Butyrylcholinesterase-Protein Interactions in Human Serum

Abstract

Measuring various biochemical and cellular components in the blood is a routine procedure in clinical practice. Human serum contains hundreds of diverse proteins secreted from all cells and tissues in healthy and diseased states. Moreover, some serum proteins have specific strong interactions with other blood components, but most interactions are probably weak and transient. One of the serum proteins is butyrylcholinesterase (BChE), an enzyme existing mainly as a glycosylated soluble tetramer that plays an important role in the metabolism of many drugs. Our results suggest that BChE interacts with plasma proteins and forms much larger complexes than predicted from the molecular weight of the BChE tetramer. To investigate and isolate such complexes, we developed a two-step strategy to find specific protein-protein interactions by combining native size-exclusion chromatography (SEC) with affinity chromatography with the resin that specifically binds BChE. Second, to confirm protein complexes' specificity, we fractionated blood serum proteins by density gradient ultracentrifugation followed by co-immunoprecipitation with anti-BChE monoclonal antibodies. The proteins coisolated in complexes with BChE were identified by mass spectroscopy. These binding studies revealed that BChE interacts with a number of proteins in the human serum. Some of these interactions seem to be more stable than transient. BChE copurification with ApoA-I and the density of some fractions containing BChE corresponding to high-density lipoprotein cholesterol (HDL) during ultracentrifugation suggest its interactions with HDL. Moreover, we observed lower BChE plasma activity in individuals with severely reduced HDL levels (≤20 mg/dL). The presented two-step methodology for determination of the BChE interactions can facilitate further analysis of such complexes, especially from the brain tissue, where BChE could be involved in the pathogenesis and progression of AD.

Keywords: ApoA-I; BChE; butyrylcholinesterase; high-density lipoprotein (HDL); protein interactions; pseudocholinesterase.

Figure 1

Isolation of the BChE–protein complexes by size-exclusion chromatography (SEC) of serum proteins. (A) Chromatogram of human serum proteins separated on the TSK gel 3000 SWXL (Tosoh, Tokyo, Japan). Protein elution was monitored at 280 nm. The first 13 fractions of 200 µL were collected every 12 s in separate tubes. (B) Determination of the BChE activity in the fractions by Ellman’s assay in the collected fractions. The BChE activity is shown as dA412/min in red numbers above corresponding bars. dA412/min stands for change in absorbance at 412 nm per minute. (C) Analysis of the protein complexes in the 13 fractions by native polyacrylamide gel electrophoresis on Mini-PROTEAN 4–15% TGX Stain-Free Gels (Bio-Rad, Hercules, CA, USA). Ten microliters of each fraction was separated under native conditions in TB buffer at 4 °C. (D) SDS-PAGE analysis of the protein complexes in the 13 fractions. Ten microliters of each fraction sample was separated on Mini-PROTEAN 4–15% precast TGX Stain-Free gels (Bio-Rad, Hercules, CA, USA). M—protein marker; 1–13—10 µL of fractions. (E) Western blot analysis of 13 fractions using mouse monoclonal anti-BChE antibody (D-5): sc-377403 Santa Cruz Biotechnology (Santa Cruz, CA, USA) (1/200 dilution). (F) Western blot analysis of the fractions using rabbit monoclonal anti-ApoA-I antibody (ab52945) (Abcam, Cambridge, UK) (1/2000 dilution). (G) Western blot analysis of the fractions using rabbit monoclonal anti-ApoB antibody (ab139401) (Abcam, Cambridge, UK) (1/2000 dilution).

Figure 2

Scheme of the isolation of the BChE–protein complexes by affinity chromatography on Hupresin AC Sepharose. Proteins from fractions 6, 8, and 10 were applied on Hupresin AC Sepharose (Chemforase, Mont-Saint-Aignan, France). The eluted proteins in complexes were identified by mass spectrometry. The tables list the first 20 most abundant proteins identified in the complexes, sorted according to the score number. For each identified protein, Mascot software calculates a score. This number reflects the combined scores of all observed mass spectra that can be matched to amino acid sequences within that protein. The score in the table is a qualitative measure, not quantitative. The higher the score, the more reliable the identification. Figure 2 was created with BioRender.com (access on 15 March 2021).

Figure 3

Fractionation of BChE–protein complexes by density iodixanol gradient ultracentrifugation (OptiPrep). (A) The separation procedure is summarized in the flow chart. (B) Picture of the centrifuged tube taken after separation. Positions of fractions are marked on the tube. (C) BChE activity of 10 uL of each fraction was estimated by Ellman’s assay as it has been described in Figure 1B. The BChE activity is shown as dA412/min. (D) Sudan black stained agarose gel electrophoresis profile of separated fractions after electrophoresis. Ellman’s reaction was developed in 100 mM PB buffer (pH 7.4) with a final concentration of 0.5 mM DTNB and 5 mM BTC. S—1 µL of serum; 4–15—1 µL of fractions. (E) SDS-PAGE; 1 µL of the fraction samples were separated on Mini-PROTEAN 4–15% precast TGX Stain-Free gels (Bio-Rad, Hercules, CA, USA). SDS-PAGE and Western blotting were performed according to the method described in Figure 1D. S—1 µL of serum; M—protein marker; 4–15—1 µL of fractions. (F) Western blot analysis of the fractions using rabbit monoclonal anti-ApoB antibody (ab139401) (Abcam, Cambridge, UK) (1/2000 dilution). (G) Western blot analysis of the fractions using rabbit monoclonal anti-ApoA-I antibody (ab52945) (Abcam, Cambridge, UK) (1/2000 dilution). (H) Western blot analysis of the fractions using mouse monoclonal anti-BChE antibody (D-5): sc-377403 Santa Cruz Biotechnology (Santa Cruz, CA, USA)(1/200 dilution). (I) Western blot analysis of the fractions using rabbit monoclonal anti-vitronectin antibody (ab46808) (Abcam, Cambridge, UK) (1/2000 dilution).

Figure 4

Scheme of the isolation of protein complexes by co-immunoprecipitation, which is a common approach to study protein–protein interactions that uses an antibody to immunoprecipitate the antigen (bait protein) and co-immunoprecipitate any interacting proteins (prey proteins). Isolation of native protein complexes from fraction 12 was performed using immobilized monoclonal antibodies on agarose support according to the instruction of the Thermo Scientific Pierce Co-Immunoprecipitation (Co-IP) Kit (Thermo Fisher Scientific, Waltham, MA, USA). The proteins in complexes were eluted and identified by mass spectrometry. The table lists the first 20 most abundant proteins identified in complexes, sorted according to the score number from the Mascot server. The score in the table is a qualitative measure, not quantitative. The higher the score, the more reliable the identification. The proteins that have also been pulled down using Hupresin AC Sepharose are listed in the table in Figure 2 in bold text. The control resin table lists proteins nonspecifically interacting with the control quenched antibody coupling resin. The proteins identified in both tables (with anti-BChE antibody and in control) are marked in red. Keratin and the immune system proteins seem to bind nonspecifically to the resins in the above pull-down experiments. Figure 4 was created with BioRender.com (access on 15 March 2021).

Figure 5

Interrelationship of BChE activity and serum HDL level. (A) Each black dot represents the enzyme activity of a single individual. BChE activity was measured by Ellman’s assay as described previously [34]. (B) Subjects were divided into 2 groups according to HDL levels. One group (n = 38) with HDL level ≤20 mg/dL, the second group (n = 35) with HDL level >20 mg/dL; mean values with error bars representing standard deviation (SD), four asterisks (****) represent p-value < 0.0001 calculated using the t-test.