Affinity Purification and Partial Characterization ... - Wiley Online Library

Journal of Neurochemistry Lippincott Williams & Wilkins, Inc., Philadelphia © 2000 International Society for Neurochemistry

Affinity Purification and Partial Characterization of the Zonulin/ Zonula Occludens Toxin (Zot) Receptor from Human Brain R. Lu, W. Wang, S. Uzzau, *R. Vigorito, *H. R. Zielke, and A. Fasano Division of Pediatric Gastroenterology and Nutrition and Center for Vaccine Development and *Division of Pediatric Research, University of Maryland School of Medicine, Baltimore, Maryland, U.S.A.

Abstract: The intercellular tight junctions (TJs) of endothelial cells represent the limiting structure for the permeability of the blood– brain barrier (BBB). Although the BBB has been recognized as being the interface between the bloodstream and the brain, little is known about its regulation. Zonulin and its prokaryotic analogue, zonula occludens toxin (Zot) elaborated by Vibrio cholerae, both modulate intercellular TJs by binding to a specific surface receptor with subsequent activation of an intracellular signaling pathway involving phospholipase C and protein kinase C activation and actin polymerization. Affinity column purification revealed that human brain plasma membrane preparations contain two Zot binding proteins of ⬃55 and ⬃45 kDa. Structural and kinetic studies, including saturation and competitive assays, identified the 55kDa protein as tubulin, whereas the 45-kDa protein represents the zonulin/Zot receptor. Biochemical characterization provided evidence that this receptor is a glycoprotein containing multiple sialic acid residues. Comparison of the N-terminal sequence of the zonulin/ Zot receptor with other protein sequences by BLAST analysis revealed a striking similarity with MRP-8, a 14kDa member of the S-100 family of calcium binding proteins. The discovery and characterization of this receptor from human brain may significantly contribute to our knowledge on the pathophysiological regulation of the BBB. Key Words: Brain—Zonulin—Zonula occludens toxin—Receptor—Tight junctions—Blood– brain barrier. J. Neurochem. 74, 320 –326 (2000).

still incompletely understood. Recently, two proteins have been described as modulators of TJs: zonulin (Fasano, 1999) and zonula occludens toxin (Zot) (Fasano et al., 1991, 1995; Baudry et al., 1992). Zonulin belongs to a family of eukaryotic proteins that modulate TJ permeability in a tissue-specific manner (Fasano, 1999). Each family member has a molecular mass of ⬃47 kDa and a distinct eight-amino acid N-terminal receptor binding motif. Amino acid substitution within the N-terminal binding site identified three amino acid residues that dictate tissue specificity (Fasano, 1999). Zot is a zonulin analogue elaborated by Vibrio cholerae that seems to be involved in the pathogenesis of cholera-associated diarrhea by opening TJs to the passage of water and electrolytes (Fasano et al., 1991, 1995). The cloning and sequencing of the zot gene have been described (Baudry et al., 1992), and the maltose binding protein–Zot fusion protein has been purified (Fasano et al., 1997). Recent evidence shows that Zot binds to a surface receptor, whose distribution varies within the intestine, being present only in the jejunum and distal ileum but not in the colon and decreasing along the villous-crypt axis (Fasano et al., 1997). This distribution coincides with the regional effect of Zot on tissue permeability (Fasano et al., 1997) and with the preferential F-actin redistribution induced by Zot at the top of the intestinal villi (Fasano et al., 1995). Zot binding protein has been recently isolated from both human and murine intestinal cell lines (S. Uzzau, R. Lu, W. Wang, C. Fiore, and A. Fasano, personal communication), whereas zonulin has been isolated from several human tissues, including the intestine, heart, and brain (Fasano, 1999). We have also

The blood– brain barrier (BBB) is composed of capillary endothelial cells, microvascular pericytes, astrocyte foot processes, and perivascular microglial cells. The intercellular tight junctions (TJs) of endothelial cells represent the only anatomical barrier that interfaces between blood and the brain. TJs, once regarded as static structures, are, in fact, dynamic and readily adapt to a variety of developmental (Revel and Brown, 1976; Magnuson et al., 1978; Schneeberger et al., 1978; Madara and Dharmsathaphorn, 1985), physiological (Gilula et al., 1976; Sardet et al., 1979; Mazariegos et al., 1984; Madara and Pappenheimer, 1987), and pathological (Milks et al., 1986; Nash et al., 1988) circumstances. The regulatory mechanisms that underlie this adaptation are

Received July 5, 1999; revised manuscript received August 27, 1999; accepted August 30, 1999. Address correspondence and reprint requests to Dr. A. Fasano at Division of Pediatric Gastroenterology and Nutrition, University of Maryland School of Medicine, 685 W. Baltimore St., HSF Bldg., Rm. 465, Baltimore, MD 21201, U.S.A. E-mail: [email protected] Abbreviations used: BBB, blood– brain barrier; BSA, bovine serum albumin; his-Zot, 6-histidine zonula occludens toxin; PBS, phosphatebuffered saline; PKC, protein kinase C; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; TJ, tight junction; Zot, zonula occludens toxin.

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PURIFICATION OF ZONULIN RECEPTOR FROM HUMAN BRAIN provided evidence that both Zot and zonulin interact with the same surface intestinal receptor (Fasano, 1999) and therefore activate the same intracellular signaling, leading to the modulation of intercellular TJs. The presence of zonulin in multiple tissues and the regional effect of Zot within the intestine raised questions regarding the tissue distribution of zonulin binding proteins and possible tissue specificity. In the present study, we report the purification to electrophoresis homogeneity of two Zot/ zonulin binding proteins from human brain tissue. Kinetic analysis and competitive binding experiments confirmed that one of the two isolated Zot/zonulin binding proteins represents the eukaryotic receptor of zonulin and its prokaryotic analogue, Zot. MATERIALS AND METHODS Purification of 6-histidine Zot (his-Zot) Plasmid pSU111, containing the clone zot gene in a pQE-30 vector with a 6-histidine tag at its N-terminus, was grown in LB medium with 20 g/L glucose, 25 ␮g/L kanamycin, and 200 ␮g/L ampicillin at 37°C with vigorous mixing until the A600 reached 0.7– 0.9. Cultures were then induced with 2 mM isopropylthio-␤-D-galactoside (Fisher), followed by an additional 2-h culture period at 37°C with vigorous shaking. The cells were harvested by centrifugation at 4,000 g for 20 min and resuspended in buffer A (6 M guanidine-HCl, 0.1 M sodium phosphate, 0.01 M Tris-HCl, pH 8.0; 5 ml/g of wet cell wt). After stirring for 1 h at room temperature, the mixture was centrifuged at 10,000 g for 30 min at 4°C. A 50% slurry of Superflow (Qiagen; 1 ml/g of wet cell wt) was added to the supernatant and stirred for 1 h at room temperature. The mixture was loaded onto a 5 ⫻ 1.5-cm column and washed sequentially with buffer A, buffer B (8 M urea, 0.1 M sodium phosphate, 0.01 M Tris-HCl, pH 8.0), and buffer C (8 M urea, 0.1 M sodium phosphate, 0.01 M Tris-HCl, pH 6.3). Each wash step was continued until the A280 of the flow-through was ⬍0.01. His-Zot was eluted by addition of 250 mM imidazole (1,3-diaza-2,4-cyclopentadiene) to buffer C. After dialysis against urea, the eluate was diluted 200 –500 times in phosphate-buffered saline (PBS), stirred with 50% slurry of Superflow (1 ml/g wet cell wt) for 2 h at room temperature, loaded onto another 5 ⫻ 1.5-cm column, washed with PBS, and eluted with 250 mM imidazole in PBS. Purity of the his-Zot protein was established by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis and western blot using polyclonal anti-Zot antibodies.

Analytical procedures SDS-PAGE was carried out on a 5–15% gradient gel, stained with Coomassie Brilliant Blue dye, destained by 7.5% acetic acid with 10% methanol, and dried with Gel Drying Film (Promega). Following SDS-PAGE, proteins were transferred onto polyvinylidene difluoride membrane (Millipore). Nonspecific binding sites were blocked by PBS with 5% milk plus 0.1% Tween 20. Primary and secondary antibodies were rabbit polyclonal anti-Zot antibody and anti-rabbit IgG (peroxidase conjugate; Sigma), respectively. Films were exposed with ECL detection reagent (Amersham) for 1 min and developed by Konica SRX101 developer.

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Immobilization of his-Zot to AminoLink Plus column One milligram of his-Zot in 4 ml of coupling buffer (0.1 M sodium phosphate, 0.15 M NaCl, pH 7.2) and 40 ␮l of 5 M sodium cyanoborohydride was added to an equilibrated AminoLink Plus column (Pierce) and gently mixed overnight at 4°C. After washing with coupling buffer, 4 ml of 1 M Tris-HCl (pH 7.4) and 40 ␮l of 5 M sodium cyanoborohydride were added to the column, followed by gentle mixing for 30 min at room temperature to block the remaining active sites. The column was washed with 1 M NaCl and stored in PBS containing 0.05% sodium azide.

Preparation of human tissue plasma membranes Adult human brain cortex, heart, and intestinal tissues were obtained from the Brain and Tissue Banks for Developmental Disorders at the University of Maryland and used under the approval of the University’s internal review board. Adult human heart and intestinal tissues were utilized for comparative analysis. Tissues were washed with buffer D (20 mM Tris-HCl, 20 mM EDTA, 250 mM sucrose, pH 7.5), homogenized in buffer E (buffer D containing 5 ␮g/ml leupeptin, 2 ␮g/ml aprotinin, 1 ␮g/ml pepstatin, 10 ␮g/ml phenylmethylsulfonyl fluoride), and centrifuged at 5,000 g and 4°C for 10 min. Supernatants were centrifuged at 12,000 g and 4°C for 45 min. Precipitates were discarded, and supernatants were centrifuged at 30,000 g and 4°C for an additional 90 min. Precipitates were dissolved in buffer E with 0.5% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), sitting on ice for 60 min with gentle mixing every 5 min.

Affinity purification of his-Zot binding proteins Membrane preparations obtained from human brain, intestine, and heart were loaded on an AminoLink Plus-Zot affinity column, washed, and equilibrated with PBS at room temperature containing 0.1% Triton X-100. The columns were incubated for 90 min at room temperature, washed with 8 volumes of PBS containing 0.1% Triton X-100, and eluted with PBS containing 0.1% Triton X-100 with 0.1, 0.3, 0.5, 0.8, and 1.0 M NaCl, respectively. Fractions were collected and subjected to SDS-PAGE.

N-terminal amino acid sequence analysis The fractions of human tissue lysates containing Zot binding proteins were resolved by 5–15% gradient SDS-PAGE and transferred onto polyvinylidene difluoride membranes using CAPS buffer [10 mM 3-(cyclohexylamino)-1-propanesulfonic acid and 10% methanol]. The protein bands were excised and subjected to N-terminal sequencing using a Perkin-Elmer Applied Biosystems Apparatus model 494. 125

I labeling of his-Zot

A pill of Iodo-beads iodination reagent (Pierce) was added to a solution containing 1 mCi of 125I (Amersham) in 0.1 ml of PBS. After incubation for 5 min at room temperature, 150 ␮g of his-Zot in 400 ␮l of PBS was added, and an additional 10-min incubation was carried out. The iodination was stopped by removing the Iodo beads. A D-salt column (Pierce) was used as the final step to purify the 125I-labeled his-Zot.

Characterization of zonulin/Zot receptor from human brain

Binding assay. One hundred microliters of 0.8 ␮M purified Zot putative receptor isolated from human brain in PBS containing 0.1% Triton X-100 was added in quadruplicate to individual wells of Stripwell plates (Corning). Control wells

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received 100 ␮l of PBS containing 0.1% Triton X-100. The wells were incubated at room temperature for 1 h with mild mixing and then washed three times with PBS containing 0.1% Triton X-100. One hundred microliters of 2.4 ␮M bovine serum albumin (BSA) solution in PBS was added to the wells and incubated for 1 h at room temperature to block nonspecific binding sites. After washing with PBS three times, 100 ␮l of increasing concentrations of 125I-Zot in PBS (pH 7.5) was added to each well and incubated at room temperature for 1 h with mild mixing. The wells were washed three times with PBS and cut for counting in an LKB gamma counter (LKB 1272 Cunigamma). Competitive assay. Competitive assays were performed as described above except that after BSA blocking, 1.8 ␮M unlabeled Zot and 11.6 nM 125I-labeled Zot were added to the same well and processed as above. Effect of pH and temperature. To estimate the effect of pH on the binding between Zot and its putative receptor, after addition of the putative receptor and BSA, 100 ␮l of 6.6 nM 125 I-labeled Zot was added to wells containing 0.15 M buffer at increasing pH (range 5–10). In another set of experiments, 100 ␮l of 6.6 nM 125I-labeled Zot was incubated at increasing temperatures (range 4 – 48°C), and the experiments were conducted as described above. Enzymatic deglycosylation of Zot/zonulin receptor. Forty microliters of a 0.3 ␮M solution of the putative receptor was treated with either peptide-N-glycosidase F (N-Glycanase, EC 3.2.2.18, recombinant; Oxford GlycoSciences), endo-␣-acetylgalactosaminidase (O-Glycanase, EC 3.2.1.97; Oxford GlycoSciences), or Sialidase (neuraminidase, EC 3.2.1.18; Oxford GlycoSciences). Deglycosylation was carried out according to the protocol supplied by the manufacturer with some modifications. N-Glycanase digestion was carried out after denaturation with 0.5% SDS, 5% ␤-mercaptoethanol in 20 mM sodium phosphate, 50 mM EDTA, and 0.02% (wt/vol) sodium azide (pH 7.5), heating at 100°C for 2 min, and adding an iodine-free detergent (Nonidet P-40) to the reaction mixture to reach a final Nonidet P-40/SDS ratio of ⬎5:1. Following the addition of 2.5 U of N-Glycanase, the mixture was incubated for 20 h at 37°C and then resolved by SDS-PAGE. O-Glycanase digestion was carried out after denaturation with 0.5% SDS, 5% ␤-mercaptoethanol in 100 mM sodium citrate phosphate, 0.1 mg/ml BSA, and 0.02% (wt/vol) sodium azide (pH 6.0), heating at 100°C for 2 min, and adding Nonidet P-40 to the reaction mixture to reach a final Nonidet P-40/SDS ratio of ⬎10:1. Following the addition of 4 mU of O-Glycanase, the mixture was incubated for 24 h at 37°C and then resolved by SDSPAGE. Sialidase treatment was carried out by incubating 0.2 U of Sialidase together with 40 ␮l of 0.3 M purified putative

FIG. 1. SDS-PAGE of Zot binding proteins isolated by affinity column chromatography from human brain cortex plasma membrane preparations. Lane 1, molecular mass standards; lane 2, whole-plasma membrane lysate; lanes 3, 4, and 5, eluate with 0.3, 0.5, and 0.8 M NaCl in PBS containing 0.1% Triton X-100, respectively.

receptor in 50 mM sodium acetate (pH 5.0) for 20 h at 37°C followed by SDS-PAGE.

RESULTS Isolation of Zot/zonulin binding proteins from human brain His-Zot was successfully immobilized to AminoLink Plus gel with immobilization yields of 89 –95%, as established by protein assay (Bio-Rad detergent-compatible protein assay). Plasma membrane preparations from human brain loaded on the Zot affinity column contained two major Zot/zonulin binding proteins with apparent molecular masses of ⬃45 kDa and 55 kDa (Fig. 1, lanes 3–5). N-terminal sequencing of Zot/zonulin binding proteins from human brain The N-terminal sequences of the two Zot/zonulin binding proteins are shown in Table 1. The two proteins were also compared with other protein sequences by BLAST search analysis. The N-terminal sequence of the 55-kDa protein was 100% identical to the N-terminal sequence of tubulin (Table 1), whereas the ⬃45-kDa

TABLE 1. N-terminal amino acid sequences of Zot binding protein (55 kDa), ␤-tubulin, Zot binding protein (45 kDa), calprotectin (MRP-8), and cystic fibrosis antigen N-terminus Sample Zot binding protein ⬃55 kDa ␤-Tubulin Zot binding protein ⬃45 kDa Calprotectin (MRP-8) Cystic fibrosis antigen


1

5

10

15

20

MREIVHIQAGQAGNQIGAKF MREIVHIQAGQAGNQIGAKF LTELEKALNXGGGVGHKY LTELEKALNSIIDVYHKY LTELEKALNSIIDVYHKY

Identity (%)

100 77 77

PURIFICATION OF ZONULIN RECEPTOR FROM HUMAN BRAIN

FIG. 2. SDS-PAGE of Zot binding proteins isolated by affinity column chromatography from human brain cortex plasma membranes, human intestine mucous membranes, and heart muscle preparations. Lane 1, molecular mass standards; lane 2, Zot binding proteins isolated from human cortex plasma membrane; lane 3, Zot binding protein isolated from human intestine mucous membrane; lane 4, Zot binding protein isolated from heart muscle.

protein band was 72% identical to the N-terminus of calprotectin, a calcium binding protein associated with chronic inflammatory processes (Odink et al., 1987) and the cystic fibrosis antigen (Dorin et al., 1987). Screening of other human tissues for presence of Zot/zonulin binding proteins To verify whether Zot binding proteins similar to those isolated from human brain were also present in other tissues, both human intestine and heart tissues were analyzed. Plasma membrane preparations of human small intestine (Fig. 2, lane 3) and heart tissue (Fig. 2, lane 4) contained an ⬃45-kDa zonulin/Zot binding protein that co-migrated with the ⬃45-kDa protein isolated from the human brain (Fig. 2, lane 2). The N-terminal sequence of these proteins revealed a distinct amino acid sequence from that obtained from human brain (Table 2). As we have previously demonstrated that the zonulins represent a family of tissue-specific modulators of TJs (Fasano, 1999), it is conceivable to hypothesize that the different forms of zonulin/Zot receptors also dictate the tissue specificity of the zonulin system. Human heart tissues and, even if in lesser amounts, the intestinal tissues also showed the ⬃55-kDa tubulin band seen in brain (see Fig. 2). These results corroborate our previous finding suggesting that Zot represents a new member of the microtubule-associated proteins (Wang et al., 1998). As Zot intracellular signaling involves the protein kinase C (PKC)-mediated polymerization of actin filaments strategically located to modulate the TJ permeability (Fasano et al., 1995), it is possible to speculate that the

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FIG. 3. Saturation analysis of 125I-Zot binding to the putative Zot receptor. Conditions were as described in Materials and Methods. Specific 125I-Zot binding was determined by subtracting control group binding from total binding. Data are means ⫾ SD (bars). Values are representative of three separate experiments, each performed in quadruplicate.

Zot microtubule binding activity is involved in the zonulin/Zot biological effect. Representation of Zot/zonulin brain receptor by 45-kDa Zot binding protein As shown in Fig. 3, we observed a dose-dependent binding of Zot to its putative receptor, the purified 45kDa Zot binding protein. The binding reached a plateau at ⬃70 nM Zot with a calculated KD of 35.28 ⫾ 2.10 nM (mean ⫾ SD). This binding was completely displaced when a 155-fold excess of unlabeled Zot was added to the well (data not shown). As purified brain zonulin was obtained starting from human tissues (Fasano, 1999), its total amount was too limited to perform the same competitive assay described with Zot. However, the analysis of human zonulins allowed us to establish the ligand structural requirements for engagement to their tissuespecific receptors and to generate synthetic peptides that specifically competed for this binding (Fasano, 1999). Therefore, competitive binding experiments were performed by using an excess of a synthetic octapeptide (named FZ1/1) that mimics the brain zonulin binding motif and competes for its binding (Fasano, 1999). As shown in Fig. 4, FZ1/1 showed 63.6% inhibitory effect on radiolabeled Zot binding. Combined, these data suggest that the 45-kDa Zot binding protein represents the brain receptor for zonulin and its prokaryotic analogue, Zot.

TABLE 2. N-terminal amino acid sequences of Zot receptor from human brain, intestine, and heart N-terminus 1 From human brain (45 kDa) From human intestine (45 kDa) From human heart (45 kDa)

5

10

15

20

XLTELEKALNXGGGVGHKY SIAFPSKXAASIG XVREQPRLFPPPSAD


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FIG. 4. Relative inhibition capability of Zot and zonulin putative synthetic binding motif octapeptide FZ1/1 on 125I-Zot binding to the purified Zot receptor. A 155-fold excess of unlabeled Zot completely displaced the binding of 125I-Zot to the target receptor, confirming the specificity of this binding. A 30-fold excess of the synthetic zonulin putative binding domain showed a 63.6% inhibitory effect on 125I-Zot binding, suggesting that zonulin also binds to the Zot receptor.

Characterization of Zot/zonulin brain receptor Temperature and pH dependence. The binding between Zot and the 45-kDa human brain zonulin/Zot receptor was both pH and temperature dependent. Optimal binding was obtained at 37°C (Fig. 5A) and at pH 8.0 (Fig. 5B). Enzymatic deglycosylation. O-Glycanase, N-Glycanase, and Sialidase were used to identify possible

FIG. 5. Effect of temperature and pH on Zot binding to its target receptor. The binding of Zot to its receptor was temperature (A) and pH (B) dependent, with optimal binding at 37°C and pH 8.


FIG. 6. SDS-PAGE of the zonulin/Zot receptor (arrow) digested by either N-Glycanase, O-Glycanase, or Sialidase. Conditions were as described in Materials and Methods. Lane 1, molecular mass standards. The receptor was either untreated (lane 2) or treated with N-Glycanase (lane 3), O-Glycanase (lane 4), or Sialidase (lane 5). Whereas digestion with either N-Glycanase or O-Glycanase was ineffective, treatment with Sialidase caused an almost complete digestion of the receptor that shifted from an apparent molecular mass of 45 kDa to ⬃33 kDa.

carbohydrate moieties of the zonulin/Zot receptor. Treatment of the purified zonulin/Zot receptor with either O-Glycanase (Fig. 6, lane 4) or N-Glycanase (Fig. 6, lane 3) did not change the apparent molecular mass of the receptor, which indicates a lack of both O-linked oligosaccharide and N-linked oligosaccharide chains at threonine, serine, or asparagine residues. Treatment of the receptor with Sialidase produced a decrease in apparent molecular mass of ⬃12 kDa (Fig. 6, lane 5). DISCUSSION The TJ or zonula occludens is the hallmark of absorptive and secretory epithelia. As a barrier between apical and basolateral compartments, the TJ selectively controls the paracellular passage of water, solutes, and immune cells between epithelial and endothelial cells. Variations in transepithelial conductance can usually be attributed to changes in the permeability of the paracellular pathway, as the resistances of eukaryotic cell plasma membranes are relatively high (Diamond, 1977). TJs represent the major barrier in this paracellular pathway, and the electrical resistance of epithelial and endothelial tissues seems to depend on the number of transmembrane protein strands and their complexity as observed by freeze-fracture electron microscopy (Madara, 1989). It has become abundantly clear that in the presence of Ca2⫹, assembly of the TJ is the result of cellular interactions that trigger a complex cascade of biochemical events that ultimately lead to the formation and modulation of an organized network of TJ elements, the composition of which has been only partially characterized (Denker and Nigam, 1998). Identification and characterization of Zot, a toxin produced by V. cholerae, has provided new information on the regulation of intercellular TJs (Fasano et al., 1991, 1995, 1997; Baudry et al., 1992). After binding to its surface receptor, Zot is internalized (Fasano, 1998) and subsequently triggers a series of intracellular events including phospholipase C and PKC␣ activation and actin polymerization, which lead to the opening of TJs (Fasano et al., 1995).

PURIFICATION OF ZONULIN RECEPTOR FROM HUMAN BRAIN Recently, we have identified the zonulins, mammalian analogues of Zot, which are involved in the regulation of TJs (Fasano, 1999). The zonulins represent a novel family of eukaryotic proteins with variable N-termini that confer tissue specificity on family members and possess a common C-terminal tau-like domain, which may be involved in cytoskeletal reorganization. Zot and zonulin act through the same intracellular effector mechanisms (Fasano, 1999), and each reversibly opens TJs (Fasano et al., 1995, 1997; Fasano, 1999), suggesting that these two agonists recognize the same receptor (Fasano, 1999). This hypothesis is further supported by the observations that Zot and zonulin exert a nonadditive effect on tissue resistance and that the permeabilizing action of both molecules is blocked by the same synthetic peptide (Fasano, 1999). As the pathological effect of V. cholerae is confined to the gastrointestinal tract (Wachsmuth et al., 1994), we initially anticipated detection of a mammalian Zot analogue only within intestinal tissues. Surprisingly, zonulins were identified in numerous extraintestinal tissues, including the brain (Fasano, 1999). N-terminal sequence and bioassay analysis revealed that brain zonulin has a distinct receptor binding motif as compared with the other zonulins and therefore lacked biological activity when tested on both rabbit and monkey intestine (Fasano, 1999). Based on this observation, we hypothesized that a distinct zonulin receptor is present in the brain that is associated with the regulation of the BBB. In the present article, we report the purification and characterization of such a receptor from human brain, although its localization in the brain has not yet been established. Affinity column purification revealed that brain plasma membrane preparations contain two main Zot binding proteins. N-terminal analysis established one of the proteins as tubulin (see Table 1). At this stage, we do not have elements to establish whether the elution of tubulin from the Zot affinity column is due either to its direct interaction with Zot or to the Zot receptor. However, our recent finding that Zot is a novel member of the microtubule binding protein family (Wang et al., 1998) supports the first hypothesis. Therefore, we concentrated our attention on the second protein as a possible candidate for the zonulin/Zot receptor. Kinetic studies, including saturation and competitive assays, confirmed that the ⬃45-kDa Zot binding protein represents the zonulin/Zot receptor. The analysis of other human tissues, including heart and intestine, revealed that this receptor is widely distributed in other organs. On the basis of this widespread tissue distribution, it is unclear whether the zonulin/zonulin receptor system exerts a systemic and/or local permeabilizing control on endothelial and epithelial cells. However, the distinct N-termini of the zonulin receptor (Table 2) and the tissue specificity of the zonulin family members (Fasano, 1999) support paracrine/ autocrine regulation. The zonulin receptor family might provide regional tissue responsiveness to a given stimulus on the basis of local requirements. This restriction of TJ disassembly to a specific organ system could prevent

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deleterious consequences in other anatomical sites (e.g., intestine versus BBB). Comparison of the N-terminal sequence of the zonulin/Zot receptor with other protein sequences by BLAST search analysis revealed a striking similarity with MRP-8 or calprotectin, a 14-kDa member of the S-100 family of calcium binding proteins (Odink et al., 1987) (Table 1). The function of these proteins remains largely unknown; however, they seem to be involved in neutrophil activation secondary to a phospholipase C-mediated PKC activation. As both zonulin and Zot act through activation of phospholipase C and PKC, we hypothesize that the zonulin/Zot receptor is directly involved in the activation of the intracellular signaling activated by both zonulin and Zot that leads to the opening of intercellular TJs. The biochemical characterization of the zonulin/Zot receptor provided evidence that the receptor is a glycoprotein containing multiple sialic acid residues (Fig. 6). The functional significance of glycosylation of the zonulin/Zot receptor remains unknown; however, sialic acid residues may contribute to stability (protection from proteolysis) or assist in translocation to the cell surface during synthesis (Hidaka and Fidge, 1992). Although the BBB has been studied for more than 100 years and is recognized as being the interface between the bloodstream and the brain, little is known about its regulation. Brain endothelial cells are coupled by TJs that are of extremely high electrical resistance, ⬎1,000 ⍀-cm2 (Rubin et al., 1998). Understanding the molecular basis of TJ regulation will be of fundamental importance to establish how BBB permeability is regulated under both normal and pathological conditions. The discovery and characterization of the zonulin/Zot receptor from human brain may significantly contribute to our knowledge on the pathophysiological regulation of the BBB. BBB dysfunction occurs in a variety of clinical conditions, including multiple sclerosis (Brosnan and Claudio, 1998), brain tumors (Stewart and Mikulis, 1998), brain edema (Fisher, 1998), traumatic brain injury (Povlishock, 1998), and human immunodeficiency virus infection (Petito, 1998). It is tantalizing to hypothesize that dysfunction of this zonulin/Zot receptor model is involved in one or more of the aforementioned clinical conditions. Studies aimed at establishing this involvement and the receptor’s localization in the brain are currently underway in our laboratories. Acknowledgment: This work was partially supported by National Institutes of Health grants DK-48373 and AI-35740 to A.F.

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