Tetramer organizing polyproline-rich peptides identified by mass spectrometry after release of the peptides from Hupresin-purified butyrylcholinesterase tetramers isolated from milk of domestic pig (Sus scrofa)

Milk of the domestic pig has 10 times more butyrylcholinesterase (BChE) per mL than porcine serum. We purified BChE from porcine milk by affinity chromatography on Hupresin-Sepharose. The pure porcine BChE (PoBChE) was a tetramer with a molecular weight of 340,000, similar to that of human BChE tetramers. The C-terminal 40 residues of PoBChE constitute the tetramerization domain. The glue that holds the 4 BChE subunits together is a polyproline-rich peptide. Mass spectrometry analysis of trypsin-digested PoBChE identified a variety of polyproline-rich peptides originating from 12 different proteins. The donor proteins exist in the nucleus or cytoplasm of cells and contribute their polyproline-rich peptides after a cell is degraded. The secreted PoBChE scavenges the polyproline-rich peptides and incorporates one polyproline peptide per PoBChE tetramer, where the polyproline peptide is bound noncovalently but very tightly with an estimated dissociation constant of 10–12 M. The most abundant polyproline-rich peptides were derived from acrosin, homeobox protein HoxB4, lysine-specific demethylase 6B, proline-rich protein 12, and proline-rich membrane anchor 1 (PRiMA). The research article associated with the data in this report can be found in Saxena et al. (2018). The Data in Brief report lists all the polyproline-rich peptides identified in PoBChE tetramers.


a b s t r a c t
Milk of the domestic pig has 10 times more butyrylcholinesterase (BChE) per mL than porcine serum. We purified BChE from porcine milk by affinity chromatography on Hupresin-Sepharose. The pure porcine BChE (PoBChE) was a tetramer with a molecular weight of 340,000, similar to that of human BChE tetramers. The C-terminal 40 residues of PoBChE constitute the tetramerization domain. The glue that holds the 4 BChE subunits together is a polyproline-rich peptide. Mass spectrometry analysis of trypsin-digested PoBChE identified a variety of polyproline-rich peptides originating from 12 different proteins. The donor proteins exist in the nucleus or cytoplasm of cells and contribute their polyproline-rich peptides after a cell is degraded. The secreted PoBChE scavenges the polyproline-rich peptides and incorporates one polyproline peptide per PoBChE tetramer, where the polyproline peptide is bound noncovalently but very tightly with an estimated dissociation Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/dib constant of 10 -12 M. The most abundant polyproline-rich peptides were derived from acrosin, homeobox protein HoxB4, lysinespecific demethylase 6B, proline-rich protein 12, and proline-rich membrane anchor 1 (PRiMA). The research article associated with the data in this report can be found in Saxena et al. (2018) Value of the data The finding that PoBChE is abundant in porcine milk [1] and in human milk [2,3] leads to new questions. What is the function of BChE in milk? Does the infant benefit from BChE in mother's milk as a consequence of the inactivation of octanoyl-ghrelin by BChE, thus reducing the infant's anxiety and stress [4]? Ghrelin has a role in regulation of neural circuits and body growth during neonatal development [5]. Is BChE in milk involved in these ghrelin-related effects?
The set of polyproline-rich peptides in PoBChE tetramers is different from the set in human BChE tetramers [6,7]. PoBChE was purified from milk. Human BChE was purified from plasma. Porcine plasma has a very low amount of BChE. Since incorporation of a polyproline-rich peptide stabilizes the BChE tetramer, is the limiting factor for the level of BChE the availability of polyproline-rich peptides? Does it mean the cells in the porcine mammary gland (source of milk BChE) undergo a higher rate of degradation and therefore produce more polyproline-rich peptides than cells in porcine liver (source of plasma BChE)?
Are excess polyproline-rich peptides toxic to cells? Does BChE incorporate polyproline peptides from degraded cells because degraded cells are a convenient source? If excess polyproline-rich peptides are toxic to cells, BChE might be protecting the organism by scavenging these peptides.
The BChE tetramer incorporates not only short polyproline-rich peptides, but also long proteins that contain a polyproline-rich region. An example is the C5 variant of human BChE whose tetrameric structure includes a 60 kDa lamellipodin protein [8]. Does the BChE tetramer, and by implication the AChE tetramer, serve as a carrier of proteins that confer the observed noncholinergic functions of BChE and AChE in bone development [9]? Examples of polyproline-rich donor proteins that could account for non-cholinergic functions of BChE and AChE include the Homeobox protein Hox-B4 which is a transcription factor involved in development, and the Formin-homology domain containing protein 1 which is involved in cell migration and adhesion.
The ability of BChE subunits 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 tag.

Data overview
We present all the polyproline-rich peptides associated with PoBChE tetramers that were identified by mass spectrometry. In addition we show the complete amino acid sequence of the full-length protein that donated each polyproline-rich peptide, the location of the polyproline-rich peptide within the full-length protein sequence, and the abundance of the polyproline-rich peptide relative to PoBChE peptides. A brief description is given of the function of the donor protein and its location within a cell.

Sample preparation for LC-MS/MS
The Hupresin-purified PoBChE was reduced in volume from 7.3 mL to 0.17 mL in a Centricon YM-30 spin filter. After the buffer was changed to 10 mM ammonium bicarbonate pH 8, the PoBChE was deglycosylated with 1 ml of PNGaseF for 1 h. Noncovalently bound polyproline-rich peptides were released from PoBChE by denaturing the protein in a boiling water bath for 5 min. The denatured 170 mg of PoBChE protein in 170 mL of 10 mM ammonium bicarbonate pH 8 was digested with 2 mg of trypsin (Promega V511C, 2 mg in 5 mL) for 20 h at 37°C in a humidified chamber. Particles that could clog the small diameter tubing in the Ultra High Pressure Liquid Chromatography column were removed by centrifuging the digest for 30 min at 14,000 rpm in a microfuge. A 10 mL aliquot from the top of the centrifuged digest was transferred to an autosampler vial. The protein concentration in the digest was estimated at 1 mg/mL.

LC-MS/MS
The protocol for liquid chromatography tandem mass spectrometry (LC-MS/MS) is described in detail in [11]. In brief, peptides in a 5 mL volume were separated on a cHiPLC Nanoflex microchip column (Eksigent, Dublin, CA) packed with ChromXP C18. The eluted peptides were electrosprayed into the 6600 Triple-TOF mass spectrometer (AB Sciex) where mass spectra were collected in positive mode. Peptides were fragmented by collision-induced dissociation with nitrogen gas. The Triple-TOF data were searched against the NCBInr 15Sep2014 database for Sus Scrofa proteins using the Paragon algorithm from Protein Pilot (AB Sciex) (Tables 1-12).
27 residues PAPPPAPPPPPPPPPPPPPPPPPPPQQ MW 2648.4. Acrosin is the major proteinase present in the acrosome of mature spermatozoa. It is a typical serine proteinase with trypsin-like specificity. It is stored in the acrosome in its precursor form, proacrosin. The active enzyme functions in the lysis of the zona pellucida, thus facilitating penetration of the sperm through the innermost glycoprotein layers of the ovum. The mRNA for proacrosin is synthesized only in the postmeiotic stages of spermatogenesis. In humans proacrosin first appears in the haploid spermatids. https://www.gtexportal.org/home/gene/ACR The human acrosin gene is expressed in testis and to a small extent in mammary breast tissue, lung, spleen, adipose tissue, and tibial artery. The HOXB4 gene is a member of the Antp homeobox family and encodes a nuclear protein with a homeobox DNA-binding domain. It is included in a cluster of homeobox B genes located on human chromosome 17. The encoded protein functions as a sequence-specific transcription factor that is involved in development. Intracellular or ectopic expression of this protein expands hematopoietic stem and progenitor cells in vivo and in vitro, making it a potential candidate for therapeutic stem cell expansion.    This gene encodes a member of a family of CCCH (C-x8-C-x5-C-x3-H type) zinc finger domaincontaining proteins. These zinc finger domains, which coordinate zinc finger binding and are characterized by three cysteine residues and one histidine residue, are nucleic acid-binding. Other family members are known to function in post-transcriptional regulation.       Total peptide count 6. 6 WAS/WASL-interacting protein family member 2 isoform X1 peptides/10987 PoBChE peptides ¼ 0.05%. 16 residues PPPPLPPPPPPPPPPR MW 1645.9. Required to anchor acetylcholinesterase (ACHE) to the basal lamina of the neuromuscular junction and to the membrane of neuronal synapses in brain. Also able to organize ACHE into tetramers.