Aminopeptidase N (CD13) Is a Molecular Target of the Cholesterol Absorption Inhibitor Ezetimibe in the Enterocyte Brush Border Membrane
2004; Elsevier BV; Volume: 280; Issue: 2 Linguagem: Inglês
10.1074/jbc.m406309200
ISSN1083-351X
AutoresWerner Kramer, Frank Girbig, Daniel Corsiero, Anja Pfenninger, Wendelin Frick, Gerhard Jähne, Matthias Rhein, Wolfgang Wendler, Friedrich Lottspeich, Elisabeth O. Hochleitner, Evelyn Orsó, Gerd Schmitz,
Tópico(s)Peptidase Inhibition and Analysis
ResumoIntestinal cholesterol absorption is an important regulator of serum cholesterol levels. Ezetimibe is a specific inhibitor of intestinal cholesterol absorption recently introduced into medical practice; its mechanism of action, however, is still unknown. Ezetimibe neither influences the release of cholesterol from mixed micelles in the gut lumen nor the transfer of cholesterol to the enterocyte brush border membrane. With membrane-impermeable Ezetimibe analogues we could demonstrate that binding of cholesterol absorption inhibitors to the brush border membrane of small intestinal enterocytes from the gut lumen is sufficient for inhibition of cholesterol absorption. A 145-kDa integral membrane protein was identified as the molecular target for cholesterol absorption inhibitors in the enterocyte brush border membrane by photoaffinity labeling with photoreactive Ezetimibe analogues (Kramer, W., Glombik, H., Petry, S., Heuer, H., Schäfer, H. L., Wendler, W., Corsiero, D., Girbig, F., and Weyland, C. (2000) FEBS Lett. 487, 293–297). The 145-kDa Ezetimibe-binding protein was purified by three different methods and sequencing revealed its identity with the membrane-bound ectoenzyme aminopeptidase N ((alanyl)aminopeptidase; EC 3.4.11.2; APN; leukemia antigen CD13). The enzymatic activity of APN was not influenced by Ezetimibe (analogues). The uptake of cholesterol delivered by mixed micelles by confluent CaCo-2 cells was partially inhibited by Ezetimibe and nonabsorbable Ezetimibe analogues. Preincubation of confluent CaCo-2 cells with Ezetimibe led to a strong decrease of fluorescent APN staining with a monoclonal antibody in the plasma membrane. Independent on its enzymatic activity, aminopeptidase N is involved in endocytotic processes like the uptake of viruses. Our findings suggest that binding of Ezetimibe to APN from the lumen of the small intestine blocks endocytosis of cholesterol-rich membrane microdomains, thereby limiting intestinal cholesterol absorption. Intestinal cholesterol absorption is an important regulator of serum cholesterol levels. Ezetimibe is a specific inhibitor of intestinal cholesterol absorption recently introduced into medical practice; its mechanism of action, however, is still unknown. Ezetimibe neither influences the release of cholesterol from mixed micelles in the gut lumen nor the transfer of cholesterol to the enterocyte brush border membrane. With membrane-impermeable Ezetimibe analogues we could demonstrate that binding of cholesterol absorption inhibitors to the brush border membrane of small intestinal enterocytes from the gut lumen is sufficient for inhibition of cholesterol absorption. A 145-kDa integral membrane protein was identified as the molecular target for cholesterol absorption inhibitors in the enterocyte brush border membrane by photoaffinity labeling with photoreactive Ezetimibe analogues (Kramer, W., Glombik, H., Petry, S., Heuer, H., Schäfer, H. L., Wendler, W., Corsiero, D., Girbig, F., and Weyland, C. (2000) FEBS Lett. 487, 293–297). The 145-kDa Ezetimibe-binding protein was purified by three different methods and sequencing revealed its identity with the membrane-bound ectoenzyme aminopeptidase N ((alanyl)aminopeptidase; EC 3.4.11.2; APN; leukemia antigen CD13). The enzymatic activity of APN was not influenced by Ezetimibe (analogues). The uptake of cholesterol delivered by mixed micelles by confluent CaCo-2 cells was partially inhibited by Ezetimibe and nonabsorbable Ezetimibe analogues. Preincubation of confluent CaCo-2 cells with Ezetimibe led to a strong decrease of fluorescent APN staining with a monoclonal antibody in the plasma membrane. Independent on its enzymatic activity, aminopeptidase N is involved in endocytotic processes like the uptake of viruses. Our findings suggest that binding of Ezetimibe to APN from the lumen of the small intestine blocks endocytosis of cholesterol-rich membrane microdomains, thereby limiting intestinal cholesterol absorption. Intestinal cholesterol absorption is a main regulator of serum cholesterol homeostasis (1Grundy S.M. Annu. Rev. Nutr. 1983; 3: 71-96Crossref PubMed Scopus (210) Google Scholar) involving digestion and hydrolysis of dietary lipids with formation of mixed micelles containing cholesterol, bile salts, fatty acids, and phospholipids (2Dawson P.A. Rudel L.L. Curr. Opin. Lipidol. 1999; 10: 315-320Crossref PubMed Scopus (96) Google Scholar). The molecular mechanisms being involved in cholesterol absorption are not understood but the findings of a strong species difference (2Dawson P.A. Rudel L.L. Curr. Opin. 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Investigations with CaCo2 cells have shown that cholesterol taken up from mixed cholesterol bile salt micelles is distributed into the brush border membrane and is moved to detergent-resistant microdomains (rafts) followed by transport from these microdomains to the endoplasmic reticulum for esterification and further assembly into chylomicron particles that are secreted by the enterocyte (11Field F.J. Born E. Murthy S. Mathur S.N. J. Lipid Res. 1998; 39: 1938-1950Abstract Full Text Full Text PDF PubMed Google Scholar). This suggests that cholesterol absorption occurs by a complex process involving several proteins rather than by a single cholesterol transporter. Consequently, to elucidate the molecular mechanisms involved in intestinal cholesterol absorption we attempted to identify the protein components of this machinery by photoaffinity labeling using photoreactive analogues of the cholesterol absorption inhibitor Ezetimibe (12Kramer W. Glombik H. Petry S. Heuer H. Schäfer H.L. Wendler W. Corsiero D. Girbig F. Weyland C. FEBS Lett. 2000; 487: 293-297Crossref PubMed Scopus (58) Google Scholar, 13Kramer W. Girbig F. Corsiero D. Burger K. Fahrenholz F. Glombik H. Heuer H. Paumgarther G. Keppler D. Leuschner U. Stiehl A. Bile Acids: From Genomics to Disease and Therapy. Kluwer Academic Publishers, Dordrecht2002: 147-160Google Scholar) (Fig. 1) and of cholesterol (14Thiele C. Hannah M.J. Fahrenholz F. Huttner W.B. Nat. Cell Biol. 2000; 2: 42-49Crossref PubMed Scopus (461) Google Scholar). With photoreactive Ezetimibe derivatives we identified an integral 145-kDa membrane protein as the target protein for cholesterol absorption inhibitors in the enterocyte brush border membrane (12Kramer W. Glombik H. Petry S. Heuer H. Schäfer H.L. Wendler W. Corsiero D. Girbig F. Weyland C. FEBS Lett. 2000; 487: 293-297Crossref PubMed Scopus (58) Google Scholar, 13Kramer W. Girbig F. Corsiero D. Burger K. Fahrenholz F. Glombik H. Heuer H. Paumgarther G. Keppler D. Leuschner U. Stiehl A. Bile Acids: From Genomics to Disease and Therapy. Kluwer Academic Publishers, Dordrecht2002: 147-160Google Scholar), whereas an integral 80-kDa membrane protein was identified as a specific cholesterol-binding protein (15Kramer W. Girbig F. Corsiero D. Burger K. Fahrenholz F. Jung C. Müller G. Biochim. Biophys. Acta. 2003; 1633: 13-26Crossref PubMed Scopus (34) Google Scholar). The 145-kDa Ezetimibe-binding protein showed an exclusive affinity for cholesterol absorption inhibitors, but did not bind cholesterol or phytosterols. Vice versa, the 80-kDa cholesterol-binding protein only bound cholesterol and plant sterols without showing affinity for cholesterol absorption inhibitors. Both binding proteins have an identical tissue distribution restricted to the anatomical site of cholesterol absorption, the small intestine (13Kramer W. Girbig F. Corsiero D. Burger K. Fahrenholz F. Glombik H. Heuer H. Paumgarther G. Keppler D. Leuschner U. Stiehl A. Bile Acids: From Genomics to Disease and Therapy. Kluwer Academic Publishers, Dordrecht2002: 147-160Google Scholar, 15Kramer W. Girbig F. Corsiero D. Burger K. Fahrenholz F. Jung C. Müller G. Biochim. Biophys. Acta. 2003; 1633: 13-26Crossref PubMed Scopus (34) Google Scholar). In the present article we localized the molecular mode of action of cholesterol absorption inhibitors to the luminal side of the small enterocyte brush border membrane and identified the 145-kDa target protein for Ezetimibe in the enterocyte brush border membrane as the ectoenzyme aminopeptidase N ((alanyl) aminopeptidase; EC 3.4.11.2; leukemia antigen CD13).Fig. 5Purification of the 145-kDa Ezetimibe-binding protein after photoaffinity labeling with the cholesterol absorption inhibitor [3H]C-1. Wheat germ lectin chromatography was used in a and b. 8 Samples of rabbit ileal BBMV (250 μg of protein) were photolabeled each with 66 nm (0.3 μCi) [3H]C-1. After washing and solubilization, the solubilized membrane proteins were bound to wheat germ lectin-agarose. After washing off unbound proteins, lectin-bound proteins were eluted with 4 portions of 300 mmN-acetylglucosamine in 10 mm Tris/Hepes buffer (pH 7.4), 100 mm NaCl, 100 mm mannitol. Aliquots from the solubilized BBMV proteins (S), the flow-through (FT), and the N-acetylglucosamine eluates (E1 and E2) were removed, proteins were precipitated and analyzed by SDS-PAGE. a, Coomassie staining. b, distribution of radioactively labeled 145-kDa binding protein. Hydroxylapatite chromatography was used in c–e. The eluates from wheat germ lectin chromatography containing the 3H-photolabeled 145-kDa Ezetimibe-binding protein were applied to a hydroxylapatite column and bound proteins were eluted with the indicated phosphate gradient. Aliquots from each fraction were removed for the determination of radioactivity, protein pattern, and distribution of radiolabeled proteins. c, elution profile. Dotted line, phosphate gradient. ▪, distribution of radioactivity. d, Coomassie staining of eluted fractions. e, distribution of radioactivity after SDS-PAGE of eluted fractionsView Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 9Photoaffinity labeling of small intestinal segments with biotin-Ezetimibe-photoaffinity probe C-5. BBMV (100 μg of protein) of intestinal segments 1–8 were incubated with 9 μm C-5 for 60 min at 20 °C in the dark followed by irradiation for 30 s at 254 nm. After washing, BBMV proteins were solubilized and proteins covalently modified with the Ezetimibe photoprobe were extracted with streptavidin beads and were eluted after extensive washing of the beads with SDS sample buffer. Equal aliquots of each extract (for in total 8 gels) were used for the various detection methods: a, Coomassie staining; b, streptavidin staining; c, staining with actin antibodies; d, staining with annexin II antibodies.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 1Structures of 2-azetidinone cholesterol absorption inhibitors, their photoreactive analogues, and an Ezetimibe affinity matrix.A, Ezetimibe; B, S 6053; C, photoaffinity probe C-1; D, photoaffinity probe C-2; E, biotin-tagged photoaffinity probe C-4; F, biotin-tagged photoaffinity probe C-5; G, Ezetimibe affinity matrix.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The cholesterol absorption inhibitors C-1, C-2, S 6053, S 6130, ezetimibel, ezetimibe glucuronide, and S 6504 were synthesized at Aventis Pharma Deutschland GmbH according to published procedures (16Kramer, W., and Glombik, H. (August 29, 2000) International Patent Application WO 02/18432Google Scholar, 17Glombik, H., Kramer, W., Flohr, S., Frick, W., Heuer, H., Jaehne, G., Linden-schmidt, A., and Schaefer, H. L. (December, 21, 2002) PCT International Patent Application WO2002050027Google Scholar). Synthesis of the biotin-tagged photoreactive cholesterol absorption inhibitor C-4 ((5-(2-oxo-hexahydro-thieno(3,4)imidazol-6-yl)-pentanoic acid-[2-(4-azido-phenyl)-1,1(4-{4-[3-(3-hydroxy-3-phenyl-propyl)-2-(4-methoxy-phenyl)-4-oxo-azetidin-1-yl]-phenylcarbamoyl}-butylcarbamoyl)-ethyl]-amide) and its methylene homologue C-5 (Fig. 1) were synthesized as described elsewhere (16Kramer, W., and Glombik, H. (August 29, 2000) International Patent Application WO 02/18432Google Scholar, 18Frick W. Bauer-Schäfer A. Girbig F. Corsiero D. Heuer H. Kramer W. Bioorg. Med. Chem. 2003; 11: 1639-1642Crossref PubMed Scopus (28) Google Scholar). Fluoresterol (22-(N-(7-nitrobenz-2-oxa-1,3-diazo)-4-yl)amino)-23,24-bisnor-5-cholen-3β-ol) was obtained from Molecular Probes (Eugene, OR). Triton X-100, Triton X-114, acrylamide, N,N′-bismethylene acrylamide, and Serva Blue R 250 were from Serva (Heidelberg, Germany), whereas Nonidet P-40, cholesterol semisuccinate, biotin, and marker proteins for the determination of the molecular masses were from Sigma. Other detergents used were purchased as follows: n-octyl glucoside from Alexis (Grünberg, Germany), n-dodecylmaltoside, n-decylmaltoside, and digitonin from Fluka (Buchs, Switzerland), n-lauroylsarcosine and CHAPS 1The abbreviations used are: CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; APA, aminopeptidase A; APN, aminopeptidase N; BBMV, brush border membrane vesicles; PBS, phosphate-buffered saline; SR-BI, scavenger receptor BI; NPC1L1, Niemann-Pick C1-like protein 1; FITC, fluorescein isothiocyanate. from ICN (Eschwege, Germany). Wheat germ lectin-agarose and streptavidin-agarose beads were purchased from Amersham Biosciences, whereas hydroxylapatite and prefilled hydroxylapatite columns CHT-II (5 ml bed volume) were from Bio-Rad. Scintillator Quickszint 501 and the tissue solubilizer Biolute S were from Zinsser Analytic GmbH (Frankfurt, Germany) and scintillator Picofluor 40 from Packard. Protein was determined with a Bradford assay kit from Bio-Rad and enzymatic activities for aminopeptidase N (EC 3.4.11.2) was measured using the Merckotest Kit 3559 (Merck kGa, Darmstadt, Germany), whereas the activity of sucrase and isomaltase was determined according to Dahlqvist (19Dahlqvist A. Anal. Biochem. 1964; 7: 18-25Crossref PubMed Scopus (1726) Google Scholar). Antibodies against rabbit aminopeptidase N were generated at Biogenes (Berlin, Germany). Antibodies were raised in chicken against amino acid sequences 34–38 (antibody APN 3624) and 848–862 (antibody APN 3625) conjugated to LPH (hemocyanine from Limulus polyphemus) followed by purification of the IgY fraction from egg yolk. Anti-actin antibodies (AC-40, antibody 11003) were purchased from Abcam (Cambridge, UK), the anti-annexin II antibody (610069) was from BD Biosciences, and the streptavidin-alkaline phosphatase conjugate (RPN 1234) from Amersham Biosciences. FITC-conjugated monoclonal anti-human CD13 antibodies, raised in mice (clone WM-47) were from Sigma. Male New Zealand White rabbits weighing 4–5 kg (Harlan Winkelmann, Borchem, Germany) were kept on Altromin® standard diet C 2023 (Altromin®, Lage, Germany) ad libitum. Brush border membrane vesicles (BBMV) from rabbit stomach, duodenum, jejunum, ileum, cecum, colon, rectum, and kidney were prepared by the Mg2+ precipitation method as described earlier (20Kramer W. Girbig F. Gutjahr U. Kowalewski S. Jouvenal K. Müller G. Tripier D. Wess G. J. Biol. Chem. 1993; 268: 18035-18046Abstract Full Text PDF PubMed Google Scholar). For preparation of BBMV from small intestinal segments, the small intestine from 2 rabbits was divided in 8 segments of equal length (numbered 1 to 8 from duodenum to ileum) and BBMV were prepared and characterized as described (20Kramer W. Girbig F. Gutjahr U. Kowalewski S. Jouvenal K. Müller G. Tripier D. Wess G. J. Biol. Chem. 1993; 268: 18035-18046Abstract Full Text PDF PubMed Google Scholar). Rat liver microsomes and rat adipocyte membranes were prepared as described elsewhere (21Arion W.J. Canfield W.K. Ramos F.C. Su M.L. Burger H.J. Hemmerle H. Schubert G. Below P. Herling A.W. Arch. Biochem. Biophys. 1998; 351: 279-285Crossref PubMed Scopus (67) Google Scholar, 22Müller G. Wetekam E.A. Jung C. Bandlow W. Biochemistry. 1994; 33: 12149-12159Crossref PubMed Scopus (33) Google Scholar). Intestinal cholesterol absorption was determined by a modification of the Zilversmit/Hughes method (23Zilversmit D.B. Hughes L.B. J. Lipid Res. 1974; 15: 465-473Abstract Full Text PDF PubMed Google Scholar) as described earlier (12Kramer W. Glombik H. Petry S. Heuer H. Schäfer H.L. Wendler W. Corsiero D. Girbig F. Weyland C. FEBS Lett. 2000; 487: 293-297Crossref PubMed Scopus (58) Google Scholar). Cholesterol monomerization activity from mixed micelles was determined by measurement of the dequenching of fluoresterol-labeled cholesterol micelles using the assay described by Cai et al. (24Cai T.Q. Guo Q. Wong B. Milot D. Zhang L. Wright S.D. Biochim. Biophys. Acta. 2002; 1581: 100-108Crossref PubMed Scopus (13) Google Scholar). The uptake of [3H]cholesterol from mixed micelles by rabbit small intestinal BBMV was determined as described by Compassi et al. (25Compassi S. Werder M. Weber F.E. Bofelli D. Hauser H. Schulthess G. Biochemistry. 1997; 36: 6643-6652Crossref PubMed Scopus (35) Google Scholar) using mixed micelles prepared by mixing 10 μm [3H]cholesterol with a solution of 4 mm taurocholate and 0.6 mm sodium oleate in 50 mm Tris/HCl buffer (pH 7.4), 150 mm NaCl buffer, followed by sonification and evaporation of chloroform. Uptake was measured by mixing BBMV (20 μg of protein) in 50 μl of 10 mm Tris/Hepes buffer (pH 7.4), 100 mm NaCl, 100 mm mannitol in the absence or presence of 150 μm of the respective Ezetimibe analogues with 50 μl of the mixed micelle suspension, followed by centrifugation at 100,000 × g for 120 s after the indicated incubation times and subsequently radioactivity in the pelleted material and the supernatant was determined by liquid scintillation counting. BBMV prepared from the indicated tissues were incubated with the indicated concentrations (see legends to figures) of Ezetimibe-photoaffinity probes in 10 mm Tris/HCl buffer (pH 7.4), 100 mm NaCl, 100 mm mannitol for 60 min at 20 °C in the dark followed by irradiation in a Rayonet-RPR-photochemical reactor RPR-100 (The Southern Ultraviolet Company, Hamden, CT) equipped with 4 RPR 2530-Å lamps for 30 s (C-1, C-4, C-5) or 120 s (C-2). Afterward, BBMV were diluted with 10 mm Tris/HCl buffer (pH 7.4), 300 mm mannitol, 1 mm Pefabloc and washed with this buffer 3 times followed by SDS-PAGE. Solubilization of BBMV was performed at a protein concentration of 1 mg/ml for 60 min at 4 °C in 10 mm Tris/Hepes buffer (pH 7.4), 75 mm KCl, 5 mm MgCl2, 1 mm EGTA, 1 mm dithiothreitol, 1% n-octyl glucoside, 1% Triton X-100, 1 mm Pefabloc ("solubilization buffer") followed by centrifugation at 100,000 × g for 30 min. Alternatively, BBMV were first solubilized in 0.25% N-lauroylsarcosine at 4 °C for 30 min, followed by a 10-fold dissolution with solubilization buffer. Wheat Germ Lectin and Hydroxylapatite Chromatography After Photoaffinity Labeling with 3H-Labeled Ezetimibe Photoprobes—Eight samples of rabbit ileal BBMV (250 μg of protein) were incubated each with 66 nm (0.3 μCi) of [3H]C-1 or 3 μm (1 μCi) [3H]C-2 in 10 mm Tris/Hepes buffer (pH 7.4), 100 mm NaCl, 100 mm mannitol for 30 min at 20 °C in the dark. After irradiation for 30 s ([3H]C-1) or 120 s ([3H]C-2) at 254 nm the samples were collected and after washing three times membrane proteins were solubilized with solubilization buffer. The supernatant containing solubilized membrane proteins was added to 0.5 ml of wheat germ lectin-agarose gel. After 60 min at 20 °C the beads were collected by centrifugation and washed 3 times with 2 ml of 10 mm PBS, 1% n-octyl glucoside. Adsorbed proteins were eluted with 4 portions of 1 ml of 10 mm PBS, 1% n-octyl glucoside, 300 mmN-acetyl-d-glucosamine each. The N-acetyl-d-glucosamine eluates were diluted to 10 ml with 10 mm sodium phosphate buffer (pH 7.4), 1% (w/v) n-octyl glucoside and applied to a hydroxylapatite column (self-filled, 10 cm height, 1 cm diameter; or Bio-Rad CHT-II columns) equilibrated with 10 mm sodium phosphate buffer (pH 7.4), 1% (w/v) n-octyl glucoside at a flow rate of 0.15 ml/min and collection of 1-ml fractions. Subsequently proteins were eluted as follows: 10 ml of 10 mm sodium phosphate buffer (pH 7.4), 1% (w/v) n-octyl glucoside followed by linear phosphate gradients in the above buffer. In each fraction the activity of aminopeptidase N, sucrase, and cholesterol monomerization as well as radioactivity was determined. From each fraction 100-μl aliquots were analyzed by SDS-PAGE with subsequent determination of the distribution of radioactively labeled proteins, after precipitation of proteins with chloroform/methanol (26Wessel D. Flügge U.J. Anal. Biochem. 1984; 138: 141-143Crossref PubMed Scopus (3191) Google Scholar). Streptavidin-biotin Chromatography after Photolabeling with C-4 or C-5—Ten samples of rabbit ileal BBMV (200 μg of protein) were incubated with the biotin-tagged cholesterol absorption inhibitors C-4 or C-5 in 10 mm Tris/Hepes buffer (pH 7.4), 100 mm NaCl, 100 mm mannitol for 30 min in the dark at 20 °C followed by irradiation at 254 nm for 30 s. After washing, proteins were solubilized and the clear supernatant was mixed with 0.5 ml of streptavidin-agarose beads and kept under stirring at 4 °C for 2 h. After centrifugation, the beads were incubated with 2 ml of 10 mm Tris/Hepes buffer (pH 7.4), 300 mm mannitol, 1% n-octyl glucoside, 4 mm phenylmethylsulfonyl fluoride, 4 mm iodacetamide, 4 mm EDTA for 10 min at 4 °C followed by centrifugation. After repeating this procedure twice, proteins were eluted from the streptavidin-agarose beads with 2 ml of the above buffer containing 6 mm biotin standing overnight at 4 °C, repeated with 2 ml for 1 h at 4 °C. Final purification was achieved by preparative SDS-gel electrophoresis (6% gel, 28 mm diameter, 5-cm gel length, Bio-Rad) at 500 V (40 mA, 6 W) and the eluates were fractionated into 0.6-ml fractions. Ezetimibe Affinity Chromatography—An Ezetimibe affinity matrix was synthesized by coupling of 1-(4-aminomethyl-phenyl)-3-(3-hydroxy-3-phenyl-propyl)-4-(4-methoxy-phenyl)-azetidin-2-on (16Kramer, W., and Glombik, H. (August 29, 2000) International Patent Application WO 02/18432Google Scholar) to a hydroxy succinimidyl-hexanoyl matrix (Hi-Trap column, Amersham Biosciences) according to the protocol of the manufacturer. N-Acetyl-d-glucosamine eluates from wheat germ agglutinin chromatography were applied to the Ezetimibe affinity column at a flow rate of 0.25 ml/min followed by elution with 10 ml of buffer and collection of 500-μl fractions. Bound proteins were eluted with 5 ml of PBS (pH 7.4), 3% Triton X-100. From all fractions aliquots were analyzed for the enzymatic activity of aminopeptidase N and sucrase as well as protein composition by SDS-gel electrophoresis. SDS-PAGE was carried out in vertical stab gels (20 × 17 × 0.15 cm) using an electrophoresis system LE 2/4 (Amersham Biosciences) with gel concentrations of 7–10.5% at a ratio of 97.2% acrylamide and 2.8% N,N-methylene bisacrylamide or in pre-casted NOVEX gels (4–12, 12, or 15%, Invitrogen, Groningen, The Netherlands) using an electrophoresis system XCell II from Novex (27Kramer W. Girbig F. Glombik H. Corsiero D. Stengelin S. Weyland C. J. Biol. Chem. 2002; 276: 36020-36027Abstract Full Text Full Text PDF Scopus (36) Google Scholar). After electrophoresis the gels were fixed in 12.5% trichloroacetic acid followed by staining with Serva Blue R 250. For determination of the distribution of radioactivity, individual gel lanes were cut into 2-mm pieces, protein was hydrolyzed with 250 μl of tissue solubilizer Biolute S, and after addition of 4 ml of scintillator Quickszint 501 radioactivity was measured by liquid scintillation counting. Western blotting and immunostaining was performed as described earlier (27Kramer W. Girbig F. Glombik H. Corsiero D. Stengelin S. Weyland C. J. Biol. Chem. 2002; 276: 36020-36027Abstract Full Text Full Text PDF Scopus (36) Google Scholar). Caco-2 cells (ATCC HTB-37) were grown at 37 °C and 10% CO2 in culture medium: Dulbecco's modified Eagle's medium (Invitrogen 41965), supplemented with 10% fetal calf serum (Invitrogen 16000), 1% l-glutamin (Invitrogen 25030), 1% nonessential amino acids (Invitrogen 11140), and 1% penicillin/streptomycin solution (Invitrogen 15140). To perform a cholesterol uptake assay, confluent Caco-2 cells from a 175-cm2 flask (BD Biosciences) were washed once with PBS (Invitrogen 14190), treated with 4 ml of trypsin-EDTA solution (Invitrogen 25300), resuspended in a 6-ml culture medium, and counted in a counting device (Schaerfe System, Casy TT). Cells were diluted in culture medium to a final concentration of 1 × 106 cells/ml. Each well of a 24-well plate (Falcon 351147) was filled up with 1 ml of culture medium and a single cell culture insert (1.0 μm pore size, Falcon 353104). 300 μl of the Caco-2 cell suspension (300,000 cells) were seeded into each cell culture insert. Cells were incubated at 37 °C and 10% CO2 up to 21 days. During that period medium was replaced every 2–3 days. Confluent CaCo-2 cells cultivated for a further 11 or 20 days were used for the transport experiments. Cells were incubated either with culture medium (see above) (control), medium containing 150 μm of the cholesterol absorption inhibitors Ezetimibe or S 6130 and kept at 37 °C overnight. After 12 h of incubation 100 μl of mixed micelles containing 100 μm [3H]cholesterol, 4 mm taurocholate, and 0.6 mm sodium oleate in 50 mm Tris/HCl buffer (pH 7.4), 150 mm NaCl were added and [3H]cholesterol uptake was measured after 0, 1, 2, 3, 4, and 5 h of incubation. Medium was removed, and cells were washed twice with 10 mm Tris/Hepes buffer (pH 7.5), 300 mm mannitol, Pefabloc. After transfer to scintillation vials, 400 μl of tissue solubilizer Biolute S (Zinsser Analytic, Frankfurt, Germany) was added followed after 2 h by scintillator Picofluor 40 (PerkinElmer Life Sciences) and radioactivity was measured with a Wallac liquid scintillation counter. For immunofluorescence staining of CD13, CaCo-2 cells (3 days after post-confluence) were cultured on Lab-Tek™ sodium borosilicate coverslips (Nunc, Wiesbaden, Germany). The cells were rinsed several times with Dulbecco's modified PBS (Biochrom, Berlin, Germany). Incubation either with vehicle (Dulbecco's PBS) or with 10 μm Ezetimibe at room temperature for 15 min was carried out before or after immunofluorescence staining of the cells with FITC-CD13 mAB. Following extensive washing the cells were fixed with methanol/acetone (1:1) at -20 °C for 10 min and processed for confocal laser scanning microscopy according to standard protocols. Fluorescence staining was recorded at 488 nm excitation with an inverted TCS 4D confocal laser scanning microscope (Leica Lasertechnik, Heidelberg, Germany), equipped with an Ar-Kr 75 mV mixed ion laser (Melles Griot Inc., Carlsbad, CA) and an acousto-optical transmission filter. Images in horizontal x-y sections were acquired by using the ScanWare instrument software. Image analysis was performed using the MetaMorph® software package (Universal Imaging Corp., Downingtown, PA). Coomassie Blue-stained protein bands were excised, destained (3 times for 30 min with 100 μl of 50% acetonitrile, 25 mm NH4HCO3, pH 8.0), and dried by acetonitrile. Gel pieces were rehydrated in 15 μl of trypsin solution (5 μg/ml) (rec., proteomics grade, Roche Diagnostics) and incubated at 37 °C overnight. Peptide extracts (50% acetonitrile, 5% trifluoroacetic acid) were pooled (3 × 30 μl), lyophilized, and reconstituted in 13 μl 2% acetonitrile, 0.1% trifluoroacetic acid. Analysis of the peptide samples was performed on a nano-ESI-LC-MS/MS system (LC Packings, Amsterdam, coupled to a LCQ Deka XP mass spectrometer, Thermo Finnigan, San Jose, CA). 13 μl of sample were desalted on a C18 precolumn (PepMap, inner diameter 300 μm, 5-mm length); loading and washing of the sample with 2% aceto
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