Polyproline-rich peptides associated with Torpedo californica acetylcholinesterase tetramers

Abstract

Acetylcholinesterase (AChE) terminates cholinergic neurotransmission by hydrolyzing acetylcholine. The collagen-tailed AChE tetramer is a product of 2 genes, ACHE and ColQ. The AChE tetramer consists of 4 identical AChE subunits and one polyproline-rich peptide, whose function is to hold the 4 AChE subunits together. Our goal was to determine the amino acid sequence of the polyproline-rich peptide(s) in Torpedo californica AChE (TcAChE) tetramers to aid in the analysis of images that will be acquired by cryo-EM. Collagen-tailed AChE was solubilized from Torpedo californica electric organ, converted to 300 kDa tetramers by digestion with trypsin, and purified by affinity chromatography. Polyproline-rich peptides were released by denaturing the TcAChE tetramers in a boiling water bath, and reducing disulfide bonds with dithiothreitol. Carbamidomethylated peptides were separated from TcAChE protein on a spin filter before they were analyzed by liquid chromatography tandem mass spectrometry on a high resolution Orbitrap Fusion Lumos mass spectrometer. Of the 64 identified collagen-tail (ColQ) peptides, 60 were from the polyproline-rich region near the N-terminus of ColQ. The most abundant proline-rich peptides were SVNKCCLLTPPPPPMFPPPFFTETNILQE, at 40% of total mass-spectral signal intensity, and SVNKCCLLTPPPPPMFPPPFFTETNILQEVDLNNLPLEIKPTEPSCK, at 27% of total intensity. The high abundance of these 2 peptides makes them candidates for the principal form of the polyproline-rich peptide in the trypsin-treated TcAChE tetramers.

Keywords: Mass spectrometry; Polyproline; Tetramer; Torpedo californica acetylcholinesterase.

Figure 1.

Schematic diagram of the structure of collagen-tailed asymmetric AChE at the neuromuscular junction. The collagen tail has a polyproline-rich region near its N-terminus whose function is to organize 4 subunits of AChE into a tetramer [9]. The polyproline-rich region of ColQ is not seen in this figure because it is embedded within the tetramers. Stoichiometry experiments demonstrated that one polyproline-rich peptide associates with 4 AChE subunits [10] to make a stable AChE tetramer. Figure taken from [10].

Figure 2.

Isolation of TcAChE tetramers by size exclusion chromatography (SEC). Fractions eluting from the SEC column were monitored at 280 nm. The figure was generated with UNICORN 5.11 (Build 407) software. The peak at 9.67 mL contains tetrameric TcAChE.

Figure 3.

Non-denaturing polyacrylamide gel stained for activity with acetylthiocholine. Lane 1) Fetal bovine serum (Gibco) is a rich source of tetrameric AChE. Lanes 2, 3, 4) The TcAChE sample migrates to the same position as the fetal bovine serum tetrameric AChE; Lane 5) HuBChE tetramers hydrolyze acetylthiocholine, but migrate to a position different from that of the AChE tetramers. The gel thus confirms that the TcAChE sample is a tetramer.

Figure 4.

SDS gel stained with Coomassie blue. The highly purified TcAChE sample displays an intense band at 75 kDa corresponding to the denatured, reduced monomer, and a barely detectable band for a non-reducible dimer at 150 kDa.

Figure 5.

ColQ sequence accession number Q03637, showing peptides originating from TcAChE tetramers. Boxed residues 64–112 include the polyproline-rich region whose function is to assemble 4 subunits into a tetramer. Boxed residues 307–341 are from the non-polyproline region of ColQ.

Figure 6.

MS/MS fragmentation spectrum of the polyproline-rich peptide SVNKCCLLTPPPPPMFPPPF. The mass, sequence, and ion type are assigned for each y-ion and b-ion peak. An internal fragment, PPFMPPP, at 764.38 has fragments at 667.33, 570.28 and 195.11. All other major peaks can be assigned to loss of water (18 Da), loss of CO (−28 Da), or to immonium ions.

Figure 7.

Cryo-EM structure of soluble HuBChE tetramers. One polyproline peptide organizes 4 subunits into a stable tetramer by non-covalent interactions with tryptophan residues in the tetramerization domain. PDB code 612T. Figure taken from [18]