In Vitro Protein Complex Formation with Cytoskeleton-anchoring Domain of Occludin Identified by Limited Proteolysis
2003; Elsevier BV; Volume: 278; Issue: 49 Linguagem: Inglês
10.1074/jbc.m302782200
ISSN1083-351X
AutoresBi-Hung Peng, J. Ching Lee, Gerald A. Campbell,
Tópico(s)Gut microbiota and health
ResumoOccludin is an essential membrane protein component of cellular tight junctions, participating in both cell-cell adhesion in the paracellular space and anchoring of the junctional complex to the cytoskeleton. The latter function is accomplished through binding of the C-terminal cytoplasmic region to scaffolding proteins that mediate binding to cytoskeletal actin. We isolated a structural domain from both the bacterial-expressed C-terminal cytoplasmic region of human occludin and native cellular occludin, extracted from epithelial (Madin-Darby canine kidney) or endothelial (human brain) cells, by limited proteolysis with trypsin. This human occludin domain contains the last 119 amino acids as identified by N-terminal sequencing and peptide mass fingerprinting using matrix-assisted laser desorption ionization-time of flight mass spectrometry. Based on the sequence and secondary structure prediction, this domain contains 4 of 5 α-helices in the C-terminal region and is linked to the fourth membrane-spanning region by a loosely structured tethering polypeptide. Comparison of circular dichroism spectra of recombinant proteins corresponding to the entire C-terminal region versus only the binding domain region also supports the interpretation that the helical structural elements are concentrated in that domain. Co-immunoprecipitation of this domain with ZO-2 demonstrated preservation of the specificity of the scaffolding protein-binding function, and binding studies with immobilized ZO-2 suggest the presence of multiple ZO-2 binding sites in this domain. These results provide a basis for development of a structural model of the ZO-binding site that can be used to investigate regulation of tight junction anchoring by intracellular signaling events. Occludin is an essential membrane protein component of cellular tight junctions, participating in both cell-cell adhesion in the paracellular space and anchoring of the junctional complex to the cytoskeleton. The latter function is accomplished through binding of the C-terminal cytoplasmic region to scaffolding proteins that mediate binding to cytoskeletal actin. We isolated a structural domain from both the bacterial-expressed C-terminal cytoplasmic region of human occludin and native cellular occludin, extracted from epithelial (Madin-Darby canine kidney) or endothelial (human brain) cells, by limited proteolysis with trypsin. This human occludin domain contains the last 119 amino acids as identified by N-terminal sequencing and peptide mass fingerprinting using matrix-assisted laser desorption ionization-time of flight mass spectrometry. Based on the sequence and secondary structure prediction, this domain contains 4 of 5 α-helices in the C-terminal region and is linked to the fourth membrane-spanning region by a loosely structured tethering polypeptide. Comparison of circular dichroism spectra of recombinant proteins corresponding to the entire C-terminal region versus only the binding domain region also supports the interpretation that the helical structural elements are concentrated in that domain. Co-immunoprecipitation of this domain with ZO-2 demonstrated preservation of the specificity of the scaffolding protein-binding function, and binding studies with immobilized ZO-2 suggest the presence of multiple ZO-2 binding sites in this domain. These results provide a basis for development of a structural model of the ZO-binding site that can be used to investigate regulation of tight junction anchoring by intracellular signaling events. Tight junctions (TJ) 1The abbreviations used are: TJtight junctionGUKguanylate kinaseMAGUKmembrane-associated guanylate kinase homologMDCKMadin-Darby canine kidneyPDZPSD95/dlg/ZO-1SH3Src homology 3CDcircular dichroismIPimmunoprecipitationMALDITOF MSmatrix-assisted laser desorption ionization-time of flight mass spectrometryGFAPglial fibrillary acidic proteinBCIP5-bromo-4-chloro-3-indolyl phosphateNBTnitro blur tetrazolium. play a key role in limiting the movement of water, ions, and macromolecules across both epithelial and endothelial surfaces (1.Powell D.W. Am. J. Physiol. 1981; 241: G275-G288PubMed Google Scholar, 2.Gumbiner B. Am. J. Physiol. 1987; 253: C749-C758Crossref PubMed Google Scholar, 3.Rubin L.L. Curr. Opin. Cell Biol. 1992; 4: 830-833Crossref PubMed Scopus (85) Google Scholar). Occludin, the first membrane protein isolated from TJ complexes, is a ∼60 kDa protein predicted to have four transmembrane regions, two extracellular loops, and N- and C-terminal cytoplasmic regions (4.Furuse M. Hirase T. Itoh M. Nagafuchi A. Yonemura S. Tsukita S. Tsukita S. J. Cell Biol. 1993; 123: 1777-1788Crossref PubMed Scopus (2181) Google Scholar). Several members of the claudin family of membrane proteins were later isolated from TJ (5.Furuse M. Fujita K. Hiiragi T. Fujimoto K. Tsukita S. J. Cell Biol. 1998; 141: 1539-1550ICrossref PubMed Scopus (1768) Google Scholar, 6.Mitic L.L. Van Itallie C.M. Anderson J.M. Am. J. Physiol. Gastrointest. Liver Physiol. 2000; 279: G250-G254Crossref PubMed Google Scholar, 7.Morita K. Furuse M. Fujimoto K. Tsukita S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 511-516Crossref PubMed Scopus (994) Google Scholar, 8.Morita K. Sasaki H. Fujimoto K. Furuse M. Tsukita S. J. Cell Biol. 1999; 145: 579-588Crossref PubMed Scopus (382) Google Scholar, 9.Morita K. Sasaki H. Furuse M. Tsukita S. J. Cell Biol. 1999; 147: 185-194Crossref PubMed Scopus (711) Google Scholar, 10.Niimi T. Nagashima K. Ward J.M. Minoo P. Zimonjic D.B. Popescu N.C. Kimura S. Mol. Cell. Biol. 2001; 21: 7380-7390Crossref PubMed Scopus (166) Google Scholar) and are thought to interdigitate with each other and occludin in linear strands to form the TJ complexes by sealing with the complementary strands on the apicolateral surfaces of adjacent cells (7.Morita K. Furuse M. Fujimoto K. Tsukita S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 511-516Crossref PubMed Scopus (994) Google Scholar, 11.Furuse M. Furuse K. Sasaki H. Tsukita S. J. Cell Biol. 2001; 153: 263-272Crossref PubMed Scopus (637) Google Scholar). Both occludin and the TJ-associated claudins also bind to the cytoplasmic scaffolding proteins, ZO-1, -2, and -3, which serve to link the TJ complexes to the cell cytoskeleton (12.Fanning A.S. Jameson B.J. Jesaitis L.A. Anderson J.M. J. Biol. Chem. 1998; 273: 29745-29753Abstract Full Text Full Text PDF PubMed Scopus (1142) Google Scholar, 13.Haskins J. Gu L. Wittchen E.S. Hibbard J. Stevenson B.R. J. Cell Biol. 1998; 141: 199-208Crossref PubMed Scopus (503) Google Scholar, 14.Itoh M. Furuse M. Morita K. Kubota K. Saitou M. Tsukita S. J. Cell Biol. 1999; 147: 1351-1363Crossref PubMed Scopus (934) Google Scholar, 15.Itoh M. Morita K. Tsukita S. J. Biol. Chem. 1999; 274: 5981-5986Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). These scaffolding proteins are members of the MAGUK (membrane-associated guanylate kinase containing) family of proteins, which contain variable numbers of PSD-95/discs-large/ZO-1 (PDZ) domains, a Src homology (SH3) domain, and a region homologous to guanylate kinase (GUK) in the same sequential order (16.Beatch M. Jesaitis L.A. Gallin W.J. Goodenough D.A. Stevenson B.R. J. Biol. Chem. 1996; 271: 25723-25726Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Occludin binds to the GUK domain of each of the ZO proteins, while the claudins bind to the first PDZ (PDZ-1) domain (12.Fanning A.S. Jameson B.J. Jesaitis L.A. Anderson J.M. J. Biol. Chem. 1998; 273: 29745-29753Abstract Full Text Full Text PDF PubMed Scopus (1142) Google Scholar, 14.Itoh M. Furuse M. Morita K. Kubota K. Saitou M. Tsukita S. J. Cell Biol. 1999; 147: 1351-1363Crossref PubMed Scopus (934) Google Scholar). The identification of the GUK domains of these three proteins is based on the amino acid comparison with GUK-containing proteins from the international protein data base (13.Haskins J. Gu L. Wittchen E.S. Hibbard J. Stevenson B.R. J. Cell Biol. 1998; 141: 199-208Crossref PubMed Scopus (503) Google Scholar, 17.Jesaitis L.A. Goodenough D.A. J. Cell Biol. 1994; 124: 949-961Crossref PubMed Scopus (401) Google Scholar). The sequence of the GUK domain in each of these proteins is different, but there is high homology between them. tight junction guanylate kinase membrane-associated guanylate kinase homolog Madin-Darby canine kidney PSD95/dlg/ZO-1 Src homology 3 circular dichroism immunoprecipitation matrix-assisted laser desorption ionization-time of flight mass spectrometry glial fibrillary acidic protein 5-bromo-4-chloro-3-indolyl phosphate nitro blur tetrazolium. The binding between occludin and the ZO proteins is determined by the structure of its C-terminal cytoplasmic region. It is highly likely that this binding occurs in a compactly folded domain within this region; however, previous studies have not elucidated the precise identity of such a domain or the manner of its linkage to the rest of the occludin molecule. It is also not determined whether the binding affinity of ZO-1 with occludin differs from ZO-2 or ZO-3. Furuse et al. (18.Furuse M. Itoh M. Hirase T. Nagafuchi A. Yonemura S. Tsukita S. Tsukita S. J. Cell Biol. 1994; 127: 1617-1626Crossref PubMed Scopus (820) Google Scholar) have shown that truncation of C-terminal segments of occludin weakens or eliminates the binding to ZO-1, while deletion of short segments near the N terminus of the C-terminal cytoplasmic region do not alter this binding function. Nusrat et al. (19.Nusrat A. Chen J.A. Foley C.S. Liang T.W. Tom J. Cromwell M. Quan C. Mrsny R.J. J. Biol. Chem. 2000; 275: 29816-29822Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar) studied a synthetic peptide, which they described as a "coiled-coil domain," corresponding to amino acids 440-469 of the C-terminal region of human occludin. This peptide was shown to bind to native ZO-1 from T84 epithelial cells. While truncation studies can localize the functional elements with regard to the primary sequence, and binding studies using synthetic peptides can identify a specific region primarily responsible for recognition, neither provides complete information regarding the structural motif responsible for binding the GUK domain of the scaffolding proteins or the identity of potential regulatory elements within the binding domain of occludin. In order to investigate both binding and regulation, the full domain of occludin that interacts with the GUK domain needs to be identified and isolated. Limited proteolysis, on the other hand, allows intact compact structural domains to be separated from more loosely coiled regions of the protein sequence, largely preserving the natural domain structure (20.Ramzan M.A. Cookson E.J. Beynon R.J. Biochem. Soc. Trans. 1991; 19: 296SCrossref PubMed Scopus (2) Google Scholar, 21.Belova G.I. Prasad R. Nazimov I.V. Wilson S.H. Slesarev A.I. J. Biol. Chem. 2002; 277: 4959-4965Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). In the study described in this report, we used this method to isolate a structural domain from the region of occludin known to bind the ZO proteins, then tested this domain for retention of the binding function using co-immunoprecipitation and estimated the stoichiometry and dissociation constant using a binding technique. This method has the advantage of preserving the natural structure of occludin, either as expressed in bacteria or extracted from cells, and isolating the functional region to an intact domain of minimal size to facilitate further structural investigation. N-terminal sequencing and peptide mass fingerprinting (22.Scheler C. Lamer S. Pan Z. Li X.P. Salnikow J. Jungblut P. Electrophoresis. 1998; 19: 918-927Crossref PubMed Scopus (162) Google Scholar) were employed to identify the precise sequence of the domain. Cell Culture—MDCK (ATCC, Manassas, VA) cells were cultured in medium containing MEM (minimum essential medium) supplemented with non-essential amino acids, 15 mm of HEPES and 10% fetal bovine serum. Cultures were maintained in a humidified CO2 incubator (5% CO2/95% air) at 37 °C. Expression Plasmid Construction—Methods followed the procedures of Sambrook et al. (23.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.2nd Ed. Vol. 1. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989: 21-84Google Scholar) with minor modifications. Two expression plasmids (for C-Occ and dN-Occ, defined below) were constructed by cloning the PCR-amplified occludin cDNA fragment into the expression vector pRSETA (Invitrogen; Carlsbad, CA). The template plasmid (pSK-hocc, a generous gift from Drs. C. M. Van Itallie and James M. Anderson) (24.Van Itallie C.M. Anderson J.M. J. Cell Sci. 1997; 110: 1113-1121Crossref PubMed Google Scholar) contains the entire coding region for human occludin (GenBank™ accession number U53823). PCR was performed to generate cDNA for the entire C-terminal cytoplasmic region of occludin (C-Occ) using two primers, 5′-TGCTGGATCCACTCGAAGAAAGATG-3′ and 5′-CAACTTGGCATCAGAATTCTATG-3′, and to generate a 121 amino acid deletion in the C-Occ cDNA between the His tag and the last four predicted helices (resultant protein designated dN-Occ) using the upstream primer, 5′-CAAAGGGAGGATCCGGAAGG-3′ and the same downstream primer as for C-Occ. The amplification cycle was programmed to denature the template at 95 °C for 1 min, anneal the primer to the template at 45 °C for 1 min and synthesize the cDNA at 68 °C for 1 min for a total of 30 cycles. The PCR fragment and the cloning vector pRSETA were digested with restriction enzymes, BamHI and EcoRI, and then the digested fragments were separated from undigested fragments by electrophoresis. The digested fragments were purified by electroelution using dialysis tubing with 12-kDa molecular mass cutoff (Spectrum Medical Industries, Houston, TX). After phenol/chloroform extraction and ethanol precipitation, these two purified fragments were ligated together at 16 °C overnight. After cloning the PCR fragment into pRSETA, the insert was sequenced from both directions to confirm its identity using T7 forward primer and pRSET reverse primer. His-tagged Protein Expression and Purification—Plasmid with cDNA insert was transformed into BL21 DE3 bacteria, and expression of the His-tagged proteins (C-Occ or dN-Occ) was stimulated by 0.1 mm of isopropyl-1-thio-β-d-galactopyranoside. Four hours after induction, bacteria were collected by centrifugation at 8630 × g for 5 min at 4 °C, and then lysed in high salt sodium phosphate buffer (20 mm sodium phosphate and 500 mm sodium chloride, pH 7.4) containing 100 μg/ml of lysozyme and 1× protease inhibitor mixture (Roche Applied Science). Protease inhibitor mixture was added as 100 μl per ml of solution of 10× inhibitor mixture prepared according to manufacturer's directions in high salt sodium phosphate buffer. Total soluble proteins were collected after centrifugation at 31,000 × g for 30 min at 4 °C, and then incubated with nickel-charged ProBond resin (Invitrogen) for 30 min. The protein was then eluted with increasing (150-400 mm) concentrations of imidazole from the ProBond column after washing with high salt sodium phosphate buffer containing 50-100 mm imidazole to remove other bacterial proteins nonspecifically bound to the nickel resin. The purified proteins were analyzed for purity using SDS-PAGE and Western blotting with occludin antibody (Zymed Laboratories Inc. South San Francisco, CA). Concentration of the resultant protein solution was ∼100 μg/ml. Full-length canine ZO-2 was expressed in Sf9 insect cells using the Baculovirus expression system (13.Haskins J. Gu L. Wittchen E.S. Hibbard J. Stevenson B.R. J. Cell Biol. 1998; 141: 199-208Crossref PubMed Scopus (503) Google Scholar). Sf9 culture supernatant, which contains viral particles encoding a His-tagged ZO-2, was a generous gift from Dr. Bruce R. Stevenson (University of Alberta, Alberta, Canada). Sf9 cells (1 × 106 cells/ml) were infected with ZO-2 cDNA containing baculovirus for 48 h, and then cells were harvested by centrifugation at 437 × g for 5 min. Procedures used to purify the His-tagged ZO-2 protein are the same as those for His-tagged C-Occ. SDS-PAGE and Western Blotting—For Western blotting analysis, proteins were separated by SDS-PAGE (10% for the in vitro binding assay, and 12.5% or 15% for limited proteolysis and co-immunoprecipitation), and transferred onto a polyvinylidene difluoride membrane (Millipore, Billerica, MA). The membrane was incubated with blocking solution containing 0.1% Tween 20, 5% nonfat milk, and 2.5% goat serum in phosphate-buffered saline for 1 h. The membrane was then transferred into fresh blocking solution containing primary antibody (1 μg/ml) to occludin or ZO-2, and incubated for 1 h. Unbound and non-specifically bound antibody was removed by three changes of washing solution (0.1% Tween 20 in phosphate-buffered saline) over a period of 15 min. Biotinylated secondary antibody (1:1000 dilution) was then incubated with the membrane for 30 min in washing solution with 5% nonfat milk added. After washing, the membrane was incubated with either alkaline phosphatase- or horseradish peroxidase-conjugated streptavidin (1:1000 dilution) for 15 min. All incubations were performed at room temperature with gentle rocking. The immunoreactive protein bands were visualized by incubating the membrane with alkaline phosphatase substrate, BCIP/NBT (Zymed Laboratories Inc.), or horseradish peroxidase substrate, stable DAB (Research Genetics Inc.; Huntsville, AL), respectively. Limited Proteolysis—Limited proteolysis was performed at room temperature using C-Occ prior to elution from the nickel resin. An aliquot of 50 μl of C-Occ (∼5 μg) bound to the resin was used for each reaction. Trypsin (Sigma, catalog no. T-8128, EC 3.4.21.4) or a broad-range protease derived from Streptomyces griseus (Sigma, cat. P-8811, EC 3.4.24.31) was added to the solution to a final enzyme concentration of 0.5, 5, 50, or 500 μg/ml, and incubated for 5 min. The reactions were stopped by adding 50 μl of solution containing 2× SDS-PAGE buffer and 1 m of dithiothreitol prior to electrophoresis. After SDS-PAGE and Western blotting with occludin antibody, the protein masses were calculated by comparison to known protein markers (Sigma) using the computer program SigmaGel (Jandel Scientific Software, San Rafael, CA) (25SigmaGel for Windows, Rel. 1.05, 1995. Jandel Scientific Software: San Rafael, CAGoogle Scholar). To demonstrate that the digestion pattern of purified C-Occ protein is identical to that of naturally occurring cellular occludin, MDCK epithelial cells and the gray matter of human brain tissue (a source of brain microvascular endothelial cells) were lysed in high salt sodium phosphate buffer containing 1% Nonidet P-40 and 0.5% sodium deoxycholate without protease inhibitors. After sonication for 1 min on ice, the solution was diluted 1:4 with 20 mm sodium phosphate solution prior to trypsin digestion (20 μg/ml for 30 min at room temperature). Protease inhibitor mixture in low salt sodium phosphate buffer (20 mm sodium phosphate, 125 mm sodium chloride, pH 7.4) was added as described above to stop the reaction after the incubation period, and cell debris was removed by centrifugation at 31,000 × g for 15 min at 4 °C. The digestion products were isolated by immunoprecipitation with a polyclonal occludin antibody (Zymed Laboratories Inc.. raised using a peptide consisting of the last 150 amino acids of occludin), separated by SDS-PAGE and identified by Western blotting with the same occludin antibody. In Vitro Binding Assay—The binding assay was performed before eluting the C-Occ protein from the ProBond nickel resin. MDCK cells were used as a source of native TJ scaffolding proteins because of their high level of expression. After binding of total soluble C-Occ protein from bacterial lysate to nickel resin and washing with high salt sodium phosphate buffer containing 100 mm imidazole, an aliquot of resin (100 μl) was transferred to a microcentrifuge tube and equilibrated with low salt sodium phosphate buffer. Tight junction proteins were extracted from confluent MDCK monolayers using high salt sodium phosphate buffer containing a high concentration of non-ionic detergents (1% Nonidet P-40 and 0.5% sodium deoxycholate). These relatively severe lysis conditions are required to extract and separate TJ proteins due to the high binding affinity of the complex to the cell cytoskeleton under native conditions. Total MDCK cell lysate was then diluted with 20 mm sodium phosphate to reduce salt and detergent concentrations to 125 mm chloride, 0.25% Nonidet P-40 and 0.125% deoxycholate to produce conditions favorable for the re-association of TJ proteins. The diluted samples were incubated with the C-Occ-charged resin for 4-6 h at 4 °C with gentle rocking. The resin was washed with low salt sodium phosphate buffer 5 times (1 ml each), and then 100 μl of 1× SDS-PAGE loading buffer was added to the nickel resin and boiled for 10 min before centrifugation to separate the supernatant for electrophoresis. The His tag region alone, expressed in bacteria containing the original pRSETA plasmid without the occludin insert, was used as a negative control for the binding assay. Immunoprecipitation (IP)—MDCK cell lysate (as described above) was diluted with 20 mm sodium phosphate to a final chloride concentration of 125 mm, and then incubated with 50 μl of protein G-conjugated agarose (Roche Applied Science) in the presence of 5 μg of antibody to either occludin, ZO-2 or GFAP (Zymed Laboratories Inc.) for 4 h at 4 °C. The antibody-bound molecules were then separated from unbound ones by centrifugation at 2940 × g for 10 s. The supernatant was used as a negative control in the binding assay, and the GFAP antibody (directed against an antigen not present in MDCK cells) was used as a non-relevant antibody control. The protein G-agarose was finally washed five times with 1 ml of buffer each time. It was followed by the addition of 100 μl of 1× SDS-PAGE loading buffer and boiling for 10 min to solubilize the attached proteins and centrifugation to remove the agarose. The supernatant was then analyzed by SDS-PAGE and Western blotting. In the co-IP experiments, the purified C-Occ was treated with trypsin (5 μg/ml) for 1 h at room temperature, and then protease inhibitor mixture was added to inactivate trypsin. The resultant solution was incubated with diluted MDCK cell lysate for 2 h at 4 °C prior to immunoprecipitation with ZO-2 antibody according to the above protocol. Amino Acid Sequencing—The purified C-Occ was digested with trypsin (50 μg/ml) for 5 min at room temperature, separated by 12.5% SDS-PAGE, transferred onto a polyvinylidene difluoride membrane, stained with Coomassie Brilliant Blue R-250 to identify the protein bands and then destained with a solution containing 30% methanol and 10% acetic acid in water. The outlined protein bands were cut out from the membrane and subjected to N-terminal amino acid sequencing by Edman degradation (26.Edman P. Begg G. Eur. J. Biochem. 1967; 1: 80-91Crossref PubMed Scopus (2450) Google Scholar) using the 494/HT PROCISE Sequencing System (Applied Biosystems). Peptide Mass Fingerprinting—After enzymatic digestion of C-Occ (∼5 μg/50 μl) with trypsin (50 μg/ml for 5 min at room temperature), the peptide fragments were separated by SDS-PAGE, stained with Coomassie Blue, and each individual protein band was eluted and digested with chymotrypsin (20 μg/ml) at 37 °C for 8 h. The enzyme was then inactivated using trifluoroacetic acid to adjust the pH between 2 and 3. The peptide samples were cleaned using a reversed-phase Zip-TipC18 (Millipore) and eluted with 50% acetonitrile in 0.1% trifluoroacetic acid, mixed with the ionization matrix and spotted on specimen grids for mass spectrometry. Mass spectrometric analysis was performed using a Voyager-DE Elite MALDI-TOF mass spectrometer (Applied Biosystems, Foster City, CA), operated in the positive ion mode using angiotensin as the calibrant. Ionization of the peptide species under study was produced by pulses from a 337-nm nitrogen laser, and an accelerating voltage of 20 kV was used. The ionization matrix for this analysis was α-cyano-4-hydroxycinnamic acid dissolved in a 1:1 water/acetonitrile mixture. A minimum of 100 laser pulses was obtained, and the results were averaged to produce the resulting output spectrum. The spectra were identified by comparing the masses of the peaks with the fingerprint database generated from the mass calculation of each predicted chymotrypsin-digested fragment using the Peptide Mass program from ExPASy (27.Wilkins M.R. Lindskog I. Gasteiger E. Bairoch A. Sanchez J.C. Hochstrasser D.F. Appel R.D. Electrophoresis. 1997; 18: 403-408Crossref PubMed Scopus (304) Google Scholar). Circular Dichroism (CD)—CD analysis of the purified His-tagged protein (in 20 mm sodium phosphate, 500 mm sodium chloride, pH 7.4) was performed using an Aviv 60DS spectropolarimeter (Aviv Associates, Lakewood, New Jersey). Measurements were made using a quartz microcell with a path length of 0.1 cm. Three scans from 185 to 260 nm were recorded at a speed of 0.5 nm/s, with a 0.5-nm interval. The scans were averaged and corrected by subtracting a buffer baseline (28.Cheng X. Lee J.C. Biochemistry. 1998; 37: 51-60Crossref PubMed Scopus (15) Google Scholar). The relative percentages of secondary structures were determined using the CDFIT program (29.Johnson W.C. Proteins. 1999; 35: 307-312Crossref PubMed Scopus (633) Google Scholar). Secondary Structural Prediction—Secondary structure prediction of the C-terminal cytoplasmic region of human occludin was performed using internet server-based programs, PHD (30.Rost B. Methods Enzymol. 1996; 266: 525-539Crossref PubMed Google Scholar), and Jpred (31.Cuff J.A. Clamp M.E. Siddiqui A.S. Finlay M. Barton G.J. Bioinformatics. 1998; 14: 892-893Crossref PubMed Scopus (924) Google Scholar). Interaction between ZO-2 and dN-Occ—In each binding reaction, purified ZO-2 was immobilized on protein A/G-agarose using 2 μg of ZO-2 antibody, and then incubated with various concentrations of dN-Occ ranging from 1 to 100 μm. This range of dN-Occ concentration was used based on preliminary studies using a broader concentration range from 10 nm to 150 μm. Irrelevant antibody to GFAP was used as a control to estimate nonspecific binding. The binding reactions were performed with gentle rocking at 4 °C for 3 h. Protein A/G-agarose with bound (ZO-2)-(dN-Occ) complexes was separated from free dN-Occ by centrifugation at 2940 × g for 2 min and washing the agarose pellet twice with low salt sodium phosphate binding buffer. The agarose with attached protein complexes was boiled for 10 min in 75 μl of 1× SDS loading buffer before separating the proteins by electrophoresis (7.5% gel for ZO-2 and 12.5% for dN-Occ). To visualize the specific protein bands, separated proteins were transferred onto a polyvinylidene difluoride membrane for Western blotting as described above. The intensity of each protein band was analyzed using SigmaGel software (Jandel Scientific Software, San Rafael, CA), and fitting of the binding data was performed using Sigmaplot (SPSS Inc., Chicago, IL) to estimate the association constants and stoichiometry, using the model shown by Equation 1,Z+On⇆Z(O)n(Eq. 1) and the association constant is defined by Equation 2.Ka=[Y]/[Z]×[X]n(Eq. 2) In Equation 2, Ka is the association constant, [Y] is the concentration of bound dN-Occ, [X] is the concentration of free dN-Occ, [Z] is the immobilized ZO-2 concentration, which is constant in each reaction as measured by Western blotting, and n is the number of dN-Occ molecules binding to each molecule of ZO-2. Concentrations of bound dN-Occ were determined by interpolation of the band intensities in a standard curve constructed from the Western blot of a series of dN-Occ dilutions of known concentration. Limited Proteolysis of the C-Occ Protein—A structural domain is, by definition, obtained as a stable proteolytic core, while interdomain sequences are generally susceptible to digestion by different proteases. Limited proteolysis can therefore be used to obtain the shortest segments of occludin that contain compact structural domains. Two different enzyme preparations, trypsin (T), and protease, were used in these experiments. Trypsin cleaves peptides at specific amino acids (lysine and arginine). Protease used here is a crude preparation from S. griseus that has a broader range of peptide cleavage. The use of these two enzyme preparations provides two independent methods of limited proteolysis and several different types of protease activity to test for shorter functional fragments than those generated by trypsin proteolysis alone. Results of limited proteolysis of the purified C-Occ protein are shown in Fig. 1. When a low concentration of trypsin (0.5 μg/ml) was used to cleave C-Occ attached to the nickel resin, the product consisted of essentially the entire C-Occ peptide without smaller fragments. With a higher concentration (5 μg/ml), however, C-Occ was cleaved into many fragments, shown in Fig. 1 as a ladder pattern. A single 19.2-kDa band resulted at the highest concentration of trypsin used (500 μg/ml). The pattern of broad range protease digestion was similar to that of trypsin digestion. However, when sequencing grade trypsin (Ts) was used instead of the cruder trypsin (T) preparation, the result was a single band at 21.5 kDa, which has the same migration distance as the top band of the 50 μg/ml trypsin digestion on the Western blot (lane 3, left panel of Fig. 1.). The latter result suggests that the cruder trypsin (T) preparation has other contaminating protease activities, ultimately resulting in the same 19.2-kDa band as the broad range protease. Both 19.2- and 21.5-kDa bands were used
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