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Enzymolysis of high density lipoprotein with a combination of membrane-immobilized esterase and trypsin
Journal of Pharmaceutical and Biomedical Analysis 71 (2012) 179–182
Contents lists available at SciVerse ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Short communication
Enzymolysis of high density lipoprotein with a combination of membrane-immobilized esterase and trypsin Takahiro Sakikawa, Youji Shimazaki ∗ Graduate School of Science and Engineering (Science Section) and Venture Business Laboratory, Ehime University, Matsuyama 790-8577, Japan
a r t i c l e
i n f o
Article history: Received 11 April 2012 Received in revised form 24 July 2012 Accepted 29 July 2012 Available online 4 August 2012 Keywords: HDL Carboxylesterase Trypsin Electrophoresis MALDI-TOF MS 6,9-Diamino-2-ethoxyacridine
a b s t r a c t Apolipoprotein A-1 (apo A-1), a major component of high density lipoprotein (HDL), was efficiently digested by membrane-immobilized trypsin after HDL was treated with membrane-immobilized esterase. Compared to treatment with membrane-immobilized trypsin alone, the relative amounts of apo A-1 polypeptides, m/z 1723.78 and m/z 1568.82, increased by 2.7- and 3.9-fold, respectively, when HDL was treated with membrane-immobilized esterase and trypsin. Furthermore, the efficient digestion of apo A-1 by trypsin was inhibited when HDL was treated with membrane-immobilized esterase in the presence of an esterase inhibitor, 6,9-diamino-2-ethoxyacridine (acrinol). The data indicate that the lipid components of lipoproteins are released by membrane-immobilized esterase. This method can be used to investigate the structure and function of other apolipoproteins. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Lipoproteins contain different kinds of lipids. For example, high density lipoprotein (HDL) is mainly composed of apolipoproteins such as apolipoprotein A-1 (apo A-1) [1]. The conformation and lipid-binding site of apo A-1 have been examined [2]. Lipid-free apo A-1 is a more accessible substrate for matrix metalloproteinase than lipid-bound apo A-1 [3]. To investigate the structure and function of HDL apolipoprotein components, it is necessary to remove lipids from the lipoproteins without destroying protein structure and function. We previously reported that phosphatidylcholine, a type of lipid, is hydrolyzed by a membrane-immobilized esterase identified as carboxylesterase 1 [4–6]. Furthermore, esterase treatment of the lipids exposes apolipoproteins, which can then be digested by trypsin. Proteins can be rapidly and efficiently digested by proteolytic enzymes such as trypsin and chymotrypsin immobilized on membranes [7–9]. Therefore, if enzymes such as esterase and trypsin are immobilized on membranes, enzymolysis of macromolecules such as HDL can be efficiently performed on the surface of the membrane. On the other hand, it has been reported
Abbreviations: 2-DE, two-dimensional electrophoresis; PVDF, polyvinylidene difluoride; MALDI-TOF MS, matrix assisted laser desorption/ionization time of flight mass spectrometry; HDL, high density lipoprotein; Tris, 2-amino-2-hydroxymethyl1,3-propanediol; Fast Red TR salt, 4-chloro-2-methylbenzene diazonium salt. ∗ Corresponding author. Tel.: +81 89 927 9617; fax: +81 89 927 9590. E-mail address: [email protected] (Y. Shimazaki). 0731-7085/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jpba.2012.07.032
that esterase activity is inhibited by an inhibitor, 6,9-diamino2-ethoxyacridine (acrinol) [10]. Thus, the efficient digestion of apo A-1 by trypsin can be inhibited, even after treatment of HDL by membrane-immobilized esterase in the presence of acrinol, because the HDL lipids are not released. We describe the efficient digestion of the apo A-1 component of HDL by membrane-immobilized trypsin following lipid removal by membrane-immobilized esterase after production of membrane-immobilized enzymes. Furthermore, digestion of apo A-1 by trypsin was inhibited when HDL was treated with the membrane-immobilized esterase in the presence of acrinol. This method can also be used for the investigation of structure and function of other apolipoproteins. 2. Materials and methods 2.1. Materials and preparation of membrane-immobilized enzymes Acronym of polyvinylidene difluoride (PVDF) membrane was purchased from Millipore (Bedford, MA, USA). Acrylamide and carrier ampholyte (Pharmalyte, pH 3–10) were purchased from Kishida Chemicals (Osaka, Japan) and GE healthcare (Uppsala, Sweden), respectively. HDL was purchased from Meridian Life Science Inc. (Cincinnati, OH, USA). Adrenocorticotropic hormone (ACTH), Ponceau S, ␣-cyano-4-hydroxycinnamic acid and bovine pancreatic trypsin were purchased from Sigma–Aldrich (St. Louis, MO, USA). Bovine trypsin (sequence grade) was purchased from
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Fig. 1. Ponceau S staining in 0.1 M acetate buffer solution (pH 5.1) (a) followed by esterase activity staining, (b) after separation of cytosolic proteins from mouse liver by non-denaturing 2-DE and membrane blotting. Ponceau S staining in 0.1 M Tris–HCl buffer solution (pH 7.0) after separation of bovine pancreas trypsin by non-denaturing electrophoresis, and membrane blotting (c). Excised spot A possesses esterase activity and spot B dose not possess esterase activity. Proteins are migrated toward an anode in the 2-DE (a and b), whereas trypsin is migrated toward a cathode of non-denaturing electrophoresis (c).
Roche (Mannheim, Germany). All other reagents were purchased from Wako Pure Chemicals (Osaka, Japan) or Nacalai Tesque (Osaka, Japan). Mouse livers (Swiss Webster) were purchased from Rockland Inc. (Gilbertsville, PA, USA) and homogenized in Tris–HCl buffer (pH 7.2; 0.1 M). The homogenate was centrifuged for 5 min at 10,000 × g to obtain the cytosolic fraction. Sucrose was added to the liver cytosolic fraction at a concentration of 40% (w/v). Proteins in the cytosolic fraction (100–300 g) were subjected to microscale non-denaturing two-dimensional electrophoresis (2DE) by a method previously reported [11]. To immobilize proteins on PVDF membranes, proteins were transferred to PVDF membranes by a semi-dry type transblotting apparatus using electrode buffers of Tris (0.05 M) – glycine (pH 8.3; 0.38 M) with a constant current of 23 mA per gel for 4 h [12]. To detect proteins on the PVDF membrane, membranes were soaked in 0.5% Ponceau S in 10 mL of acetate buffer (pH 5.1; 0.1 M). For preparation of membrane-immobilized trypsin, sucrose was added to bovine pancreatic trypsin to a concentration of 40% (w/v). Trypsin (20 g) was then subjected to non-denaturing electrophoresis on a 5% acrylamide (0.25% Bis) gel containing Tris–HCl (pH 6.9; 0.13 M). The electrode buffer consisted of Tris (0.05 M) – glycine (pH 8.3; 0.38 M). Because trypsin is a type of basic proteins, it migrates toward a cathode. When the top and bottom of the gel were connected to anode and cathode, respectively, trypsin was migrated from the top to the bottom of the gel. The protein was then transferred to a PVDF membrane on the cathode using a semi-dry-type blotting apparatus to immobilize it. The immobilized enzyme was detected by staining with 0.001% Ponceau S in 10 mL of Tris–HCl buffer (pH 7.0; 50 mM) and destained with H2 O. To analyze esterase activity, membranes were incubated in 10 mL of phosphate buffer (pH 7.1; 0.2 M), containing 0.2 mL of 1% ␣-naphthylacetate and 4 mg of 4chloro-2-methylbenzene diazonium salt (Fast Red TR salt). Regions of membrane containing esterase or trypsin enzymes were excised and used to digest HDL samples.
37 ◦ C. The obtained polypeptides were collected and analyzed by MALDI-TOF MS. One microlitre of the liquid containing polypeptides was mixed with 1 L of a solution containing saturated ␣-cyano 4-hydroxycinnamic acid, 0.1% trifluoroacetic acid and 70% acetonitrile. The sample mixture and matrix was placed on a stainless steel sample plate (sample plate for Voyager DE PRO; Applied Biosystems, Framingham, MA, USA) and dried. Mass analysis was performed using MALDI-TOF MS (Voyager DE PRO; Applied Biosystems) in positive ion reflector or linear mode. Monoisotopic or average peak of ACTH 18-39 (m/z 2465.1989) or (m/z 2466.72)
2.2. Digestion by membrane-immobilized trypsin and MALDI-TOF MS analysis HDL (25 L of 1.5 g/L solution) was first incubated with either membrane-immobilized esterase (Fig. 1a, spot A) or membrane without esterase enzyme (Fig. 1a, spot B) for 4 h at 37 ◦ C. Following this, HDL digestion products were incubated with membrane-immobilized trypsin for 0–6 h at 37 ◦ C. To inhibit esterase activity on the membrane, HDL was incubated with membrane-immobilized esterase (Fig. 1a, spot A) in the absence or presence of 0.1 mM acrinol, or without membrane-immobilized esterase (Fig. 1a, spot B) in the absence of 0.1 mM acrinol for 4 h at 37 ◦ C. Following these treatments, HDL digestion products were incubated with membrane-immobilized trypsin for 6 h at
Fig. 2. MALDI-TOF MS spectra of HDL polypeptides following incubation with membrane-immobilized trypsin for 0 h (a) or 6 h (b and c) at 37 ◦ C following incubation with (b, spot A in Fig. 1a) or without (c, spot B in Fig. 1a) membrane-immobilized esterase for 4 h at 37 ◦ C. * Polypeptides from homo sapiens apo A-1. IS indicates internal standard of ACTH 18-39 (m/z 2465.1989).
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Fig. 3. Changes in the relative amounts of apo A-1 polypeptides m/z 1302.68 (a), 1568.82 (b), 1585.69 (c) and 1723.78 (d) following HDL digestion with membrane-immobilized trypsin for 0–6 h at 37 ◦ C with or without prior esterase treatment. Black bar treated with membrane-immobilized esterase; white bar treated with membrane lacking esterase activity. Each datum indicates mean ± standard error of more than eight individual measurements. *P < 0.05, **P < 0.02, ***P < 0.005 (Student’s t-test).
was used for internal calibration respectively. Spectra were analyzed using the mass values for monoisotopic peaks that were used for searches (Mascot, http://www.matrixscience.com/) against the Swiss-Prot database. The database was searched using the following terms: taxonomy (Homo sapiens), trypsin digest (one missed cleavage allowed), cysteine modified by carbamidomethylation, and mass tolerance of 50 ppm, using internal calibration and oxidation of methionines. The criteria used to accept identification included the extent of sequence coverage, the number of peptides matched, and the probability score (the required probability for a random match was <0.05). Relative amounts of peak intensity of polypeptides were estimated by the average peak intensity of ACTH 18-39 (m/z 2466.72). MS data were analyzed using the Student’s t-test.
3. Results and discussion 3.1. HDL digestion by membrane-immobilized trypsin after treatment with membrane-immobilized esterase Mouse liver cytosolic proteins were separated by nondenaturing 2-DE, blotted onto membranes, stained with Ponceau S (Fig. 1a) and assayed for esterase activity (Fig. 1b). We previously reported that the spot A in Fig. 1a was identified as mouse carboxylesterase 1 [4], and the esterase activity was retained even after non-denaturing electrophoresis, membrane immobilization and Ponceau S staining [12]. We purified and immobilized bovine pancreatic trypsin (Fig. 1c). Therefore, this method can be used to produce several types of membrane-immobilized enzymes such as carboxylesterase and trypsin after separation by non-denaturing electrophoresis, immobilization, staining and excision. Fig. 2 shows the MALDI-TOF MS spectra of the obtained
polypeptides of HDL when HDL samples were incubated with or without membrane-immobilized esterase for 4 h at 37 ◦ C followed by membrane-immobilized trypsin for 0 h (a) and 6 h (b and c) at 37 ◦ C. As shown in Fig. 2b, the main monoisotopic m/z peaks of 1302.68, 1568.82, 1585.69, 1380.74, 1283.65, 1157.71 and 1723.78 corresponded to polypeptides of apo A-1 polypeptides from Homo sapiens, (Swiss Prot: http://web.expasy.org/docs/userman.html): however, only one polypeptide peak (m/z 1723.78) was obtained following HDL digestion by membrane-immobilized trypsin without prior esterase digestion (Fig. 2c).
3.2. Efficient digestion of HDL by trypsin after treatment of HDL with membrane-immobilized esterase Different relative amounts of apo A-1 polypeptides (m/z 1302.68, 1568.82, 1585.69 and 1723.78) were obtained after HDL digestion by membrane-immobilized trypsin with or without prior digestion by membrane-immobilized esterase (Fig. 3). Specifically, there was a significant increase in apo A-1 polypeptides of m/z 1568.82 and 1723.78 following a 4-h digestion with trypsin along with prior esterase treatment compared with samples without esterase treatment (Fig. 3b and d). In addition, following a 6-h digestion of HDL with membrane-immobilized trypsin, there was a 3.9- and 2.7-fold increase in the amounts of these peptides in samples previously treated with esterase relative to controls without esterase treatment (Fig. 3b and d). Similarly, significantly more m/z 1302.68 polypeptide was obtained after a 6-h HDL digestion with membrane-immobilized trypsin following esterase treatment than in controls without esterase treatment (Fig. 3a). In contrast, the amount of m/z 1585.69 polypeptide obtained after a 6-h HDL digestion with trypsin was not significantly higher after esterase treatment (Fig. 3c).
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Thus, our results indicate that HDL lipid removal by membraneimmobilized esterase exposes apo A-1, which can then be digested by membrane-immobilized trypsin. 4. Conclusion Membrane-immobilized enzymes such as esterase and trypsin were produced after separation, immobilization and staining under non-denaturing conditions. The apolipoprotein component of HDL was efficiently digested by membrane-immobilized trypsin following lipid digestion by membrane-immobilized esterase. This method provides a novel approach for examination of the structure and function of apolipoproteins. References Fig. 4. Changes in the relative amounts of apo A-1 polypeptides (m/z 1302.68, 1568.82, 1585.69 and 1723.78) after HDL digestion by membrane-immobilized trypsin for 6 h at 37 ◦ C with prior esterase treatment in the absence (black bar) or presence of acrinol (gray bar), or without prior esterase treatment (white bar). Each datum indicates mean ± standard error of more than eight individual measurements. *P < 0.05 (Student’s t-test).
3.3. Inhibition of digestion by trypsin after treatment of HDL with membrane-immobilized esterase in the presence of an inhibitor Fig. 4 shows changes in relative amounts of apo A-1 polypeptides (m/z 1302.68, 1568.82, 1585.69 and 1723.78) after HDL digestion by membrane-immobilized trypsin with prior digestion by membrane-immobilized esterase in the absence or presence of acrinol, or without prior digestion by membrane-immobilized esterase in the absence of acrinol. The amounts of polypeptides (m/z 1302.68, 1568.82 and 1723.78) from apo A-1 significantly increased after HDL digestion by membrane-immobilized trypsin with prior digestion by membrane-immobilized esterase in the absence of acrinol. On the other hand, the increase of these polypeptides (m/z 1302.68, 1568.82 and 1723.78) was clearly inhibited after HDL digestion by membrane-immobilized trypsin with prior digestion by membrane-immobilized esterase in the presence of acrinol. These data indicate that efficient digestion of apo A-1 by trypsin was inhibited when lipid digestion by esterase was inhibited by acrinol. These results indicate that removal of lipids from HDL by esterase digestion increases the efficiency of tryptic proteolysis. Previous reports indicate that lipids bind directly to apo A-1, and that lipid-bound apo A-1 within HDL is resistant to proteolysis, whereas lipid-free apo A-1 is susceptible to proteolysis [2,13].
[1] A. Iglesias, M. Arranz, J.J. Alvarez, J. Perales, J. Villar, E. Herrera, M.A. Lasuncion, Choleteryl ester transfer activity in liver disease and cholestasis, and its relation with fatty acid composition of lipoprotein lipids, Clin. Chim. Acta 248 (1996) 157–174. [2] H.L. Zhu, D. Atkinson, Conformation and lipid binding of a C-terminal (198–243) of human apolipoprotein A-1 (apo A-1), Biochemistry 46 (2007) 1624–1634. [3] J.H. Park, S.M. Park, K.H. Park, K.H. Cho, S.T. Lee, Analysis of apolipoprotein A-I as a substrate for matrix metalloproteinase-14, Biochem. Biophys. Res. Commun. 409 (2011) 58–63. [4] Y. Shimazaki, S. Watanabe, Silver staining of an esterase compatible with activity and mass spectrometry analysis after separation using non-denaturing two-dimensional electrophoresis, Clin. Chim. Acta 390 (2008) 145–147. [5] Y. Shimazaki, T. Kuroda, Production of enzyme reactors after separation by non-denaturing two-dimensional electrophoresis and immobilization on membrane, Biotechnol. Lett. 31 (2009) 1545–1549. [6] T. Sakikawa, Y. Shimazaki, Reversible inhibition of esterase activity after separation and immobilization, Appl. Biochem. Biotechnol. 165 (2011) 69–74. [7] Y. Liu, H. Lu, W. Zhong, P. Song, J. Kong, P. Yong, H.H. Girault, B. Lui, Multilayerassembled microchip for enzyme immobilization as reactor toward low-level protein identification, Anal. Chem. 78 (2006) 801–808. [8] F. Xu, W.H. Wang, Y.J. Tan, M.L. Bruening, Facile trypsin immobilization in polymeric membranes for rapid, efficient protein digestion, Anal. Chem. 82 (2010) 10045–10051. [9] Y. Zhou, T. Yi, S.S. Park, W. Chadwick, R.F. Shen, W.W. Wu, B. Martin, S. Maudsley, Rapid and enhanced proteolytic digestion using electric-field-oriented enzyme reactor, J. Proteomics 74 (2011) 1030–1035. [10] S. Bencharit, C.L. Morton, J.L. Hyatt, P. Kuhn, M.K. Danks, P.M. Potter, M.R. Redinbo, Crystal structure of human carboxylesterase 1 complexed with the Alzheimer’s drug tacrine: from binding promiscuity to selective inhibition, Chem. Biol. 10 (2003) 341–349. [11] Y. Shimazaki, Y. Sugawara, Y. Ohtsuka, T. Manabe T, Analysis of the activity and identification of enzymes after separation of cytosol proteins in mouse liver by microscale nondenaturing two-dimensional electrophoresis, Proteomics 3 (2003) 2002–2007. [12] Y. Shimazaki, T. Sakikawa, Determination of malic acid using a malate dehydrogenase reactor after purification and immobilization in non-denaturing conditions and staining with Ponceau S, Protein Peptide Lett. 17 (2010) 1048–1052. [13] M.A. Hossain, S. Ngeth, T. Chan, M.N. Oda, G.A. Fracis, Lipid-bound apolipoproteins in tyrosyl radical-oxidized HDL stabilize ABCA1 like lipid-free apolipoprotein A-I, BMC Biochem. 13 (2012) 1–11.
Contents lists available at SciVerse ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Short communication
Enzymolysis of high density lipoprotein with a combination of membrane-immobilized esterase and trypsin Takahiro Sakikawa, Youji Shimazaki ∗ Graduate School of Science and Engineering (Science Section) and Venture Business Laboratory, Ehime University, Matsuyama 790-8577, Japan
a r t i c l e
i n f o
Article history: Received 11 April 2012 Received in revised form 24 July 2012 Accepted 29 July 2012 Available online 4 August 2012 Keywords: HDL Carboxylesterase Trypsin Electrophoresis MALDI-TOF MS 6,9-Diamino-2-ethoxyacridine
a b s t r a c t Apolipoprotein A-1 (apo A-1), a major component of high density lipoprotein (HDL), was efficiently digested by membrane-immobilized trypsin after HDL was treated with membrane-immobilized esterase. Compared to treatment with membrane-immobilized trypsin alone, the relative amounts of apo A-1 polypeptides, m/z 1723.78 and m/z 1568.82, increased by 2.7- and 3.9-fold, respectively, when HDL was treated with membrane-immobilized esterase and trypsin. Furthermore, the efficient digestion of apo A-1 by trypsin was inhibited when HDL was treated with membrane-immobilized esterase in the presence of an esterase inhibitor, 6,9-diamino-2-ethoxyacridine (acrinol). The data indicate that the lipid components of lipoproteins are released by membrane-immobilized esterase. This method can be used to investigate the structure and function of other apolipoproteins. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Lipoproteins contain different kinds of lipids. For example, high density lipoprotein (HDL) is mainly composed of apolipoproteins such as apolipoprotein A-1 (apo A-1) [1]. The conformation and lipid-binding site of apo A-1 have been examined [2]. Lipid-free apo A-1 is a more accessible substrate for matrix metalloproteinase than lipid-bound apo A-1 [3]. To investigate the structure and function of HDL apolipoprotein components, it is necessary to remove lipids from the lipoproteins without destroying protein structure and function. We previously reported that phosphatidylcholine, a type of lipid, is hydrolyzed by a membrane-immobilized esterase identified as carboxylesterase 1 [4–6]. Furthermore, esterase treatment of the lipids exposes apolipoproteins, which can then be digested by trypsin. Proteins can be rapidly and efficiently digested by proteolytic enzymes such as trypsin and chymotrypsin immobilized on membranes [7–9]. Therefore, if enzymes such as esterase and trypsin are immobilized on membranes, enzymolysis of macromolecules such as HDL can be efficiently performed on the surface of the membrane. On the other hand, it has been reported
Abbreviations: 2-DE, two-dimensional electrophoresis; PVDF, polyvinylidene difluoride; MALDI-TOF MS, matrix assisted laser desorption/ionization time of flight mass spectrometry; HDL, high density lipoprotein; Tris, 2-amino-2-hydroxymethyl1,3-propanediol; Fast Red TR salt, 4-chloro-2-methylbenzene diazonium salt. ∗ Corresponding author. Tel.: +81 89 927 9617; fax: +81 89 927 9590. E-mail address: [email protected] (Y. Shimazaki). 0731-7085/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jpba.2012.07.032
that esterase activity is inhibited by an inhibitor, 6,9-diamino2-ethoxyacridine (acrinol) [10]. Thus, the efficient digestion of apo A-1 by trypsin can be inhibited, even after treatment of HDL by membrane-immobilized esterase in the presence of acrinol, because the HDL lipids are not released. We describe the efficient digestion of the apo A-1 component of HDL by membrane-immobilized trypsin following lipid removal by membrane-immobilized esterase after production of membrane-immobilized enzymes. Furthermore, digestion of apo A-1 by trypsin was inhibited when HDL was treated with the membrane-immobilized esterase in the presence of acrinol. This method can also be used for the investigation of structure and function of other apolipoproteins. 2. Materials and methods 2.1. Materials and preparation of membrane-immobilized enzymes Acronym of polyvinylidene difluoride (PVDF) membrane was purchased from Millipore (Bedford, MA, USA). Acrylamide and carrier ampholyte (Pharmalyte, pH 3–10) were purchased from Kishida Chemicals (Osaka, Japan) and GE healthcare (Uppsala, Sweden), respectively. HDL was purchased from Meridian Life Science Inc. (Cincinnati, OH, USA). Adrenocorticotropic hormone (ACTH), Ponceau S, ␣-cyano-4-hydroxycinnamic acid and bovine pancreatic trypsin were purchased from Sigma–Aldrich (St. Louis, MO, USA). Bovine trypsin (sequence grade) was purchased from
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Fig. 1. Ponceau S staining in 0.1 M acetate buffer solution (pH 5.1) (a) followed by esterase activity staining, (b) after separation of cytosolic proteins from mouse liver by non-denaturing 2-DE and membrane blotting. Ponceau S staining in 0.1 M Tris–HCl buffer solution (pH 7.0) after separation of bovine pancreas trypsin by non-denaturing electrophoresis, and membrane blotting (c). Excised spot A possesses esterase activity and spot B dose not possess esterase activity. Proteins are migrated toward an anode in the 2-DE (a and b), whereas trypsin is migrated toward a cathode of non-denaturing electrophoresis (c).
Roche (Mannheim, Germany). All other reagents were purchased from Wako Pure Chemicals (Osaka, Japan) or Nacalai Tesque (Osaka, Japan). Mouse livers (Swiss Webster) were purchased from Rockland Inc. (Gilbertsville, PA, USA) and homogenized in Tris–HCl buffer (pH 7.2; 0.1 M). The homogenate was centrifuged for 5 min at 10,000 × g to obtain the cytosolic fraction. Sucrose was added to the liver cytosolic fraction at a concentration of 40% (w/v). Proteins in the cytosolic fraction (100–300 g) were subjected to microscale non-denaturing two-dimensional electrophoresis (2DE) by a method previously reported [11]. To immobilize proteins on PVDF membranes, proteins were transferred to PVDF membranes by a semi-dry type transblotting apparatus using electrode buffers of Tris (0.05 M) – glycine (pH 8.3; 0.38 M) with a constant current of 23 mA per gel for 4 h [12]. To detect proteins on the PVDF membrane, membranes were soaked in 0.5% Ponceau S in 10 mL of acetate buffer (pH 5.1; 0.1 M). For preparation of membrane-immobilized trypsin, sucrose was added to bovine pancreatic trypsin to a concentration of 40% (w/v). Trypsin (20 g) was then subjected to non-denaturing electrophoresis on a 5% acrylamide (0.25% Bis) gel containing Tris–HCl (pH 6.9; 0.13 M). The electrode buffer consisted of Tris (0.05 M) – glycine (pH 8.3; 0.38 M). Because trypsin is a type of basic proteins, it migrates toward a cathode. When the top and bottom of the gel were connected to anode and cathode, respectively, trypsin was migrated from the top to the bottom of the gel. The protein was then transferred to a PVDF membrane on the cathode using a semi-dry-type blotting apparatus to immobilize it. The immobilized enzyme was detected by staining with 0.001% Ponceau S in 10 mL of Tris–HCl buffer (pH 7.0; 50 mM) and destained with H2 O. To analyze esterase activity, membranes were incubated in 10 mL of phosphate buffer (pH 7.1; 0.2 M), containing 0.2 mL of 1% ␣-naphthylacetate and 4 mg of 4chloro-2-methylbenzene diazonium salt (Fast Red TR salt). Regions of membrane containing esterase or trypsin enzymes were excised and used to digest HDL samples.
37 ◦ C. The obtained polypeptides were collected and analyzed by MALDI-TOF MS. One microlitre of the liquid containing polypeptides was mixed with 1 L of a solution containing saturated ␣-cyano 4-hydroxycinnamic acid, 0.1% trifluoroacetic acid and 70% acetonitrile. The sample mixture and matrix was placed on a stainless steel sample plate (sample plate for Voyager DE PRO; Applied Biosystems, Framingham, MA, USA) and dried. Mass analysis was performed using MALDI-TOF MS (Voyager DE PRO; Applied Biosystems) in positive ion reflector or linear mode. Monoisotopic or average peak of ACTH 18-39 (m/z 2465.1989) or (m/z 2466.72)
2.2. Digestion by membrane-immobilized trypsin and MALDI-TOF MS analysis HDL (25 L of 1.5 g/L solution) was first incubated with either membrane-immobilized esterase (Fig. 1a, spot A) or membrane without esterase enzyme (Fig. 1a, spot B) for 4 h at 37 ◦ C. Following this, HDL digestion products were incubated with membrane-immobilized trypsin for 0–6 h at 37 ◦ C. To inhibit esterase activity on the membrane, HDL was incubated with membrane-immobilized esterase (Fig. 1a, spot A) in the absence or presence of 0.1 mM acrinol, or without membrane-immobilized esterase (Fig. 1a, spot B) in the absence of 0.1 mM acrinol for 4 h at 37 ◦ C. Following these treatments, HDL digestion products were incubated with membrane-immobilized trypsin for 6 h at
Fig. 2. MALDI-TOF MS spectra of HDL polypeptides following incubation with membrane-immobilized trypsin for 0 h (a) or 6 h (b and c) at 37 ◦ C following incubation with (b, spot A in Fig. 1a) or without (c, spot B in Fig. 1a) membrane-immobilized esterase for 4 h at 37 ◦ C. * Polypeptides from homo sapiens apo A-1. IS indicates internal standard of ACTH 18-39 (m/z 2465.1989).
T. Sakikawa, Y. Shimazaki / Journal of Pharmaceutical and Biomedical Analysis 71 (2012) 179–182
181
Fig. 3. Changes in the relative amounts of apo A-1 polypeptides m/z 1302.68 (a), 1568.82 (b), 1585.69 (c) and 1723.78 (d) following HDL digestion with membrane-immobilized trypsin for 0–6 h at 37 ◦ C with or without prior esterase treatment. Black bar treated with membrane-immobilized esterase; white bar treated with membrane lacking esterase activity. Each datum indicates mean ± standard error of more than eight individual measurements. *P < 0.05, **P < 0.02, ***P < 0.005 (Student’s t-test).
was used for internal calibration respectively. Spectra were analyzed using the mass values for monoisotopic peaks that were used for searches (Mascot, http://www.matrixscience.com/) against the Swiss-Prot database. The database was searched using the following terms: taxonomy (Homo sapiens), trypsin digest (one missed cleavage allowed), cysteine modified by carbamidomethylation, and mass tolerance of 50 ppm, using internal calibration and oxidation of methionines. The criteria used to accept identification included the extent of sequence coverage, the number of peptides matched, and the probability score (the required probability for a random match was <0.05). Relative amounts of peak intensity of polypeptides were estimated by the average peak intensity of ACTH 18-39 (m/z 2466.72). MS data were analyzed using the Student’s t-test.
3. Results and discussion 3.1. HDL digestion by membrane-immobilized trypsin after treatment with membrane-immobilized esterase Mouse liver cytosolic proteins were separated by nondenaturing 2-DE, blotted onto membranes, stained with Ponceau S (Fig. 1a) and assayed for esterase activity (Fig. 1b). We previously reported that the spot A in Fig. 1a was identified as mouse carboxylesterase 1 [4], and the esterase activity was retained even after non-denaturing electrophoresis, membrane immobilization and Ponceau S staining [12]. We purified and immobilized bovine pancreatic trypsin (Fig. 1c). Therefore, this method can be used to produce several types of membrane-immobilized enzymes such as carboxylesterase and trypsin after separation by non-denaturing electrophoresis, immobilization, staining and excision. Fig. 2 shows the MALDI-TOF MS spectra of the obtained
polypeptides of HDL when HDL samples were incubated with or without membrane-immobilized esterase for 4 h at 37 ◦ C followed by membrane-immobilized trypsin for 0 h (a) and 6 h (b and c) at 37 ◦ C. As shown in Fig. 2b, the main monoisotopic m/z peaks of 1302.68, 1568.82, 1585.69, 1380.74, 1283.65, 1157.71 and 1723.78 corresponded to polypeptides of apo A-1 polypeptides from Homo sapiens, (Swiss Prot: http://web.expasy.org/docs/userman.html): however, only one polypeptide peak (m/z 1723.78) was obtained following HDL digestion by membrane-immobilized trypsin without prior esterase digestion (Fig. 2c).
3.2. Efficient digestion of HDL by trypsin after treatment of HDL with membrane-immobilized esterase Different relative amounts of apo A-1 polypeptides (m/z 1302.68, 1568.82, 1585.69 and 1723.78) were obtained after HDL digestion by membrane-immobilized trypsin with or without prior digestion by membrane-immobilized esterase (Fig. 3). Specifically, there was a significant increase in apo A-1 polypeptides of m/z 1568.82 and 1723.78 following a 4-h digestion with trypsin along with prior esterase treatment compared with samples without esterase treatment (Fig. 3b and d). In addition, following a 6-h digestion of HDL with membrane-immobilized trypsin, there was a 3.9- and 2.7-fold increase in the amounts of these peptides in samples previously treated with esterase relative to controls without esterase treatment (Fig. 3b and d). Similarly, significantly more m/z 1302.68 polypeptide was obtained after a 6-h HDL digestion with membrane-immobilized trypsin following esterase treatment than in controls without esterase treatment (Fig. 3a). In contrast, the amount of m/z 1585.69 polypeptide obtained after a 6-h HDL digestion with trypsin was not significantly higher after esterase treatment (Fig. 3c).
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Thus, our results indicate that HDL lipid removal by membraneimmobilized esterase exposes apo A-1, which can then be digested by membrane-immobilized trypsin. 4. Conclusion Membrane-immobilized enzymes such as esterase and trypsin were produced after separation, immobilization and staining under non-denaturing conditions. The apolipoprotein component of HDL was efficiently digested by membrane-immobilized trypsin following lipid digestion by membrane-immobilized esterase. This method provides a novel approach for examination of the structure and function of apolipoproteins. References Fig. 4. Changes in the relative amounts of apo A-1 polypeptides (m/z 1302.68, 1568.82, 1585.69 and 1723.78) after HDL digestion by membrane-immobilized trypsin for 6 h at 37 ◦ C with prior esterase treatment in the absence (black bar) or presence of acrinol (gray bar), or without prior esterase treatment (white bar). Each datum indicates mean ± standard error of more than eight individual measurements. *P < 0.05 (Student’s t-test).
3.3. Inhibition of digestion by trypsin after treatment of HDL with membrane-immobilized esterase in the presence of an inhibitor Fig. 4 shows changes in relative amounts of apo A-1 polypeptides (m/z 1302.68, 1568.82, 1585.69 and 1723.78) after HDL digestion by membrane-immobilized trypsin with prior digestion by membrane-immobilized esterase in the absence or presence of acrinol, or without prior digestion by membrane-immobilized esterase in the absence of acrinol. The amounts of polypeptides (m/z 1302.68, 1568.82 and 1723.78) from apo A-1 significantly increased after HDL digestion by membrane-immobilized trypsin with prior digestion by membrane-immobilized esterase in the absence of acrinol. On the other hand, the increase of these polypeptides (m/z 1302.68, 1568.82 and 1723.78) was clearly inhibited after HDL digestion by membrane-immobilized trypsin with prior digestion by membrane-immobilized esterase in the presence of acrinol. These data indicate that efficient digestion of apo A-1 by trypsin was inhibited when lipid digestion by esterase was inhibited by acrinol. These results indicate that removal of lipids from HDL by esterase digestion increases the efficiency of tryptic proteolysis. Previous reports indicate that lipids bind directly to apo A-1, and that lipid-bound apo A-1 within HDL is resistant to proteolysis, whereas lipid-free apo A-1 is susceptible to proteolysis [2,13].
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