentage of the most intense peak. The maximum value has been set to 40% to accentuate the minor peaks. Consequently, the most intense peaks (those at high mass) have been truncated. The right-hand y-axis indicates the signal intensity in counts-per-second. Ions from sodium-promoted fragmentation are identified by an asterisk (*). The immonium ion of proline is at 70 Da and a characteristic ion for proline is at 126 Da. The mass at 23 Da is sodium.

Figure 3

MALDI MSMS spectrum of the 1721.9 Da peptide PPPQP PPPPP PPPPP PP Na⁺ isolated from boiled FBS AChE by HPLC. The right-hand y-axis indicates the signal intensity in counts-per-second. Ions from sodium-promoted fragmentation are identified by an asterisk (*). The immonium ion of proline is at 70 Da. The sodium atom is at 23 Da.

Figure 4

Orbitrap MSMS spectrum of peptide PPPPP PPPPP PPP isolated from boiled FBS AChE by HPLC. The doubly-charged parent ion has a mass of 640.85 m/z. Amino acid sequences are marked to show singly-charged y-ion series, singly-charge b-ion series, a doubly-charged y-ion, and a doubly-charged b-ion series. The y-axis shows the signal intensity relative to the most intense peak in the spectrum.

Figure 5

Four identical subunits of fetal bovine serum AChE (accession number gi 108493 and gi 115497516) are linked by interactions with a polyproline peptide in the center of the tetramer. Dimers of AChE are formed by interchain disulfide bonds at Cysteine 580. The close contact between the globular subunits is intended to show that interactions between subunits contribute to the stability of the tetramer. Each AChE subunit contains 583 amino acids. The MW of the glycosylated tetramer is 340 kDa.

Figure 6

Model of the AChE tetramer in the presence of the full-length ColQ protein. AChE monomers are displayed as globular units with their tryptophan-rich, C-terminal, amphiphilic α-helices protruding out from the top of each unit. The ColQ protein, or alternatively the tetramer organizing polyproline peptide (yellow), projects through the middle of the AChE tetramer complex. The N-terminus of ColQ interacts with the C-terminal tetramerization domain of AChE (at the top) and extends as a single chain through the bottom of the complex. Figure reproduced from [5].