Polyproline-rich Peptides Organize 4 Cholinesterase 2 Subunits into A Tetramer; BChE and AChE Scavenge 3 Polyproline Peptides Released during Metabolic 4 Turnover 5

: The genes for acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) encode the 11 proteins responsible for enzyme activity. Additional gene products, PRiMA and ColQ, anchor 12 AChE and BChE proteins into membranes. Soluble AChE and BChE tetramers are composed of 4 13 identical subunits plus one polyproline-rich peptide. Dilution does not release the polyproline-14 rich peptide from tetramers. However, protein denaturation, for example heating in a boiling 15 water bath, dissociates the polyproline-rich peptide. Using mass spectrometry to sequence 16 peptides released from soluble AChE and BChE tetramers, we find sequences that correspond to 17 proline-rich regions from a variety of proteins. A typical peptide sequence contains 20 consecutive 18 prolines in a 23-residue peptide LPPPPPPPPPPPPPPPPPPPPLP. There is no single, common 19 consensus sequence i.e., no specific gene appears to be responsible for the polyproline-rich peptides 20 found in soluble AChE and BChE tetramers. We propose that during metabolic turnover, protein 21 fragments containing polyproline-rich sequences are scavenged by AChE and BChE dimers, to 22 make stable AChE and BChE tetramers. The 40-residue, alpha-helical C-terminus of AChE or 23 BChE is the tetramerization domain that binds the polyproline-rich peptide. Four parallel alpha 24 helices wrap around a single antiparallel polyproline peptide to lock the tetramer in place. This 25 organization was established by classical X-ray crystallography for isolated C-termini in complex 26 with a proline-rich peptide. The organization was confirmed for intact, tetrameric human BChE 27 using cryoelectron microscopy. When 40 amino acids are deleted from the carboxy terminus, 28 monomeric enzymes are created that retain full enzymatic activity. 29


Introduction
Butyrylcholinesterase (P06276) in human plasma is stable in the circulation with a half-life of 11 days [1].Its stability is attributed to several factors including a) its large size of 340 kDa, b) the fact that it is sugar coated with 36 N-linked glycans per tetramer [2,3], c) it is resistant to proteolysis, and d) it is a tetramer.The focus of this review is the tetramer organization of butyrylcholinesterase (BChE).Soluble BChE and acetylcholinesterase (AChE) are assembled into tetramers through interaction of 4 tetramerization domains with one polyproline-rich peptide [4,5].This motif for tetramerization is unique for the cholinesterases as of the year 2020, but future studies may find it in other protein tetramers.

Tetramers are the product of more than one gene
The coding sequence for the 85 kDa monomer of human BChE (P06276) is on chromosome 3q26 [6] and for the 70 kDa monomer of human AChE (P22303) on chromosome 7q22 [7].Monomeric proteins with these sequences have full enzyme activity, but they are unstable in the circulation because they are not tetramers.Assembly into tetramers requires additional gene products.The membrane bound forms of BChE and AChE use polyproline-rich regions of ColQ and PRiMA to assemble into tetramers.The tail end of these polyproline-rich proteins anchor BChE and AChE into the basal lamina at neuromuscular junctions or to membranes in the brain [8,9].In contrast, no specific gene encodes the polyproline-rich peptides found in soluble BChE and AChE tetramers.
The soluble BChE and AChE tetramers assemble around any polyproline-rich peptide, regardless of its origin or length as long as the peptide has at least 12 residues.An example is the 15 residue LLTPPPPPLFPPPFF of ColQ [10].Polyproline peptides purchased from Sigma-Aldrich with molecular weights from 2000 to 5000 convert recombinant BChE monomers and dimers into tetramers [11,12].

Tetramerization domain
The tetramerization domain of soluble BChE and AChE tetramers is located at the C-terminus and is encoded by a separate exon.The sequence of the 40-residue BChE tetramerization domain is NIDEAEWEWKAGFHRWNNYMMDWLNQFNDYTSKKESC571VGL.The tetramerization domain forms an alpha helix [4,13].Two alpha helices are linked through a disulfide bond at Cysteine 599 (C571 in the mature secreted BChE).This disulfide bond is the only disulfide bond between subunits [14].The BChE tetramer is a dimer of two disulfide-linked dimers containing a 4-helix bundle at the interface between 2 monomers [4].Four tetramerization domains assemble in a superhelical, coiled-coil structure around a central polyproline II helix, as in Figure 1.The polyproline peptide is tightly bound via hydrophobic stacking with tryptophans and by hydrogen bonds [4,13] .

Mass spectrometry identification of tetramer organizing peptides
We have identified polyproline-rich peptides in BChE tetramers isolated from human plasma, equine plasma, porcine milk, and from recombinant human BChE expressed in Chinese Hamster Ovary Cells [15][16][17][18][19].In all cases the polyproline peptides were bound noncovalently.Polyproline peptides remained tightly bound in dilute protein solutions, but were released when the proteins were denatured in a boiling water bath.The sequences of the released polyproline peptides were determined by mass spectrometry.Figure 2 shows the masses and sequences of 10 polyproline-rich peptides released from human BChE tetramers.Reproduced from [18].

Polyproline-rich peptides in soluble BChE tetramers.
Table 1 shows that the polyproline peptides in human plasma BChE tetramers originate from 13 different proteins [18].Lamellipodin contributes 70% of the polyproline peptides.Lamellipodin donates 39 polyproline peptides, ranging in length from 11 to 29 residues, to human plasma BChE tetramers.Short peptides can be derived from longer peptides by losing amino acids through the action of N-and C-terminal aminopeptidases and carboxypeptidase [20].The longest observed peptide associated with a specific donor protein is listed in Table 1.In some cases polyproline peptides could be matched to more than one donor protein.The short LPPPP PPPPP P peptide was matched to 27 different proteins.Polyproline-rich peptides consist predominantly of prolines and often include leucine, alanine, serine or glutamine, but never tryptophan.Recombinant human BChE tetramers expressed in Chinese Hamster Ovary cells (Cricetulus griseus) were purified and analyzed for polyproline peptides.The goal was to determine whether polyproline peptide sequences are specific to the BChE protein or to the cells that synthesize BChE.
We identified 60 protein donors of the polyproline peptides in recombinant BChE tetramers [15].
The 60 donor proteins are all Chinese Hamster Ovary (Cricetulus griseus) proteins.Despite their origin from a nonhuman species, the polyproline peptides were incorporated into recombinant human BChE.Five donor proteins from Chinese Hamster Ovary cells were also donor proteins for human plasma BChE synthesized in the liver.The names and accession numbers of the 5 common donor proteins are listed in Table 3. (Q6ZPZ3 Mus musculus) From [15].Two proteins have accession numbers for Mus musculus because the Cricetulus griseus database is incomplete.
No donor protein contributed the majority of polyproline-rich peptides to recombinant human BChE tetramers expressed in Chinese Hamster Ovary cells.This contrasts with BChE tetramers purified from human plasma, where 70% of the tetramer-organizing peptides were traced to lamellipodin.It was concluded that polyproline peptide sequences in human BChE tetramers are specific to the cells that synthesize BChE and are not specific to the BChE protein.

Polyproline-rich peptides in soluble AChE tetramers.
Purified fetal bovine serum AChE tetramers released polyproline-rich peptides [22] from the 5 donor proteins listed in Table 4.All 5 of these proteins are also donors for the peptides in human plasma BChE tetramers.

BChE and AChE scavenge polyproline peptides released from proteins in the cytoplasm, nucleus, endoplasmic reticulum, extracellular space, and cell membrane
Tetramer-organizing polyproline-rich peptides derive from a large number of proteins that reside in a variety of cell compartments including the cytoplasm, nucleus, endoplasmic reticulum, extracellular space, and cell membrane.For example, lamellipodin resides on the cytoplasm side of the cell membrane.Homeobox protein Hox-B4 resides in the nucleus.BChE is secreted through the Golgi apparatus and is never in the cytoplasm or the nucleus.Another fact to consider is that human BChE dimers are converted to human BChE tetramers upon addition of polyproline peptides from Sigma-Aldrich [23].This was demonstrated for mouse plasma.The human BChE dimers had been produced in mouse plasma by injecting mice with an adenovirus vector encoding human BChE [23].
Exogenously added polyproline peptides became incorporated to form BChE tetramers.
The AChE tetramer in fetal bovine serum, like the BChE tetramer in human serum, incorporates polyproline peptides from a variety of protein donors.These observations lead to the conclusion that polyproline peptides are released from cellular proteins during metabolic turnover.The peptides circulate in the blood.Before the peptides reach the kidney they are taken up by newly synthesized BChE and AChE subunits.This process defines a new function for BChE and AChE, that of scavenging polyproline-rich peptides.

Conclusions
Soluble BChE and AChE are peptide scavengers.They scavenge polyproline-rich peptides that are released during cell degradation.This is a newly defined function of soluble BChE and AChE.If The BChE tetramer incorporates not only short polyproline-rich peptides, but also long protein fragments that contain a polyproline-rich region.An example is the C5 variant of human BChE whose tetrameric structure includes a 60 kDa lamellipodin fragment [24].The ability of BChE monomers to assemble into stable, long-lived tetramers by binding the polyproline-rich region of a protein, suggests that BChE could serve as a delivery vehicle for any protein that has been engineered to include a polyproline-rich peptide tag.
AChE and BChE have non-cholinergic functions in bone development [25].A possible explanation for their non-cholinergic function is that AChE and BChE tetramers serve as carriers of proteins that confer the non-cholinergic function.
Author Contributions: OL and LMS; writing-review and editing.All authors have read and agreed to the published version of the manuscript.

Figure 1 .
Figure 1.Cryo-EM structure of the BChE tetramer purified from human plasma.PDB code 6i2t.

Figure
Figure from reference[4].Four identical subunits, each composed of 574 amino acids and 9 N-linked glycans, assemble into a tetramer in the presence of a polyproline-rich peptide.Assembly into tetramers does not occur when polyproline peptides are unavailable.

Figure 2 .
Figure 2. MALDI-TOF spectrum of polyproline-rich peptides released from human plasma BChE tetramers by denaturing the pure BChE protein in a boiling water bath.All ten peptides match human lamellipodin (Q70E73).Reproduced from [18].

Figure 3 .
Figure 3. MS/MS fragmentation spectrum of the 29-residue peptide PSPPL PPPPP PPPPP PPPPP PPPPP LPSQ released from the human plasma BChE tetramer.The quadruply-charged parent ion excess polyproline-rich peptides are toxic to cells, then scavenging activity protects the cells.Polyproline-rich peptides in BChE and AChE tetramers originate from a variety of proteins that reside in the cytoplasm, nucleus, endoplasmic reticulum, and cell membrane.Secreted BChE and AChE have no access to proteins in the cytoplasm and nucleus.During cell degradation peptides are released to the circulation, where they are scavenged by newly synthesized BChE and AChE monomers.Soluble BChE and AChE tetramers are not degradation products of membrane bound BChE and AChE.The evidence for this statement is that their polyproline peptides derive primarily from lamellipodin and not from ColQ and PRiMA polyproline peptides.

Table 1 .
[16]n protein donors for polyproline-rich peptides released from serum BChE A. A) Data from[18].B)The longest observed peptide in a family of related peptides is listed for each protein donor.Two peptides are listed when a protein has two different polyproline-rich regions.C) Pept# is the number of different observed peptides that match to fragments from the Observed Peptide.D) Spectral Count is the total number of times that polyproline peptides from this Protein Donor appeared in the mass spectral data.E) Prot Match indicates the number of proteins that contain the Observed Peptide.Polyproline peptides in equine plasma BChE tetramers originate from 12 proteins, of which 8 proteins have a match in the mammalian taxonomy, but 4 have no perfect match[16].Some polyproline sequences could be matched to more than one protein.For example a string of 21 contiguous prolines fits both UDP-N-acetylglucosamine transferase subunit ALG13 homolog and formin-like protein 2-like in the Equus caballus taxonomy.Polyproline peptides originating from lamellipodin were present in both equine and human plasma BChE tetramers.
[19,21].The most frequent donors are lysine-specific demethylase 6B, acrosin, proline-rich protein 12, and homeobox protein hox-B4.No polyproline peptides from lamellipodin were found in BChE tetramers of porcine milk.The protein donors of polyproline-rich peptides in BChE tetramers from porcine milk are not identical to those in BChE tetramers from human plasma, though 3 protein donors appear in both Tables1 and 2. They are homeobox protein HoxB4, Zinc finger homeobox protein 4, and Zinc finger CCCH domain-containing protein 4.

Table 2 .
Protein [19]rs for polyproline-rich peptides released from porcine milk BChE A. A) Data from[19].B) A composite of observed peptides from a family of related peptides for each protein donor.Two peptides are listed when two different proline-rich peptides appear in one protein.C) Pept# is the number of different peptides that match to fragments from the Observed Peptide.D) Spectral Count is the total number of times that polyproline peptides associated with this Protein Donor appeared in the mass spectral data.

Table 3 .
Five donor proteins in common between recombinant human BChE tetramers expressed inChinese Hamster Ovary cells (Cricetulus griseus) and human plasma BChE tetramers synthesized in human liver.