WO2008138495A1

WO2008138495A1 - Method for purifying brush border membrane (bbm) proteins

Info

Publication number: WO2008138495A1
Application number: PCT/EP2008/003502
Authority: WO
Inventors: Axel Ducret; Eirini Tsirogianni
Original assignee: F Hoffmann La Roche AG
Current assignee: F Hoffmann La Roche AG
Priority date: 2007-05-11
Filing date: 2008-04-30
Publication date: 2008-11-20
Anticipated expiration: 2009-11-11

Abstract

The present invention relates to a method for purifying brush border membranes comprising the following steps: a) providing tissue comprising brush border membranes; b) adding an inhibitor of microvillar membrane peptidases to the tissue; c) homogenizing the tissue; d) subjecting the homogenate to a low speed centrifugation resulting in a pellet and a supernatant; e) subjecting the supernatant of step d) to a high speed centrifugation resulting in a pellet and a supernatant; f) resuspending the pellet of step e); g) subjecting the resuspended pellet of step f) to a CaCl2 or MgCl2 precipitation resulting in a suspension; h) subjecting the suspension of g) to a low speed centrifugation resulting in a pellet and a supernatant; i) subjecting then the supernatant of step h) to a high speed centrifugation resulting in a pellet and supernatant; and k) washing the pellet of step i).

Description

METHOD FOR PURIFYING BRUSH BORDER MEMBRANE (BBM) PROTEINS

A brush border is the name for the microvilli-covered surface of pseudostratified columnar epithelium found in multiple locations of the body. They are found in two main locations: the small intestine tract and the kidney. The brush borders of the intestinal lining are the site of terminal carbohydrate digestions. The microvilli which constitute the brush border have enzymes for this final part of digestion anchored into their apical plasma membrane as integral membrane proteins. These enzymes are found near to the transporters which will then allow absorption of the digested nutrients. In the kidney, the brush border is useful in distinguishing the proximal tubule (which possesses the brush border) from the distal tubule (which does not).

Many proteolytic enzymes are constitutionally located in the Brush Border

Membrane and cleave protein for transport through the small intestine. Many of those peptidases, such as Aminopeptidase N, Aminopeptidase A, Neprylisin, Di-Peptidyl- Protease IV, and Trehalase, are not inhibited by the more common (Ser-, Cys-) proteinase inhibitors. These peptidases could be inhibited chemically but only in anhydrous conditions. Protein degradation, however, is a major problem for relative quantification: Even a western blot will provide erroneous answers if the target protein is degraded.

These problems may be avoided with the present invention. Therefore, the present invention provides a method for purifying brush border membranes and thereby enriching the brush border membrane proteins. The method comprises the following steps: a) providing tissue which comprises brush border membranes; b) adding an inhibitor of microvillar membrane peptidases to the tissue; c) homogenizing the tissue; d) subjecting the homogenate to a low speed centrifugation resulting in a pellet and a supernatant; e) subjecting the supernatant of step d) to a high speed centrifugation resulting in a pellet and a supernatant; f) resuspending the pellet of step e); g) subjecting the resuspended pellet of step f) to a CaCl₂ or MgCl₂ precipitation resulting in a suspension; h) subjecting the suspension of g) to a low speed centrifiαgation resulting in a pellet and a supernatant; i) subjecting then the supernatant of step h) to a high speed centrifugation resulting in a pellet and supernatant; and k) washing the pellet of step i).

Preferably, an inhibitor of microvillar membrane peptidases (MMPs) is additionally added in step f) before CaCl₂ or MgCl₂ precipitation. The MMPs inhibitor may be added for example to the buffer with which the pellet of step e) is resuspended in step f). Most preferably, the MMPs inhibitor is added to each processing step (steps b) to k)) of the method of the invention.

The preferred MMPs inhibitor is amastatin.

The preferred concentration of the MMPs inhibitor is higher than O.lmM. More preferably, the concentration of the MMPs inhibitor is between 0.5mM to l.lmM. Most preferably the concentration is ImM. When the MMPs inhibitor is added at more than one step of the method (e.g. at step b) and step f); or at each one of step b) to step k)), the concentration of added MMPs inhibitor may be different for the individual steps. However, preferably, the MMPs inhibitor is added in the same concentration.

Peptidase substrates may also be added to various steps of the method of the invention. Such substrate may for example be Angiotensin 1-5, Ala-Pro-PNA or Met- Bradykinin, or a mixture thereof. Preferably, the substrates are added additionally to the inhibitor. Also preferably, a peptidase substrate is added in step b) and/or step f). The peptidase substrate may be added in step b) for example dissolved in a buffer to the tissue. In step f) the peptidase substrate may be added for example to the buffer with which the pellet of step e) is resuspended.

Furthermore, in preferred embodiment, a chelating agent is added to the supernatant after the low speed centrifugation of step h) (supernatant resulting from step h)). The preferred chelating agent is EDTA.

A tissue which comprises brush border membranes is e.g. the mucosa tissue. The term "mucosa tissue" as used herein refers to the mucous tissue or mucous membrane of the small intestine. The mucous tissue/membrane is the innermost layer of the gastrointestinal tract, surrounding the lumen, or space within the tube. It comprises the epithelium, the lamina propria and a muscle layer. The epithelium is folded and forms so called villi wherein each of the surfaces of the villi is also folded to form microvilli. The epithelium of these microvilli consists essentially of brush border cells (see also Figure 1). Brush border membrane is the cell membrane of said epithelial cells.

The term "intestine" refers to the portion of the alimentary canal extending from the stomach to the anus and, in humans and other mammals, consists of two segments, the small intestine and the large intestine. The small intestine is the portion of the intestine closest to the stomach. The small intestine has three structures to handle absorption: villi, microvilli and the circular folds.

Mucosa tissue may provided by scrapping the inner lining of the small intestine. The tissue may be isolated from proximal, central or distal section of the small intestine whereby the proximal section is the part nearest to the stomach and the distal section farthest from the stomach. For example, the provided mucosa tissue may derive from a pool of proximal, central and distal section of the small intestine or it may derive from only one or two of those sections.

The small intestine may be the small intestine of any animal. Preferably, it is the small intestine of a rodent or a bovine. More preferably, it is the intestine of a mouse or a cow.

The term "peptidase" refers to an enzyme which hydrolyzes peptide bonds between amino acids. Such a peptidase catalyzes the splitting of proteins or peptide into smaller peptide fractions and amino acids.

Microvillar membrane peptidases (MMPs) are peptidases which located within the cells of the brush border membrane. These peptidases are set free when the cell membrane is damaged, e.g. when the mucosa tissue is homogenized. Examples for such MMPs are Aminopeptidase N, Aminopeptidase A, Neprylisin, Di-Peptidyl-Protease IV, and Trehalase.

The term "inhibitor" refers to a molecule which represses or prevents another molecule from engaging in a reaction. The term "inhibitor of MMPs" refers to a peptidase inhibitor which represses or prevents to proteolysis of proteins by MMPs wherein said inhibitor is not a Serine- or a Cysteine-proteinase inhibitor.

A Serine-proteinase is a proteinase that is characterised by the presence of a serine residue in the active site of the enzyme. A Cysteine- proteinase is a proteinase that is characterised by the presence of a cysteine residue in the active site of the enzyme. - A -

The term "homogenization" refers to a process that involves breaking apart cells and thereby releasing organelles and cytoplasm.

Methods for homogenization of membranes are well known to the skilled in the art. Such methods comprise for example osmotic alteration of the media in which the cells are or they comprise the use of physical force to disrupt cell structure. The physical means encompass use of mortars and pestles, blenders, compression and/or expansion, or ultrasonification (see e.g. Kessler M., Acuto O., Storelli C, Murer H., Mϋller M., and Semenza, G. Biochimica and Biophysica Acta, 506 (1978) 136-154.). Usually, tissue is homogenized in the presence of a buffer.

The term "low speed centrifugation" refers to a centrifugation at a speed of 1000 x g to 5000 x g Preferred speed is 200Ox g to 4000 x g. An even more preferred speed is 3000 x g-

The term "high speed centrifugation" refers to a centrifugation at a speed of 20'0OO x g to 70'000xg. Preferred speed is 30'OOOxg to 60000xg. An even more preferred speed is 50'OOOxg.

The term "buffer" refers to a solution containing either a weak acid and its salt or a weak base and its salt, which is resistant to changes in pH. Weak acids are not completely ionized, and a solution of a weak acid has a relatively low concentration of hydrogen ions. A weak base is a base that reacts only partially in an aqueous solution to produce an OH^" ion and the conjugate acid. A buffer is for a example a Tris buffer which comprises trishydroxymethylaminomethane (Tris) a buffer compound.

Preferably, pellet of step i) is washed with a high salt solution and high pH solution, both without calcium. Preferably, the salt solution comprises a chelating agent. The preferred chelating agent is EDTA. High salt solution means a salt concentration equal or higher than IM for salts with monovalent cations and equal or higher than 0.5M for divalent cations. Preferably, the high salt solution is a KCl solution. A high pH solution means a solution with a pH value between 10 and 12. Preferably the pH value is 11. The preferred high pH solution is a Na₂CO₃ solution. Preferably, the pellet of step i) is washed first with a high salt solution and then with a high pH solution. In a preferred embodiment, the pellet is washed 1 to 3 times with each of the solutions, more preferably the pellet is washed twice. The washed pellets of step k) comprise purified BBM membranes. These membranes, usually available as BBM vesicles, comprise enriched BBM proteins. The protein may be isolated from membranes with methods used for isolating other membrane proteins. Such methods are well known to the skilled in the art. A preferred method uses detergents such SDS or CHAPS.

The BBM proteins may be identified and relatively quantified by methods well known to the skilled in the art. For example, the BBM proteins are separated by ID SDS PAGE and then digested with a site-specific endopeptidase, preferably Trypsin. The digested peptides are extracted and measured by LC-MS/MS.

The method of invention allows to purify the brush border membrane and thereby to enrich the brush border membrane proteins with less loss of proteins to degradation by peptidases. The method is a valuable tool for identifying brush border membrane proteins which are present in a low concentration and for the relative quantification of the membrane proteins.

The present invention further provides purified brush border membrane obtainable by the methods as described above. In addition, the present invention provides enriched brush border membrane proteins obtainable by the above described methods.

Furthermore, the present invention provides the use of amastatin for purifying brush border membrane.

Having now generally described this invention, the same will become better understood by reference to the specific examples, which are included herein for purpose of illustration only and are not intended to be limiting unless otherwise specified, in connection with the following figures.

Figures:

Figure 1 shows a schematic representation of a mucosa membrane. The mucous membrane comprises an epithelium. Said epithelium is folded and forms so called villi wherein each of the surfaces of the villi is also folded to form microvilli. The epithelium of these microvilli consists essentially of brush border cells.

Figure 2 shows a flow chart of the purifying method. Tissue comprising mucosa tissue is provided (preferably "total mucosa" = pooled from proximal, central and distal sections of the small intestine). Peptidase inhibitor is added and the tissue homogenated (step 1). The homogenized tissue is centrifuged at low speed (step 2) and then the supernatant resulting from the low speed centrifugation is centrifuged a high speed (step 3). The pellet resulting from the high speed centrifugation is resuspended, said resuspended pellet is subjected to a CaCl₂ precipitation or MgCl₂ precipitation and then the resulting suspension of the precipitation is centrifuged at low speed (step 4). The supernatant resulting from the low speed centrifugation is centrifuged a high speed (step 5) and the resulting pellet is washed (step 6).

Figure 3 shows a flow chart of a preferred embodiment of the purifying method.

Figure 4 shows a ID SDS PAGE of purified brush border membrane (BBM) proteins. Lane M = Marker, Lane I = BBM control (without MMPs inhibitor), Lane II = with MMPs Inhibitor and EDTA, Lane III = BBM control washed (without MMPs inhibitor with washing step), Lane IV = with MMPs inhibitor and EDTA and washing step.

Figure 5 shows monitoring protein degradation by western blot of Aminopeptidase N. The primary antibody was a polyclonal antibody against the whole protein. Figure 5A shows the Aminopetidase N signal at the starting homogenized sample. Figure 5 B and C show respectively the Aminopeptidase N (APN) signal in a dilution series of the control BBM fraction and the BBM fraction in the presence of MMPs inhibitors. The APN signal is indicated by an arrow.

Lane M = Marker, Lane 1 = homogenized control (without MMPs inhibitor), lμg; Lane 2= homogenized sample treated with MMPs inhibitor and peptidase substrates, lμg Lane 3-6= BBM control, Lane 3: 0.5μg, Lane 4 = O.lμg; Lane 5:0.05μg; Lane 6: O.Olμg; Lane 7-11: BBM with MMPs inhibitor and substrates, Lane 7: 5μg, Lane 8 = lμg; Lane 9: 0.5μg; Lane 10: O.lμg; Lane 11: 0.05μg Examples

Commercially available reagents referred to in the examples were used according to manufacturer's instructions unless otherwise indicated.

Example 1: Isolation of membrane vesicles.

Brush Border Membrane (BBM) vesicles were isolated by calcium precipitation using a modified method of Kessler et al. (Kessler M., Acuto O., Storelli C, Murer H., Mϋller M., and Semenza, G. Biochimica and Biophysica Acta, 506 (1978) 136-154). Scrapped mucosa from the small intestine of wild type C57B/6J-Bom male mice (Taconic) was suspended in 50 mM Mannitol, 2mM Tris (pH: 7.1) in presence of protease inhibitor cocktail tablets (Roche product Cat. No. 11080733001), 0.1 mM PMSF, 1 mM Amastatin (Bachem) (Hooper N.M., Hesp R.J., and Tieku S., Biochem. ]., 298 (1994) 635-639) and the peptide substrates Angiotensin 1-5, Ala-Pro-PNA, and Met- Bradykinin, at 1 mM concentration each. The control conditions are without the addition of Amastatin, peptide substrates and EDTA (see Figure 3: flow diagram of BBM preparation). The suspension was homogenized four times 20 seconds using a Polytron (Kinemetica GmbH) at maximum speed with intervals of 40 seconds in ice. The homogenized solution was then centrifuged at 3,000 x g for 15 min at 4 ⁰C and the cell debris was discarded. The supernatant was re-centrifuged at 27,000 x g for 30 min at 4 ⁰C. The resulting pellet was then suspended in the above buffer and CaCl₂ was added to a concentration of 10 mM. After incubation on ice for 20 min, a low spin centrifugation was repeated as described above. EDTA was added to the supernatant to a concentration of 40 mM and was re-centrifuged at 27000 x g for 30 min at 4 ⁰C. The pellet was successively washed with 1 M KCl in presence of 40 mM EDTA, with 1 M KCl, and twice with 0.1 M Na₂CO₃, with 30 min centrifugation at 27,000 g at 4 ⁰C after each washing step. The purified BBM pellet was finally solubilized in storage buffer (100 mM Mannitol, 1 mM Heppes-Tris pH: 7.5, 1% SDS and 1% CHAPS) and stored at -20 ⁰C.

A comparison of the degradation between control and the method with amastatin, peptide substrates are shown in Figure 4 (SDS PAGE) and 5 (Amonopeptidase N in dilution series).

Example 2: Gel electrophoresis and protein digestion

The control and the inhibitor/EDTA BBM preparations (before and after the washes) were analyzed by SDS-PAGE using a NuPAGE 10% Bis-Tris gel (Invitrogen). The gels were stained by colloidal Coomassie Blue (Invitrogen). The gel lanes were systematically excised into sections (see: Figure 4). Proteins present in each gel piece were digested with trypsin and tryptic peptides were extracted with acetonitrile (ACN) and formic acid. The extracted peptide samples were dried out in a speed vac and were re- dissolved in aqueous solution that contains 2% ACN and 0.5% acetic acid, prior to analysis.

Example 3: Reverse phase liquid chromatography electrospray tandem mass spectrometry (LC-MS/MS)

Each peptide mixture was separated by nanoLC (Diomex)_and analyzed on-line by electrospray tandem mass spectrometry using an a LTQ-Orbitrap mass spectrometer (ThermoJ. The system consisted of a nano flow HPLC (Ultimate 3000, DIONEX). The column (75 μm x 100 mm) was packed in house with Reprosil 70 C18, 3 μm material.

Mobile phase A was 2% ACN and 0.5% acetic acid and mobile phase B was 80 % ACN and 0.5% acetic acid. Gradient elution was carried out from 5% B to 60% B in 75 min. Eluting peptides were detected and sequenced using an LTQ Orbitrap instrument (Thermo). The full scan survey was performed in the Orbitrap detector at 30,000 resolution while daughter spectra were acquired in the LTQ mass spectrometer.

Example 4: Data analysis

MS/MS spectra were processed through the SEQUEST software package (Thermo) against the a database comprising all mouse proteins using the following criteria: parent mass ± 10 ppm, daughter ions ± 0.5Da, tryptic cleavage specificity with one miss cleavage allowed. Peptides that had Xcorr of 1.5/2.0/2.5 for single/double/triple charged precursor and dCn>0.1 were accepted for further processing. A protein that was represented by at least two different peptides was considered confidently identified.

Example 5: Western Blot analysis

The separated proteins in ID SDS-PAGE gel (see Example 2), were transferred to a PVDF membrane (Perkin-Ellmer) and probed with a goat polyclonal primary antibody against mouse aminopeptidase N (aa69-966), (R&D Systems). The secondary antibody was a donkey anti-goat antibody, HRP-conjugated (Santa Cruz Biotechnology). Blots were developed with eclectrochemiluminescence (ECL) detection reagent according to manufacturer's instructions (Amersham Biosciences, France)

Results:

The modified CaCl₂ precipitation in combination with the high salt and high pH washes (Zhao Y., Zhang W., Kho Y., and Zhao Y., Anal Chem., 76 (2004) 1817-1823) was very effective for the enrichment of very hydrophobic proteins, for example, some of the important transporters and receptors that have been described to play critical role in cholesterol absorption (table 1). The addition of Amastatin (a general aminopeptidase inhibitor) in combination with peptide substrates specifically chosen to compete for the highly abundant peptidases of the BBM membrane fraction (table 2), help to decrease protein degradation (see figure 5: western blot of Aminopeptidase N).

The partial inhibition of membrane peptidases such as Aminopeptidase N, DPP IV, Neprilysin, is also visible in the comparison of the number of identified different peptides for each protein and the number of times that this protein has been identified in each gel lane (see examples on table 3).

The protein identification of BBM vesicles has been the target of many recent publications (Babusiak M., Man P., Petrak J., and Vyoral D., Proteomics, 7 (2007) 121- 129; Nguyen H.T.T., Amine A.B., Laffite D., Waheed A.A., Nicoletti C, Villard C, Letisse M., Deyris V., Roziere M., Tchiakpe L., Danielle C, and Comeau L., Hiol A., Biochemical and Biophysical Research Communication, 342 (2006) 236-244; Blonder J., Hale M.L., Lucas D.A., Schaefer C.F., Yu L., Conrads T.P, Issaq HJ, Stiles B.G, and Veenstra T.D., Electrophoresis, 25 (2004) 1307-1318) in the proteomic field, but so far there is none that shows identification of lower abundant proteins such as receptors and transporters that participate in cholesterol absorption. The additional steps and the washes in the membrane preparation were essential for the identification of low abundant proteins. The inhibition of the BBM peptidases is critical for further studies in cholesterol absorption, where different cholesterol inhibitors are used and the protein abundance and distribution needs to be defined and compared quantitatively.

Even the partial inhibition of these resistant peptidases can enhance the reliability of a western blot technique. As an example, if an antibody is raised against a specific epitope, the protein signal in a western blot can decrease or disappear and/or degradation products maybe detected. This might lead to a false interpretation of the results if protein degradation is not considered.

Table 1: purified BBM proteins according method of invention (possible candidates of cholesterol absorption) Mass = mass in kDa of proteins, Coverage = the amino acid sequence coverage of the protein that is covered by the identified peptide, PepCount = sum of the times a petide has been identified for a given protein; DifferentPeps = number of different peptides that have been identified in a given protein; Occurrence = number of gel bands that a given protein has been identified The rank order is representative of the level of abundance Rank 1 is the most abundant protein with the highest peptide count and the most different peptides.

Table 2: Most abundant proteins in the BBM fraction. Peptidases are highlighted in grey. Mass = mass in kDa of proteins, Coverage = the amino acid sequence coverage of the protein that is covered by the identified peptide; PepCount = sum of the times a peptide has been identified for a given protein; DifferentPeps = the number of different peptides that have been identified in a given protein; Occurrence = The number of gel bands that a given protein has been identified The rank order is representative of the level of abundance. Rank 1 is the most abundant protein with the highest peptide count and the most different peptides

Claims

1. Method for purifying brush border membranes comprising the following steps: a) providing tissue comprising brush border membranes; b) adding an inhibitor of microvillar membrane peptidases to the tissue; c) homogenizing the tissue; d) subjecting the homogenate to a low speed centrifugation resulting in a pellet and a supernatant; e) subjecting the supernatant of step d) to a high speed centrifugation resulting in a pellet and a supernatant; f) resuspending the pellet of step e); g) subjecting the resuspended pellet of step f) to a CaCl₂ or MgCl₂ precipitation resulting in a suspension; h) subjecting the suspension of g) to a low speed centrifugation resulting in a pellet and a supernatant; i) subjecting then the supernatant of step h) to a high speed centrifugation resulting in a pellet and supernatant; and k) washing the pellet of step i).

2. Method according to claim 1 wherein the peptidase inhibitor is amastatin.

3. Method according to claim 1 or 2, wherein a peptidase substrate is added in step b).

4. Method according to any one of claims 1 to 3, wherein a peptidase substrate is added in step f).

5. Method according to claim 3 or 4, wherein, the peptidase substrate is Angiotensin 1-5, Ala-Pro-PNA, or Met-Bradykinin, or a mixture thereof.

6. Method according to any one of claims 1 to 5, wherein the peptidase inhibitor is additionally added in step f)-

7. Method according to any one of claims 1 to 5, wherein the peptidase inhibitor is additionally added at each one of steps c) to k) of the method.

8. Method according to any one of claims 1 to 7, wherein a chelating agent is added to the supernatant resulting from step h).

9. Method according to any one of claims 1 to 8, wherein in step k) the pellet of step i) is washed with a high salt solution and a high pH solution.

10. Purified brush border membranes obtainable by the methods according to any one of claims 1 to 9.

11. Use of amastatin for purifying brush border membranes.

12. Methods, use and proteins substantially as described herein before especially with reference to the foregoing examples.