Functional interaction of Escherichia coli heat-labile enterotoxin with blood group A-active glycoconjugates from differentiated HT29 cells Estela M. Galva ´ n, German A. Roth and Clara G. Monferran Departamento de Quı ´ mica Biolo ´ gica – CIQUIBIC (CONICET), Facultad de Ciencias Quı ´ micas, Universidad Nacional de Co ´ rdoba, Argentina The type I heat-labile toxin produced by enterotoxi- genic Escherichia coli (LT-I), and cholera toxin (CT) secreted by Vibrio cholerae, are responsible for the diarrhea observed in traveller’s diarrhea and cholera, respectively. These enterotoxins are the closest struc- tural and functionally related members of the CT fam- ily [1,2]. LT-I and CT are AB 5 toxins, in which the pentameric B subunit [B subunit of E. coli heat labile toxin (LT-B), B subunit of cholera toxin (CT-B)] medi- ates toxin binding to membrane receptors on polarized intestinal epithelial cells. Upon binding, the holotoxin enters the cell and moves by retrograde transport to the trans-Golgi and the endoplasmic reticulum [3,4]. The A subunit, responsible for the toxic activity, undergoes controlled proteolytic cleavage and reduc- tion in the endoplasmic reticulum, giving rise to the fully active A 1 -peptide, which is translocated to the cytoplasm [5]. ADP ribosylation of the a subunit of the heterotrimeric GTP-binding protein by the A 1 -pep- tide renders adenylylate cyclase irreversibly activated and, consequently, increases cyclic AMP production, leading to net fluid secretion [6,7]. Keywords ABH glycoconjugates; differentiated HT29 cells; Escherichia coli heat-labile toxin; glycosphingolipids; toxin receptors Correspondence C. G. Monferran, Departamento de Quı ´ mica Biolo ´ gica, Facultad de Ciencias Quı ´ micas, Universidad Nacional de Co ´ rdoba, Ciudad Universitaria, Co ´ rdoba X5000HUA, Argentina Fax: +54 351 4334074 Tel: +54 351 4334168 ⁄ 4334171 E-mail: cmonfe@mail.fcq.unc.edu.ar (Received 1 March 2006, revised 28 April 2006, accepted 22 May 2006) doi:10.1111/j.1742-4658.2006.05368.x Human colon adenocarcinoma cells (HT29-ATCC) and the clone HT29- 5F7 were cultured under conditions that differentiate cells to a polarized intestinal phenotype. Differentiated cells showed the presence of junctional complexes and intercellular lumina bordered by microvilli. Intestinal brush border hydrolase activities (sucrase, aminopeptidase N, lactase and mal- tase) were detected mainly in differentiated HT29-ATCC cells compared with the differentiated clone, HT29-5F7. The presence of non-GM1 recep- tors of Escherichia coli heat-labile enterotoxin (LT-I) on both types of dif- ferentiated HT29 cells was indicated by the inability of cholera toxin B subunit to block LT-I binding to the cells. Binding of LT-I to cells, when GM1 was blocked by the cholera toxin B subunit, was characterized by an increased number of LT-I receptors with respect to undifferentiated control cells. Moreover, both types of differentiated cells accumulated higher amounts of cyclic AMP in response to LT-I than undifferentiated cells. Helix pomatia lectin inhibited the binding of LT-I to cells and the subsequent production of cyclic AMP. LT-I recognized blood group A-active glycosphingolipids as functional receptors in both HT29 cell lines and the active pro-sucrase form of the glycoprotein carrying A-blood group activity present in HT29-ATCC cells. These results strongly suggest that LT-I can elicit an enhanced functional response using blood group A-active glycoconjugates as additional receptors on polarized intestinal epithelial cells. Abbreviations CT, cholera toxin; CT-B, B subunit of cholera toxin; LT-I, type I heat-labile toxin produced by enterotoxigenic Escherichia coli; LT-B, B subunit of E. coli heat labile toxin; TEM, transmission electron microscopy. 3444 FEBS Journal 273 (2006) 3444–3453 ª 2006 The Authors Journal compilation ª 2006 FEBS LT-I and CT bind with high affinity to the ganglio- side GM1 in cell membranes and other systems [1]. Despite the high amino acid sequence and structural homology, LT-B and CT-B are bacterial lectins that also recognize non-GM1 carbohydrate structures with different specificity. Numerous studies have shown that LT-I binds glycosphingolipids and glycoproteins from intestinal mucosal cells of several animal species [8–14], although most of these interactions have no recognized biological function. We have previously reported a dif- ferential ability of glycosphingolipids and glycoproteins obtained from pig and rabbit gastrointestinal tract tis- sue to interact with LT-I, depending on the type of ABH blood group determinant carried by these glyco- conjugates. Conversely, CT showed almost no inter- action with either blood group-active glycolipids or glycoproteins [12–14]. Furthermore, LT-I recognized ABH glycoconjugates among the abundant non-GM1 receptor population on rabbit intestinal brush border membranes and was demonstrated to activate adenylate cyclase, suggesting that ABH glycolipids and glycopro- teins are LT-I functional receptors in rabbit intestine [15]. Recently, we have demonstrated that LT-I binding to blood group A-active glycosphingolipids from the plasma membrane of human adenocarcinoma HT29 cells elicits a signal transduction pathway, resulting in an increase of the cellular cyclic AMP levels [16]. HT29 cells and other few intestinal cell lines undergo morphological and functional differentiation in vitro. Under standard culture conditions, HT29 cells are covered by irregular microvilli and devoid of tight junctions. When HT29 cells are cultured under specific conditions [17–21], they develop some features of distinct pathways of enterocyte differentiation, charac- terized basically by cell polarization. The plasma mem- brane of enterocyte-like cells differentiated in vitro exhibits two structural and functionally different domains - apical and basolateral - separated by tight junctions. The apical membrane is characterized by the presence of microvilli containing peptidase and glyco- hydrolase digestive enzymes, whereas the basolateral membrane displays distinct surface protein markers [22–25]. It is well known that enterocyte-like differenti- ation overcomes the impaired glycosylation and rapid degradation of the glycoprotein observed in the undifferentiated stage, allowing the expression of sucrase-isomaltase, which carries ABH blood group determinants [26,27]. Because the natural target of LT-I is a polarized intestinal cell, the purpose of this study was to investi- gate the interaction of LT-I with non-GM1 receptors of polarized HT29 cells. Toxic activity, triggered by LT-I binding to additional receptors, was measured as intracellular cyclic AMP accumulation. We also inves- tigated the nature of alternate LT-I receptors in differ- entiated cells. Results Characterization of differentiated HT29 cells In order to analyze the interaction of LT-I with cells that resemble the polarized enterocyte, HT29 cells from American type culture collection (ATCC) (HT29- ATCC), and the clone HT29-5F7, were grown under conditions appropriate for stimulating intestinal differ- entiation. Some structural and biochemical features of the differentiated cells have been studied. Contrary to that observed in undifferentiated HT29-ATCC cells, cells at late confluence clearly showed, by phase-con- trast microscopy, intercellular lumina that were visible as vesicles or cysts between cells (Fig. 1A). By trans- mission electron microscopy (TEM), it was clearly evident that intercellular lumina were bordered by abundant microvilli provided by surrounding cells facing the medium, and junctional complexes between cells were frequently observed (Fig. 1B–D). TEM sec- tions also showed that confluent differentiated HT29- ATCC cells were formed by three to four cell layers, while undifferentiated cells at confluence had five to Fig. 1. Morphological studies of differentiated HT29 cells. Phase contrast micrograph (A) and thin sections (B–D) of postconfluent cultures of HT29-ATCC cells (day 21) grown in RPMI-1640 contain- ing 10% fetal bovine serum. Note the presence of intercellular lumina (ICL) (A and B), apical brush border (arrows in C) and junc- tional complexes between adjacent cells facing the lumen (arrows in D). Magnification: A, ·40; B, ·4000; C, ·12 000; and D, ·20 000. E. M. Galva ´ n et al. Interaction of LT-I with differentiated HT29 cells FEBS Journal 273 (2006) 3444–3453 ª 2006 The Authors Journal compilation ª 2006 FEBS 3445 seven cell layers (data not shown). Similar structural characteristics were also observed on HT29-5F7 cells at late confluence (data not shown). In order to characterize biochemically the differen- tiated stage of HT29-ATCC and HT29-5F7 cells, intestinal enzyme activities were measured in brush border-rich membrane fractions. Table 1 shows that maltase (EC.3.2.1.20), lactase (EC.3.2.1.23), sucrase (EC.3.2.1.48) and aminopeptidase N (EC.3.4.11.2) were active in differentiated HT29-ATCC cells, and that lower amounts of maltase and aminopeptidase N activity were present in polarized HT29-5F7. Together, these results indicated that HT29 cells, when growing under appropriate conditions, could acquire some mor- phological and biochemical characteristics of entero- cytes. LT-I binding to differentiated HT29 cells and cyclic AMP-induced production Several concentrations of nontoxic LT-B and CT-B were assayed for competitive inhibition on 125 I-labelled LT-I binding to differentiated HT29 cells. Figure 2 shows that complete inhibition of LT-I binding to HT29-ATCC cells was dependent on the LT-B concen- tration, indicating the specificity of the 125 I-labelled LT-I preparation. When CT-B was assayed at concen- trations similar to those used for LT-B, toxin binding was not blocked. From these results it is apparent that most of the LT-I receptors are not shared with CT-B. CT-B was able to block 125 I-labelled CT binding to both differentiated HT29 cell types in a concentration- dependent manner (results not shown). In order to determine the number of LT-I receptors, additional to GM1, on the cell membrane of polarized and nonpolarized HT29-ATCC and HT29-5F7 cells, we measured the binding of 125 I-labelled LT-I in the absence and in the presence of CT-B. Saturation curves performed at steady state showed that in polar- ized and nonpolarized cells, there was little difference in the binding of 125 I-labelled LT in the presence and in the absence of CT-B (Fig. 3A,B). Moreover, Fig. 3 shows that the binding capacity for non-GM1 recep- tors was approximately four times higher on differenti- ated HT29-ATCC cells than on undifferentiated control cells (Fig. 3A). Differentiated HT29-5F7 cells also exhibited a significantly higher number of addi- tional LT-I receptor sites with respect to the undiffer- entiated stage (1600 versus 800 fmolÆ10 )6 cells) (Fig. 3B). Helix pomatia lectin, which recognizes the carbohydrate structure of blood group A, inhibited 125 I-labelled LT-I binding to differentiated HT29 and HT29-5F7 cells in a dose-dependent manner (Fig. 4). These results indicate that the differentiation process increased the expression of non-GM1 receptors for LT-I and that blood group A-active glycoconjugates may be alternate LT-I receptors in both cell lines. The functional response of differentiated cells to LT-I was determined in terms of the cyclic AMP Table 1. Activity of brush border membrane-associated enzymes (mUÆmg )1 protein). Sucrase (EC.3.2.1.48), maltase (EC.3.2.1.20), lactase (EC.3.2.1.23) and aminopeptidase N (EC.3.4.11.2) activities were measured in brush border membranes (P2 fractions) from un- differentiated and differentiated HT29-ATCC and HT29-5F7 cells, as described in the Experimental procedures. Values are the mean ± SD of two experiments. ND, not detected. Undifferentiated Differentiated HT29-ATCC Sucrase ND 6.75 ± 1.59 Maltase ND 29.2 ± 7.9 Lactase ND 4.41 ± 0.14 Aminopeptidase N ND 6.75 ± 2.09 HT29-5F7 Sucrase ND ND Maltase ND 2.56 ± 0.52 Lactase ND ND Aminopeptidase N ND 1.28 ± 0.72 Fig. 2. Effect of B subunits of cholera toxin (CT-B) and Escheri- chia coli heat-labile toxin (LT-B) on the binding of 125 I-labelled heat-labile enterotoxin (LT-I) to cells. HT29-ATCC cells grown at confluence for 18 days were incubated with different concentra- tions of CT-B or LT-B for 30 min at 4 °C and then further incubated with 125 I-labeled LT-I (5.0 nM) for 60 min at 4 °C. Binding of 125 I- labelled LT-I was determined as indicated in the Experimental pro- cedures. Each point is the mean of triplicate determinations, with a standard deviation 610%. Interaction of LT-I with differentiated HT29 cells E. M. Galva ´ n et al. 3446 FEBS Journal 273 (2006) 3444–3453 ª 2006 The Authors Journal compilation ª 2006 FEBS content, measured after incubation with increasing concentrations of toxin. Figure 5 shows that differenti- ated HT29-ATCC cells increased the cyclic AMP con- tent compared with control cells, reaching a maximum at a toxin concentration of  12 nm. Table 2 shows that both differentiated HT29-ATCC and HT29-5F7 cell lines accumulated twice the con- tent of cyclic AMP, with respect to control cells, in response to 10 nm LT-I acting on either the total or the non-GM1 receptor population. When cells were pre-incubated with H. pomatia lectin before the addi- tion of LT-I, the cyclic AMP level was significantly diminished. These results strongly suggest that LT-I alternate receptors can elicit a functional response in both polarized HT29-ATCC and HT29-5F7 cells and, furthermore, that blood group A-active glyco- conjugates could represent a major proportion of the functional additional receptors to LT-I in both cell lines. Fig. 3. Binding of Escherichia coli heat-labile enterotoxin (LT-I) to HT29 cells in culture. Differentiated and control HT29-ATCC (A) and HT29-5F7 (B) cells were incubated with increasing concentra- tions of 125 I-labelled LT-I for 60 min at 4 °C in the absence or in the presence of 1.0 l M unlabelled cholera toxin B subunit (CT-B). The bound 125 I-labelled toxin was determined as described in the Experimental procedures. Results have been corrected for the nonspecific binding of 125 I-labelled LT-I. The levels of nonspecific binding were not greater than 10% of total binding for each toxin concentration. In all panels, each point represents the mean ± SD of three experiments. Fig. 4. Concentration-dependent effect of Helix pomatia lectin on 125 I-labelled LT-I binding to differentiated cells. Lectin was pre-incu- bated with differentiated HT29-ATCC and HT29-5F7 cells for 30 min at 4 °C before the addition of 125 I-labelled LT-I (10 nM)and then further incubated for 60 min at 4 °C. Bound toxin was meas- ured as indicated in the Experimental procedures. Each point repre- sents the mean of triplicate determinations ± SD. Fig. 5. Intracellular cyclic AMP stimulated by Escherichia coli heat- labile enterotoxin (LT-I) in HT29-ATCC cells. Undifferentiated and differentiated HT29-ATCC cell monolayers were incubated with increasing concentrations of LT-I in the presence of 1.0 l M CTB for 90 min at 37 °C. Cyclic AMP was assayed as described in the Experimental procedures. Each point is the mean of triplicate deter- minations. E. M. Galva ´ n et al. Interaction of LT-I with differentiated HT29 cells FEBS Journal 273 (2006) 3444–3453 ª 2006 The Authors Journal compilation ª 2006 FEBS 3447 Presence of blood group A-active glycoconjugates on differentiated HT29 cells and interaction with LT-I Because cell polarization involves marked changes in the cell architecture as well as in the expression and sorting of new membrane molecules, we investigated the nature of ABH glycoconjugates able to bind LT-I in the HT29-ATCC and HT29-5F7 differentiated cells. Brush border membrane preparations (P2) from differ- entiated cells were examined by western blotting for the interaction with LT-I and for the presence of blood group A activity. Figure 6A shows that LT-I only recognized one blood group A-active glycoprotein, which was identified as pro-sucrase-isomaltase by reac- tion with the corresponding antibody and the expected relative migration after SDS ⁄ PAGE. In the P2 frac- tions from differentiated HT29-5F7, no glycoprotein with the ability to bind LT-I (data not shown), and no glycoprotein carrying the blood group A determinant, were detected. Total lipid extracts from both differentiated HT29- ATCC and HT29-5F7 cells were separated by HPTLC and assayed for binding of the blood group A mAb and LT-I by the TLC-overlay technique. Figure 6B shows that LT-I recognized GM1 and several blood group A-active glycosphingolipids, migrating more slowly than GM1, from lipid extracts of both differen- tiated cells. The ability of LT-I to interact more effi- ciently with the complex glycosphingolipids carrying the blood group A determinant from polarized cells is similar to that previously observed with lipid extracts from undifferentiated HT29 cells [16]. Discussion LT-I is a major virulence factor of enterotoxigenic E. coli, which colonizes human and animal intestines. The toxic activity of LT-I on the target cell is mediated by permanent activation of adenylate cyclase, which increases the cyclic AMP level in intestinal mucosa cells. Consequently, alteration in Na + and Cl – fluxes in villus and crypt cells has been involved in the char- acteristic symptoms of diarrhea. The polarized HT29 cell model was used, in this work, to study the interaction of LT-I with non-GM1 receptors. The undifferentiated HT29 parental cell line contains a very small proportion of differentiated cell types, which, under a pressure selection process, emerge as one of mainly two differentiated polarized enterocyte-like or mucus-secreting phenotypes [17]. The mechanisms by which biochemical conditions or drug pressure induce survival of colon carcinoma cells are currently under study [28–31]. In the present work, enterocytic differentiation was induced in HT29-ATCC parental cells and clone HT29-5F7, as detected by ultrastructural and functional studies. At late conflu- ence, cells were polarized, had well developed brush border at the apical membrane and expressed several intestinal enzymes from the mature enterocyte. The Table 2. Cyclic AMP production elicited by Escherichia coli heat- labile enterotoxin (LT-I) on HT29 cells. Effect of CT-B and Helix po- matia lectin. Undifferentiated and differentiated HT29-ATCC and HT29-5F7 cells were pre-incubated at 4 °C with 1.0 l M CTB or 10 l M H. pomatia lectin and then cells were further incubated with 10 n M LT-I at 37 °C for 90 min. Intracellular cyclic AMP was meas- ured as described in the Experimental procedures. Values are the mean ± SD of two experiments. Cells Cyclic AMP (pmol ⁄ 10 6 cells) LT-I LT-I + CT-B LT-I + HP HT29-ATCC 670 ± 70 400 ± 50 48.0 ± 11 HT29 differentiated 1260 ± 130 915 ± 90 112.0 ± 28 HT29-5F7 1170 ± 120 830 ± 80 5.2 ± 0.4 HT29-5F7 differentiated 2270 ± 120 1600 ± 130 2.1 ± 0.3 AB Fig. 6. Blood group A-active glycoconjugates from differentiated HT29 cells and their ability to interact with Escherichia coli heat- labile enterotoxin (LT-I). (A) Blood group antigenic activity and LT- I-binding properties of brush border-enriched P2 fractions from dif- ferentiated HT29-ATCC-P2 fractions were separated by SDS ⁄ PAGE and electrotransferred to nitrocellulose. Nitrocellulose strips were incubated with mouse monoclonal anti-(blood group A) or anti-(suc- rase-isomaltase) (SI) Ig and then with horseradish peroxidase (HRP)-conjugated monoclonal anti-mouse Ig. For LT-I binding, nitro- cellulose strips were incubated with 5.0 n M LT-I followed by incu- bation with rabbit anti-LT-I Ig and HRP-conjugated Protein A. In all cases, peroxidase was revealed by a chemiluminescent reaction. (B) Blood group A activity and LT-I binding to glycosphingolipids from HT29-ATCC and HT29-5F7 differentiated cells. HPTLC plates were overlaid with anti-(blood group A) IgM and then with a secon- dary HRP-conjugated antibody. Peroxidase was revealed with 0.05% 4-chloro-1-naphtol and 0.01% hydrogen peroxide as sub- strate solution. Interaction of LT-I with differentiated HT29 cells E. M. Galva ´ n et al. 3448 FEBS Journal 273 (2006) 3444–3453 ª 2006 The Authors Journal compilation ª 2006 FEBS morphological features of differentiated HT29-ATCC and HT29-5F7 cells observed in this work closely resembled that previously reported [21,22,29,30]. Func- tionally, differentiation was accompanied by the expression of aminopeptidase N, lactase, maltase and sucrase activities. Sucrase-isomaltase is localized at the apical brush border membranes of HT29 cells differen- tiated in RPMI [22] and by glucose deprivation [26,27]. LT-I binds to the high-affinity receptor, GM1, and to alternate receptors (glycosphingolipid and gycopro- teins) from several cell membranes [8–10,12–16]. We have previously described that ABH-active glycoconjugates could act as alternate LT-I receptors on intestinal brush border membranes from pig and rabbits and in undifferentiated HT29-ATCC cells [12–16]. In the present work, we found that specific LT-I binding to differentiated intestinal cells is not sig- nificantly diminished in the presence of a molar excess of CT-B (Fig. 3), which may reflect a very low contri- bution of GM1 to LT-I binding on cells. Saturation curves performed on differentiated HT29-ATCC and HT29-5F7 cells showed that 125 I-labelled CT maximally bound 74 and 28 fmolÆ10 )6 cells, respectively (data not shown), supporting the idea of the existence of an unbalanced ratio between alternate ⁄ GM1 receptor sites in HT29 cells. By using the polarized HT29-ATCC cell line and the HT29-5F7 clone we demonstrated an increased expression (two- to four-fold) of non-GM1 LT-I receptor sites with respect to the undifferentiated control cells. The dose-dependent inhibition of LT-I binding by H. pomatia lectin clearly indicates that LT-I recognized blood group A-active glycoconjugates on the cell sur- face of undifferentiated [16] and differentiated HT29 cells (Fig. 4). Although no direct quantification of blood group A-active glycoconjugates on the cell sur- face was performed, we assumed that the higher num- ber of LT-I receptor sites on differentiated cells should result from a greater number of blood group A-active glycoconjugates on the cell surface. Differentiation of adenocarcinoma cell lines (e.g. HT29, Caco-2) to an enterocyte like-status involves a change in morphologi- cal features, such as the development of brush border membranes. A great increase of the brush border membrane surface in differentiated cells (Fig. 1) may increase the number of receptor sites provided by blood group A-glycosphingolipid and blood group A-glycoprotein sucrase-isomaltase (the latter on the HT29-ATCC plasma membrane). Polarized cells were also capable of inducing an increase in the intracellular cyclic AMP level in response to LT-I concentrations higher than 6 nm. This effect was observed, even at 10 nm toxin, when the number of occupied binding sites of differentiated and control cells were similar. We have no clear explanation for this observation and further studies are necessary to add new insight into the mechanism of the toxin action on these cells. However, we speculate that the enhanced cyclic AMP production can be rela- ted to the polarized status of cells, which may allow a more efficient coupling of the secondary signal path- ways triggered by the toxin in respect to nonpolarized HT29 cells. For CT, it has been shown that a small percentage of the cell-bound toxin is converted to A1 peptide over a period of time during which the full activation of adenylate cyclase is reached [6]. Because CT binding to differentiated cells was completely blocked by 100 nm CT-B in the present work (results not shown), we attributed cyclic AMP accumulation in polarized cells to the action of LT-I on low-affinity non-GM1 LT-I receptors. Apparently, these alternate receptors account for 70% of the total cyclic AMP response to LT-I in both polarized cell lines (Table 2). Using toxin overlay assays, we found that several blood group A-glycosphingolipids from HT29-ATCC and HT29-5F7 cell lines, migrating more slowly than GM1, efficiently bound LT-I. These results, together with the inhibitory effect of H. pomatia on toxin action, indicated that glycoconjugates bearing the blood group A determinant are additional receptors to LT-I in HT29-ATCC and HT29-5F7 cells. We have recently reported that blood group A-active glycosp- hingolipids, migrating more slowly than GM1, are additional LT-I receptors in parental HT29 cells and that these non-GM1 receptors may account for  50% of the cyclic AMP response elicited by the toxin in these cells [16]. The results from this work indicate that blood group A-active glycosphingolipids are major functional LT-I alternate receptors in HT29-ATCC and HT29-57 cells. Even though glycosphingolipid distribution in polarized cells was not investigated in this work, we speculate that polarized HT29 cells have glycosphingo- lipid-enriched brush border membranes resembling the mature enterocyte [32]. Interestingly, a glycoprotein band present in the brush border-enriched membrane preparation from HT29-ATCC cells bound LT-I. This glycoprotein was identified as the glycosylated blood group A-active pro-sucrase-isomaltase by western blot assays (Fig. 5). The glycosylated pro-form of sucrase- isomaltase has been clearly detected in the enterocytic differentiated HT29 cells carrying the A blood group of the human donor [26,27]. Furthermore, the results of the present work suggest that this glycoprotein may function as an LT-I receptor on human intestinal brush border membranes. Sucrase-isomaltase has already E. M. Galva ´ n et al. Interaction of LT-I with differentiated HT29 cells FEBS Journal 273 (2006) 3444–3453 ª 2006 The Authors Journal compilation ª 2006 FEBS 3449 been postulated as a glycoprotein receptor of LT-I on intestinal brush border membranes from several animal species, but we have detected this interaction between LT-I and blood group-A active-sucrase-isomaltase in porcine and rabbit intestines [13,14]. The present results, together with our earlier findings, support the idea that the blood group A determinant (mostly of the type 2 oligosaccharide chain) from glycosphingolipids and glycoproteins may actually be involved in the car- bohydrate structure recognized by LT-I. Recently, the fine structural basis of the interaction of a hybrid between CT and LT-I and a type 2 blood group A pen- tasaccharide, which involves a novel binding site at the toxin molecule, was established [33]. Several epidemiological studies have demonstrated a relationship between ABH blood group status and high risk of developing cholera [34–37]. Recently, a study was carried out to eilucidate the relationship of the ABH blood group, immunity and susceptibility to symptomatic and asymptomatic infections with V. cholerae [38]. An association has also been observed in the occurrence of diarrhea after ingestion of E. coli- producing LT-I in volunteers [39]. LT-I and, to a much lesser degree, CT, interacted with ABH glyco- conjugates from human and animal intestinal mucosa [12–14], and furthermore, some of these interactions have proved to be functional [15,16]. These interac- tions may have relevance in the clinical outcome of diarrhea caused by LT-I and CT in relation to the blood group of the patient. Regarding differentiated HT29 cells as intestinal model system, it is apparent that enterocyte-like differ- entiated HT29 cells provide a useful in vitro model to evaluate the functional role of interactions between bacterial virulence factors and intestinal polarized cells. Experimental procedures Cell culture The human colon adenocarcinoma HT29 parental cell line (HT29-ATCC) was grown in Dulbecco’s modified Eagle’s medium (D-MEM) containing 10% heat-inactivated fetal bovine serum. Enterocytic differentiation was performed as described by Hekmati et al. [21]. Briefly, cells were switched to RPMI-1640 containing 10% inactivated fetal bovine serum, replated four times in this medium and then exam- ined at late confluence (18–21 days). After HT29-ATCC cells reached confluence, RPMI-1640 was changed every day. Clone HT29-5F7, which was selected by resistance to 5-fluoruracil (kindly donated by Dr T. Lesuffleur, INSERM U560, Lille, France) was usually grown in D-MEM, con- taining 10% inactivated fetal bovine serum, and examined at early confluence (undifferentiated) or at late confluence (12 days) when the cells exhibit a polarized phenotype [23,30]. Antibiotics (100 UÆmL )1 penicillin, 100 lgÆmL )1 streptomycin) were added to both D-MEM and RPMI- 1640. Cell lines were maintained at 37 °C in a humidified atmosphere containing 5% CO 2 . Cell number was deter- mined by Trypan blue exclusion in a hemocytometer. Toxin-binding assay LT-I was iodinated by a stoichiometric method with chlor- amine T [40], as described previously [11], and the specific activity for the iodinated LT-I was 3.0 lCiÆlg )1 . Biological activity of the 125 I-labelled LT-I preparation was 90%, measured as the percentage of 125 I-labelled LT-I total pro- tein able to specifically bind to GM1-containing membranes (rat red blood cells or NHI 3T3 fibroblasts). Toxin binding to cells in culture was assayed as previ- ously described [16]. Briefly, cells were incubated in serum- free D-MEM buffered with 25 mm Hepes or RPMI-1640 containing 0.01% BSA without (total binding) or with unlabeled LT-B (1.0 lm) before the addition of 125 I-labelled toxin (3.0 lCiÆlg )1 ). After 60 min at 4 °C, cells were washed, solubilized with NaOH and the radioactivity was counted. Nonspecific binding was measured as the binding of 125 I-labelled toxin in the presence of an excess of unlabe- led LT-B. To assay nonspecific binding of 125 I-labelled LT-I or competitive inhibition by unlabelled LT-B or CT-B, the B subunits of toxin were incubated with cells for 30 min at 4 °C and then further incubated with 125 I-labelled LT-I for 60 min at 4 °C. The blocking effect of 125 I-labelled LT binding by H. pomatia lectin was also determined by pre- incubation of cells with lectin, as indicated for B subunits of toxins. Toxin-stimulated accumulation of intracellular cyclic AMP The toxin-stimulated accumulation of intracellular cyclic AMP was determined as described previously [16]. Briefly, cells were pre-incubated without or with CT-B (1.0 lm), or H. pomatia lectin (10 lm), at 4 °C. LT-I was then added for 90 min at 37 °C. Finally, cells were treated with 0.1 m HCl and the dried acid extracts were assayed for cyclic AMP by RIA (Immunotech SA, Marseille, France), accord- ing to instructions of the manufacturer. Electron microscopy TEM was performed as follows. Cell monolayers were fixed in 2% glutaraldehyde and then postfixed in 1% OsO 4 . After dehydration in graded ethanol solutions, the cells were embedded in Epon. Ultrathin sections were contrasted Interaction of LT-I with differentiated HT29 cells E. M. Galva ´ n et al. 3450 FEBS Journal 273 (2006) 3444–3453 ª 2006 The Authors Journal compilation ª 2006 FEBS with uranyl acetate and lead citrate. Thin sections were examined in a Jeol EX 1220 transmission electron micro- scope (Jeol, Tokyo, Japan). Hydrolase assays Brush border-enriched membrane fractions (P2) were pre- pared according to Trugnan et al. [27]. Briefly, cells were scraped in Tris-mannitol buffer, pH 7.1, containing prote- ase inhibitors (1.0 lgÆmL )1 antipain, 17.5 lgÆmL )1 benzami- dine, 1.0 mm phenylmethylsulfonyl fluoride, 1.0 lgÆmL )1 pepstatin, 10 lgÆmL )1 aprotinin and 1.0 lgÆmL )1 leupep- tin). Cells were disrupted by sonication and then CaCl 2 was added (to 18 mm). The homogenate was centrifuged (950 g, 10 min; Rotor Type 50, Beckman Instruments, Fullerton, CA, USA) and the supernatant was centrifuged again (33 500 g, 30 min) to yield the P2 fraction. Proteins were measured by the method of Lowry et al. [41]. Glycohydrolases (sucrase, maltase and lactase) and ami- nopeptidase N activities were determined in P2 fractions according to Messer and Dalqvist [42] and Maroux et al. [43], respectively. The enzyme activities are expressed as milli- units (mU) per mg of protein. One unit is defined as the acti- vity that hydrolyzes 1.0 l mol of substrate per min at 37 °C. ABH phenotyping of cellular glycoconjugates and toxin-binding assays To detect blood group-active and toxin-binding glycopro- teins, P2 fractions were separated by 7.5% SDS ⁄ PAGE, electrotransferred to nitrocellulose sheets, and immuno- stained as previously described [16]. The sucrase–isomaltase complex was identified using a mouse anti-(human sucrase- isomaltase) IgG (kindly donated by Dr A. Quaroni, Ithaca, NY, USA) followed by a horseradish peroxidase-conju- gated secondary antibody. Peroxidase was detected with an enhanced chemiluminiscence immunodetection system (Amersham Biosciences, Uppsala, Sweden). Total lipids from cells were extracted and separated using HPTLC. Glycolipids that bind either the toxins or the anti- (blood group) IgM were immunodetected, essentially as previously described [16]. Acknowledgements We thank Dr W. S. Dallas (Glaxo Wellcome Research, NC, USA) for providing the LT-I producing- bacterial strains, Dr J. D. Clements (Tulane University, New Orleans, LA, USA) for kindly donating LT-B, Dr The ` cla Lesuffleur (INSERM U560, France) for provi- ding the HT29-5F7 clone and Dr Andrea Quaroni (Cornell University, NY, USA) for providing mouse monoclonal anti-human intestinal hydrolases. 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Binding of Escherichia coli heat-labile enterotoxin (LT-I) to HT29 cells in culture. Differentiated and control HT29- ATCC (A) and HT29- 5F7 (B) cells were incubated with increasing concentra- tions of 125 I-labelled