Glycobiology, 2016, vol. 26, no. 1, 63–73 doi: 10.1093/glycob/cwv080 Advance Access Publication Date: 24 September 2015 Original Article

Microbial Biology

Novel GM1 ganglioside-like peptide mimics prevent the association of cholera toxin to human intestinal epithelial cells in vitro Robert K Yu1,2,3, Seigo Usuki2,3,5, Yutaka Itokazu2,3, and Han-Chung Wu4 2

Department of Neuroscience and Regenerative Medicine, Medical College of Georgia, Georgia Regents University, Augusta, GA 30912, USA, 3Charlie Norwood VA Medical Center, Augusta, GA 30904, USA, and 4Institute of Cellular and Organismic Biology, Academia Sinica, Nankang, Taipei 115, Taiwan 1

To whom correspondence should be addressed: Tel: +1-706-721-8931; Fax: +1-706-721-8727; e-mail: [email protected]

5

Present address: Laboratory of Biomembrane and Biofunctional Chemistry, Graduate School of Advanced Life Science, Frontier Research Center for Post-Genome Science and Technology, Hokkaido University, Sapporo 001-0021, Japan.

Received 11 June 2015; Revised 9 September 2015; Accepted 11 September 2015

Abstract Cholera is an acute diarrheal disease caused by infection in the gastrointestinal tract by the gramnegative bacterium, Vibrio cholerae, and is a serious public health threat worldwide. There has not been any effective treatment for this infectious disease. Cholera toxin (CT), which is secreted by V. cholerae, can enter host cells by binding to GM1, a monosialoganglioside widely distributed on the plasma membrane surface of various animal epithelial cells. The present study was undertaken to generate peptides that are conformationally similar to the carbohydrate epitope of GM1 for use in the treatment of cholera and related bacterial infection. For this purpose, we used cholera toxin B (CTB) subunit to select CTB-binding peptides that structurally mimic GM1 from a dodecamer phage-display library. Six GM1-replica peptides were selected by biopanning based on CTB recognition. Five of the six peptides showed inhibitory activity for GM1 binding to CTB. To test the potential of employing the peptide mimics for intervening with the bacterial infection, those peptides were examined for their binding capacity, functional inhibitory activity and in vitro effects using a human intestinal epithelial cell line, Caco-2 cells. One of the peptides, P3 (IPQVWRDWFKLP), was most effective in inhibiting cellular uptake of CTB and suppressing CT-stimulated cyclic adenosine monophosphate production in the cells. Our results thus provide convincing evidence that GM1-replica peptides could serve as novel agents to block CTB binding on epithelial cells and prevent the ensuing physiological effects of CT. Key words: bacterial infection, cAMP, cholera toxin, epithelial cell, GM1 ganglioside

Introduction Cholera is an acute diarrheal disease that can kill within hours if left untreated (Sack et al. 2004). It remains a worldwide threat to public health and is a serious social problem, particularly in the unsanitary conditions of under-developed countries. It has been reported that an estimated 3–5 million cholera infection cases and 100,000– 120,000 deaths occur due to cholera every year (King et al. 2008;

Ali et al. 2012). Cholera toxin (CT), produced by Vibrio cholerae, is the major factor responsible for the severe acute watery diarrhea with severe dehydration by toxicating the intestinal epithelial cells (Spangler 1992). Structurally, CT is an A1B5-subunit type toxin composed of a monomeric A1 subunit and a pentameric B subunit. Each B monomeric subunit (cholera toxin B, CTB) possesses binding activity to GM1 by recognizing a specific epitope of the oligosaccharide portion of the GM1 molecule (Holmgren et al. 1973). To initiate the

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64 pathogenic effect, CT must cross the intestinal epithelial barrier, which is normally impermeable to macromolecules. Subsequent to binding of CT to GM1 in the microdomain of the intestinal cell plasma membrane, a CT–GM1 complex is formed and endocytosed and transported to the endoplasmic reticulum (ER) (Tsai et al. 2002; Lencer and Tsai 2003). After a series of intracellular processing steps of the cholera toxin A (CTA) subunit, CTA catalyzes the adenosine diphosphate (ADP)-ribosylation of the Gαs subunit. The increased Gαs activation leads to increased adenylate cyclase activity, which elevates the intracellular concentration of cyclic adenosine monophosphate (cAMP) to more than 100-fold over normal and over-activates cytosolic protein kinase A (PKA) activity (Wernick et al. 2010). The activated PKA then catalyzes the phosphorylation of cystic fibrosis transmembrane conductance regulator (CFTR), leading to the excretion of a high concentration of Cl− into the intestine. The excretion of Cl− creates an osmotic and electrolyte gradient for the extrusion of Na+ and water (Welsh et al. 1982; Sundaram et al. 1991). The massive secretion of water and electrolytes results in life-threatening severe diarrhea, severe dehydration and death. Since the identification of the CT-receptor GM1, prevention of attachment of CT to the cell surface GM1 has been studied by use of the oligosaccharide head group of GM1 or other CT antagonists (Sixma et al. 1992; Thompson and Schengrund 1998; Minke et al. 1999; Mitchell et al. 2004). A major obstacle has been the difficulty in synthesizing large quantities of these oligosaccharides and antagonists. In principle, it is possible to mimic the carbohydrate epitope with a suitable polypeptide, which can be easily and economically synthesized in large quantities. Identification of a suitable polypeptide mimic, for example, for ganglioside GD3, can

R K Yu et al. be conveniently achieved by phage-display technology (Simon-Haldi et al. 2002; Taki et al. 2008). To our knowledge, GM1-replica peptides have never been developed. In this study, we showed that GM1-replica peptides can mimic GM1 by inhibiting CTB binding to GM1. Using the technology of a phage-displayed peptide library displayed on the envelope protein PIII of filamentous phages, we obtained six peptides that were active in neutralizing CTB binding to GM1. These peptides exhibited functional activities similar to GM1. In particular, one peptide (P3: IPQVWRDWFKLP) possessed functional activity to inhibit CT-induced cAMP production in a human intestinal epithelial cell line, Caco-2 cells. The peptides thus show promise to be useful for competing with CTB for GM1 and have great potentials for the treatment of cholera and other bacterial infections using GM1 as a receptor. The translational aspect of this avenue of research should prove beneficial to patients suffering from this and similar microbial infections.

Results Identification of novel peptides that bind to CTB In this study, we used a phage-displayed random peptide library to isolate phages that were able to bind to CTB. After four rounds of affinity selection (biopanning), the titer of bound phage increased by up to 68-fold (Figure 1A). Through enzyme-linked immunosorbent assay (ELISA) screening and DNA sequencing, we identified five phage clones with unique peptide sequences that bound to CTB, P1–P5 (Figure 1B and Table I). Inserted nucleotides of selected phage clones

Fig. 1. Identification of peptides binding to CTB using phage display. (A) A phage-display random peptide library was used to identify peptides that bind to CTB. After four rounds of biopanning, the titer of bound phage had increased 68-fold relative to the first round of selection. PFU, plaque-forming units. (B) A phage-displayed random peptide library was screened by CTB. After four rounds of biopanning, 13 phage clones from 32 selected clones showed significant reactivity to CTB but not to control NMIgG.

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GM1-replica peptides block cholera toxin binding were sequenced. All contained 36 inserted nucleotides (translated to 12 amino acid residues) (Table I). Inserted oligonucleotide sequences of phage DNAs were selected and translated to peptide sequences (Wu et al. 2015). Another peptide, P6, had very weak binding affinity for CTB. P6, on the other hand, had weak affinity for rabbit polyclonal anti-GM1 antibodies and was used to evaluate the ability of the peptide mimics to interact with those antibodies (see below).

P4, 77.4 (25.4) and P5, 34.4 pmol/mL (11.3 pmol/mL). P6 did not exhibit any appreciable inhibitory activity toward CTB. The overall data indicate that P3 has the best fit with a GM1 structure that interferes with the binding of GM1 to CTB. We then investigated the cross-inhibitory properties of these peptides with other CTB-reactive ligands, such as fucosyl-GM1 (F-GM1), GM1-like lipooligosaccharide (LOSGM1), GD1b and Lipid A-F (Figure 3). At the IC50 concentration for the GM1 binding assay, the peptides were compared for their interference to CTB binding of F-GM1, LOSGM1, GD1b and Lipid A-F, respectively (Figure 4). Since P6 did not show any appreciable binding to CTB, P6 treatment was done at 100 pmol/mL for the binding experiment for the other four ligands. When the inhibitory strength at the IC50 concentrations of peptides for GM1 was set as 100%, P4 treatment for F-GM1 was 1.24-fold higher than for GM1, but P1 and P3 exhibited a slightly weaker inhibition (91.6, 87.8%) of P3 treatment for GM1. P2 and P5 exhibited about the same levels of the inhibition for F-GM1. For LOSGM1 bindings, P1–P6 indicated similar levels of the inhibition as compared with GM1. For GD1b binding, P1, P3 and P5 exhibited

The inhibitory activity of GM1-replica peptides on CTB binding The phage-displayed peptides, P1–P6, were tested for their inhibitory activities for GM1–CTB binding based on ELISA, and the strengths of their inhibitory activities were expressed as IC50 values. For comparison, the corresponding Ki values, calculated using the equation of Cheng–Prussoff (Cheng and Prusoff 1973; Dawson 2005), are included in parentheses. As shown in Figure 2, five peptides showed remarkable inhibition with the following IC50 and Ki values (in parentheses), respectively: P1, 38.0 (12.5); P2, 46.1 (15.1); P3, 9.6 (3.1);

Table I. Alignment of phage-displayed peptide sequences selected by CTB Peptide

Clone

P1 P2 P3 P4 P5 P6

1, 3 4, 21, 24, 26, 28, 32 8 27 18 29

Q D

I A

N S

I

Y P

V S Q

S P V

T K

A A

K L

W R

W F W F W S

K H R P K S

T K D A T G

W W W W H M

F F F F F P

P P K T

N S L K

L M P L

A H

V

Y

P

R

T

Phage-displayed consensus amino acid residues are shown in boldface.

Fig. 2. Inhibition profile of GM1-replica peptides on GM1 binding to CTB. Six GM1-replica peptides (P1, P2, P3, P4, P5 and P6; at 10, 50, 100, 200, 300 or 500 pmol/mL) were screened from a phage display and were tested for HRP-CTB (1:20,000 dilution) binding to GM1 (25 ng) coated on an ELISA plate. The IC50 values of P1–P6 were determined by curve-fitting analysis of semi-logarithmic concentration vs. ELISA absorbance value plots.

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Fig. 3. Structure of GM1, F-GM1, LOSGM1, GD1b and Lipid A-F. It is known that the CTB-binding activity with its ligand is not limited to GM1; in fact, several other glycolipids and Lipid A also can bind with CTB. The relative activities of GM1, LOSGM1, F-GM1, GD1b and Lipid A-F were compared (see Figure 4). The common carbohydrate epitope is denoted by dashed lines. Lipid A-F is a specific Lipid A that has CTB-binding activity and is derived from the lipooligosaccharide fraction of C. jejuni (strain HS19).

Fig. 4. Inhibitory activity of GM1-replica peptides on CTB binding of different ligands. Peptides were tested for CTB binding of the following ligands: F-GM1, LOSGM1, GD1b and Lipid A-F. The relative inhibition activity of P1–P5 peptide was calculated as the ratio of a value at the IC50 concentration of peptides for GM1. Columns above or below the dotted line at 1.0 show higher or lower inhibitory activities as compared with GM1. As P6 peptide had no inhibitory effect on GM1–CTB binding, inhibitions of other ligand– CTB bindings were tested at a concentration of 100 pmol/mL (nM).

75.8–79.8% of the inhibition; P2 and P5 37.8−43.8% of the inhibition and P6 had no inhibitory effect for interaction with GM1, respectively. For Lipid A-F binding, no peptide that we tested (P1–P6) showed any inhibition at the IC50 concentrations that were obtained by GM1 binding to CTB (Figure 4).

The inhibitory activity of GM1-replica peptides on anti-GM1 and other anti-glycolipid antibodies To evaluate the specificity of the six peptides for CTB and anti-GM1 antibodies and other cross-reacting antibodies and ligands, we performed inhibition assays using ELISA. As expected, the six peptides

R K Yu et al. also showed an inhibitory effect for an anti-GM1 rabbit polyclonal antibody (Ab1). The inhibition profile was completely different from that for CTB binding, suggesting that different binding modes may account for the different inhibitory capabilities. As shown in Figure 5, P1–P6 generally showed a much weaker inhibitory effect for the antibodies than the corresponding inhibitory potency for CTB, as indicated by the following IC50 values: P1, 273.1; P2, no inhibition; P3, 75.6; P4, 175.3; P5, 481.2 and P6, 293.4 pmol/mL, respectively. These results suggest that these peptides also have inhibitory activity for the binding of GM1 and anti-GM1 antibodies. We did not find evidence for direct interaction between the peptides and the antigen itself, such as GM1. Taken together, these observations suggest that the mode of interaction between the antigen GM1 and its monomeric antibody may be different from that between GM1 and CTB. To investigate the possibility that the rabbit polyclonal anti-GM1 antibody may recognize a unique epitope of GM1, we tested four other preparations of rabbit polyclonal anti-GM1 antibodies (Ab2, Ab3, Ab4 and Ab5) that were generated using GM1 as the antigen and compared their IC50 with that of Ab1. As shown in Figure 6, P1–P6 treatment presented different inhibition profiles compared with that for Ab1, suggesting that inhibition of anti-GM1 activities by different peptide mimics may result from the polyclonal diversity of anti-GM1 antibodies, that is, each polyclonal antibody may recognize a slightly different antigenic epitope of GM1 that is mimicked by a different peptide. To further investigate the specificity of P1–P6 to anti-GM1 antibodies, the peptides were tested for their cross-inhibitory capability of other rabbit polyclonal anti-glycolipid antibodies, such as AbGM2, AbGM4, AbGD1a, AbGD1b, AbGD3, AbF-GM1 and Absulfoglucuronosyl paragloboside (AbSGPG). P1–P6 did not exhibit any inhibitory activity for these antibodies at the IC50 concentrations of Ab1 (Figure 7). Crossinhibitory activities of peptides, however, were observed for AbGM2 with P1 and P5, AbGD1b with P1, P2, P4 and P6 and AbF-GM1 with P1, P4 and P6. In addition, P3 had no reactivity for AbGM2, AbGD1b and AbF-GM1. P5 had no reactivity for AbGD1b and AbF-GM1. Based on the above results, we conclude that P1, P2, P4, P5 and P6 were capable of inhibiting, albeit not completely, the interactions between the antigenic epitope of GM1 and its antibodies. P3, on the other hand, appeared to be the most potent inhibitor for binding to the antigenic epitope of GM1 (Figure 7).

Efficacy of CTB adsorption by peptide-affinity matrices To investigate the efficacy of GM1-replica peptides for elimination of CT, we prepared peptide matrices using the coupling resin kit according to the manufacturers’ instructions (AminoLink Coupling Resin, Pierce, Rockford, IL). The six peptide mimics, P1–P6, were converted into peptide matrices, and each was filled into a small cartridge. To validate their capability to adsorb and eliminate CT, biotinylatedcholera toxin B (b-CTB) was diluted 1:5000 in phosphate-buffered saline (PBS, Sigma-Aldrich) and was passed through each of the peptide matrix-containing cartridge. The first-pass aliquots were recovered as “passed” samples. The binding activity of GM1-replica peptide matrices to b-CTB was calculated based on the formula given in the “Materials and Methods” section. As shown in Figure 8, P3 absorbed up to 68.1% of the b-CTB, followed by P1 and P2 at 54.5 and 52.5%, respectively. P4, P5 and P6 had relatively low affinities for b-CTB, showing 23.5, 28.1 and 25.9%, respectively, compared with the control matrix that had no immobilized peptides. P1, P2 and P3 showed statistically significant differences in their ability to bind to b-CTB as compared with control CTB (P < 0.001, n = 3, Dunnet’s test).

GM1-replica peptides block cholera toxin binding

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Fig. 5. Inhibition profile of GM1-replica peptides on GM1 binding to anti-GM1 antibody. P1–P6 (10, 50, 100, 200, 300 or 500 pmol/mL) were tested for their inhibitory potency of a rabbit polyclonal anti-GM1 antibody (Ab1, 1:500 dilution in 1% BSA/PBS) binding to GM1 (25 ng) coated on the ELISA plate. The IC50values of P1–P6 were determined by curve-fitting analysis of semi-logarithmic concentration vs. ELISA absorbance value plots. P3 and P4 were found to be the most potent inhibitors.

Fig. 6. Inhibitory activity of GM1-replica peptides on anti-GM1 antibodies. Peptides were tested for anti-GM1 antibodies: Ab2, Ab3, Ab4 and Ab5. The relative inhibitory activity of P1 and P3–P6 peptides was calculated as the ratio of a value at the IC50 concentration of the particular peptide for Ab1. Bars more or less than dotted line 1.0 show higher or lower inhibitory activities, respectively, as compared with Ab1. As P2 peptide had no inhibitory effect for GM1–Ab1 binding, inhibitions of Ab2–Ab5 were tested at the concentration of 100 pmol/mL, then expressed as the relative ratio of 100 pmol/mL values of P2 for Ab1.

cAMP production To investigate functional inhibition of CT by GM1-replica peptides, the effect of GM1-replica peptides on cAMP induction was examined using CT-sensitive Caco-2 cells that were derived from a human colonic adenocarcinoma and resembled small intestinal enterocytes. These cells are used widely as a model for studying the mechanism of action of CT (Orlandi et al. 1993). We treated the cells with P3, which exhibited the strongest inhibitory activity for GM1–CTB

Fig. 7. Inhibitory activity of GM1-replica peptides on CTB–ligand binding. GM1-replica peptides were tested for CTB binding of various ligands: F-GM1, LOSGM1, GD1b and Lipid A-F. The relative inhibition activity of P1–P5 peptide was calculated as the ratio of a value at IC50 concentration of peptides for GM1. Columns high or lower than the dotted line at 1.0 show higher or lower inhibitory activities, respectively, as compared with GM1. Since P6 had no inhibitory activity for GM1–CTB binding, inhibitions of other ligand–CTB bindings were tested at the concentration of 100 pmol/mL.

binding among the six peptides. A standard curve of cAMP concentration is shown in Figure 9. Curve-fitting resulted in the following convergent parameters: IC50 = 7.383, H = 0.6595. According to the above formula, the cAMP concentrations in the sample ( pmol/mg of cell protein) were determined. Figure 9 shows that CT treatment induced a remarkable production of cAMP (228.6 pmol/mg cell protein). Treatment of the cells with P3 at 50 nM resulted in inhibition of up to 27% of the CT-induced cAMP production. At higher concentrations of P3, such as 50 µM, there was complete suppression of the CT-induced cAMP production to the control level. It has been

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Fig. 8. Comparison of the ability of GM1-replica peptide matrices to adsorb CTB. GM1-replica peptide matrices were prepared using synthetic peptides P1–P6, using AminoLink Coupling Resin. The amount of b-CTB in the pass-through sample vs. the amount of b-CTB in the applied sample was then determined using the ABC reagent. “Control” refers to the first column using a matrix without peptides. P1–P3 matrices showed statistically significant differences as compared with Control. **Means were significantly different (P < 0.001, n = 3, Dunnet’s test).

R K Yu et al. (Figure 10A). Caco-2 cells were treated with 1–1000 µM, P3 and a GD3-like peptide mimic, followed by analyzing b-CTB incorporation into the cells. The GD3-like peptide mimic (PGD3: RHAYRSMAEWGF) was first developed by phage-display technology (Taki et al. 2008) and was reported as a useful reagent for studying GD3-related diseases such as chronic inflammatory demyelinating neuropathies (CIDP) (Usuki et al. 2010). In this study, the GD3-like peptide mimic was used as a negative control for GM1-replica peptide. Blot analysis indicated that peptides P1 and P3 (100 and 1000 µM) significantly lowered the b-CTB uptake by the cells (P < 0.05, n = 4, Dunnet’s test). The inhibitory effect was observed even at the low concentration of 9.6 nM, with P3 being more potent than P1. The efficacy of P3 was consistent with the IC50 values of P1 and P3, as shown in Figure 2. Confocal microscopy confirmed that P3 inhibited CTB binding to GM1 on the cell surface (Figure 10B and C). These results clearly demonstrated that P3 was capable of neutralizing CTB in the culture medium and preventing it from interacting with GM1 on the cell surface.

Discussion

Fig. 9. Inhibition of CT-stimulated cAMP production in Caco-2 cells by GM1-replica peptide. According to the standard curve of cAMP, the amount of CT-stimulated cAMP production was estimated. The effect of P3 on cAMP induction was examined for CT-sensitive Caco-2 cells. Cells were exposed for 30 min to GM1 (0.5 µg/mL) or in the absence of GM1. CT was added to culture medium in the presence of P3 (50 nM or 50 µM) or in the absence of P3. Cells were incubated at 37°C for 6 h, then sonicated briefly, and the supernatants were collected and the protein concentration was adjusted to 1.0 mg/mL. The cAMP level in the supernatants was quantified using a cAMP EIA kit. Values are means ± SE for nine replicates. *Means were significantly different (P < 0.001, n = 9, Dunnet’s test).

reported by Orlandi et al. (1993) that GM1 enhanced CT-inducedcAMP production. In our study, GM1 treatment caused a 1.6-fold amplification of CT-stimulated cAMP production. Under GM1 treatment, P3 completely inhibited the stimulatory effect of CT and reduced the intracellular cAMP concentration to the control level. This observation is consistent with retardation of CT from attaching to the cell surface GM1 to allow for the entry of CTA to the intracellular space.

Peptides P1 and P3 blocked CTB uptake by Caco-2 cells To investigate the effects of peptide mimics on CT uptake by cells, P1 and P3 were tested for their effects on b-CTB internalization

The purpose of this study was to develop GM1-replica peptides to inhibit the attachment of CT to GM1 on the cell membrane surface. Since peptide mimics can simulate complex carbohydrate structures and are easy to make employing current peptide synthesis technologies, they should be useful as effective antagonists to “block” the cytotoxic effect of CT. Additionally, they have potential clinical applications in preventing other similar microbial infections, such as Escherichia coli heat-labile enterotoxin, Clostridium perfringens enterotoxin, that depend on carbohydrate-based receptors on the host cell surface for infectivity. In this study, we used a panning procedure to identify phage-display peptides that could bind to the GM1 binding site of the CTB subunit of CT. Here, the size of the library was approximately 2.5 × 108 peptides. Six dodecamer peptides, P1–P6, were generated using this technology. Among those six peptides, P3 exhibited the strongest inhibitory effect on GM1 binding to CTB. The six peptides inhibited CTB binding to different degrees, with the following potency: P3 > P1 = P5 > P2 > P4. P6, on the other hand, had very weak affinity to CTB. Unexpectedly, it was later found that it had a weak affinity for anti-GM1 and anti-F-GM1 polyclonal antibodies. This observation suggests that there are subtle structural characteristics between the carbohydrate-binding sites of CTB and anti-GM1 antibodies. Binding of CTB with its ligand is not limited to GM1; several other glycolipids also bind to CTB with less avidity (1- to 90-fold) compared with GM1 with the following order: GM1 > F-GM1 >> GM2 > GD1a > GM3 > GT1b > GD1b > asialo-GM1 (Kuziemko et al. 1996). We investigated the inhibitory activities of the six peptide mimics for their potency in inhibiting binding of different glycolipids with CTB. As shown in Figure 4, P1–P6 showed a similar IC50 potency for F-GM1 as GM1. However, inhibition of GD1b binding to CTB showed 10–30% less potency than that by GM1. It is interesting to note that binding of CTB is not limited to glycosphingolipids (GSLs). Recently, it has been reported that lipooligosaccharides (LOSs) of certain gram-negative bacteria also possess binding affinity to CTB to form a CTB–LOS complex, although their dissociation constants have not yet been compared with those of GM1 (Usuki et al. 2007). For this reason, CTB-binding activity involved in LOS components was investigated for their potency to bind these peptides. Two LOS components, LOSGM1 and Lipid A-F, were tested for the CT binding assay. Peptides P1–P5 showed similar IC50

GM1-replica peptides block cholera toxin binding

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Fig. 10. Reduced CTB binding on Caco-2 cells with GM1-replica peptides. (A) Cells were incubated with b-CTB (100 nM) with or without the peptide (0, 1, 10, 100 and 1000 nM) for 1 h. An equal amount of protein (40 µg) was loaded per lane. P3, P1 and GD3-replica peptide (negative control) were used. P1 and P3 inhibited CTB binding but GD3-replica peptide (PGD3) did not. Values are means ± SE for four replicates. *The means were significantly different (P < 0.05, n = 4, Dunnet’s test). (B) Confluent monolayers of Caco-2 cells were labeled with AlexaFluor 488 conjugated CTB (CTB-488). (C) GM1-replica peptide P3 inhibited CTXB-488 binding to GM1 on the cell surface. Images were processed identically for the two micrographs shown for each fluorophore so that the intensities could be compared. This figure is available in black and white in print and in colour at Glycobiology online.

potency for LOSGM1, but did not show any inhibition for Lipid A-F. We speculate that these peptides did not inhibit Lipid A-F binding to CTB, as compared with GM1, due to a completely different binding mechanism. Since those peptide mimics were selected based on their specificity for CTB binding, we reasoned that they were also effective in inhibiting other ligand-binding interactions with GM1. For this reason, anti-GM1 and other polyclonal anti-glycolipid antibodies were investigated for their reactivity with peptides P1–P6. As expected, P1–P6 showed cross inhibitions for anti-GM1 antibodies with GM1, but the avidity was approximately 1/10th of that for CTB-binding inhibition (Figure 5). The inhibition potency of anti-GM1 antibody binding

to GM1 was of the following order: P3 > P4 > P1 = P6 > P4. A notable difference for inhibition was the finding that P6 was not active for CTB, and P2 was not active for anti-GM1 antibody (Ab1). In addition, as shown in Figure 7, these peptides were highly specific for antibodies against gangliosides with a GM1-like epitope. These results clearly indicated that these peptide mimics could also inhibit binding of GM1 to anti-GM1 antibodies. However, the peptide-binding site of anti-GM1 antibodies is distinctly different from that for CTB binding, most likely owing to the different recognizing elements of CTB and anti-GM1 antibodies. Further studies for the structural basis of those differences are currently underway. We then examined the ability of the peptide mimics for reducing or preventing CT binding to cells in order to determine their potential for

70 clinical application in preventing cholera infection. Thus, the obtained peptide mimics were investigated for their adsorption/elimination capacity, functional inhibitory activity and in vitro effects. First, peptides P1–P6 were prepared as peptide matrices, and as expected the P3-matrix proved to be the most effective for adsorption and elimination for up to 68.1% of CTB from 0.4 mL of a liquid sample containing CTB (Figure 8). Second, to address whether peptide P3 was active in inhibiting CT-stimulated cAMP production, we used the wellestablished adenylyl cyclase assay using Caco-2 cells. Caco-2 cell monolayers were treated with CT and P3 for 6 h. As shown in Figure 8, CT-stimulated cAMP production was suppressed by treatment with P3 (50 nM and 50 µM). In addition, we also confirmed that GM1 stimulated CT-responsive cAMP production, as previously reported by Orlandi et al. (1993). This stimulation was abolished by treatment of the cells with P3. The results clearly demonstrated that the functional target of P3 was associated with GM1. Third, to investigate the uptake of CTB into Caco-2 cells, the cells were treated with CTB and peptide P3 or P1. As shown in Figure 9, GM1-like peptides (P3 and P1) showed suppression of uptake of CTB into cells. This effect was dosedependent based on the b-CTB incorporation experiment (Figure 10). Despite the considerable variations in binding mode and affinity, our study clearly revealed the validity and efficacy of using GM1-like replica peptides, selected by the phage-display technology, to inhibit CTB binding to GM1 and GM1-containing cells. We have successfully obtained several useful peptide inhibitors, the most potent one being P3, to achieve functional inhibition of CT–GM1 interactions in vitro. Because of the ease in preparing large quantities of these peptides as the result of recent advances in peptide synthesis, the use of peptide mimics should represent an effective and economic means for the treatment of other similar diseases that rely on carbohydrate-based receptors for disease development. Moreover, peptide mimics should have the potential to be employed as a useful tool to probe carbohydrate–protein interactions in a variety of biological systems. At present, the exact mode of interaction between GM1-like peptide mimics and CTB is still unclear. It is known, however, that CTB binds to a variety of ligands. In addition to GM1, it is capable of binding to F-GM1, GD1b and, to a lesser degree, asialo-GM1 (Yanagisawa et al. 2006; Usuki et al. 2007). It is interesting to note that CTB also has been reported to interact with Kdo2-lipid A (Horstman et al. 2004). We have confirmed this finding and further shown that the binding of CTB to lipid A requires the phosphorylated form of lipid A (Usuki et al. 2007). These observations clearly illustrate a far more complex mode of interaction between CTB with the various ligands. Effort is underway to examine the detailed mode of interaction between CTB with the GM1-like peptides. In conclusion, our present investigation has demonstrated the efficacy of using peptide mimics to interfere with the binding of CTB with GM1, which may represent a novel treatment strategy for the treatment of cholera, for which there is still no cure. We are exploring the possibility of testing those bioactive peptides for the prevention of cholera in animal models. Additionally, the use of specific peptide mimics to target removal of specific pathogenic anti-GM1 antibodies in patients with autoimmune-mediated peripheral neuropathies, such as Guillain–Barré Syndrome and chronic inflammatory demyelinating polyneuropathies, should warrant particular attention (Usuki et al. 2010; Yu et al. 2012).

Materials and methods Materials The following GSLs were prepared in our laboratory based on published procedures (Ledeen and Yu 1982): GM1, GM2, GM4,

R K Yu et al. GD1a, GD1b and GT1b from bovine brain; GD3 from bovine butter milk; F-GM1 from PC12 cells (Ariga, Kobayashi, et al. 1987); and sulfoglucuronosyl paragloboside (SGPG) from bovine cauda equina (Ariga, Kohriyama, et al. 1987). LOSGM1 and Lipid A-F were also prepared in our laboratory from Campylobacter jejuni HS:19 (Usuki et al. 2007). Rabbit anti-GM1 polyclonal antibodies with the following lot numbers, 431A (Ab1), 431B (Ab2), 101J (Ab3), 104H (Ab4) and 105T (Ab5), were prepared in our laboratory from bovine brain gangliosides by a previously published conventional method (Usuki et al. 2006). Other anti-GSL antibodies with the lot numbers D710-2 (AbGM2), GMR6 (AbGM4), 109H (AbGD1a), D710-6 (AbGD1b) and D710-5 (AbSGPG) were also prepared by us. Biotin-conjugated CTB (b-CTB) subunit was purchased from Sigma-Aldrich (St. Louis, MO), and AlexaFluor 488-cholera toxin B subunit (CTB-488) was from Life Technologies (Carlsbad, CA). Azide-free CT from V. cholere Inaba 569B was obtained from List Biological Laboratories (Campbell, CA). 3-Isobutyl-1-methylxanthine (IBMX) was purchased from Cayman Chemical Company (Ann Arbor, MI). CT was reconstituted and then dissolved in 0.01% bovine serum albumin (BSA) in 2.5 mM HEPES buffer.

Phage-display biopanning procedure The ELISA plate was coated with 10 µg/mL of protein in 0.1 M NaHCO3 ( pH 8.6) buffer for 2 h at room temperature (RT) and blocked with blocking buffer at 4°C overnight. A phage-displayed peptide library (New England Biolabs, Ipswich, MA) was diluted to 4 × 1010 phages and incubated with the protein-coated plate for 50 min at RT. After washing with PBS, the bound phages were eluted with 0.2 M glycine, pH 2.2. The eluents were neutralized with 1 M Tris–HCl, pH 9.1. The eluted phages were amplified in ER2738 (New England Biolabs) overnight, and the culture was vigorously shaken for 4.5 h at 37°C. The amplified phages were precipitated with 20% polyethylene glycol-8000 in 2.5 M NaCl (PEG/NaCl) at 4°C overnight. The phages were centrifuged for 20 min at 8000 × g at 4° C and resuspended in PBS. The phages were re-precipitated with PEG/NaCl, isolated by centrifugation at 4°C for 10 min and resuspended in PBS. The amplified phages were titered on LB/IPTG/ X-Gal plates. The second round was identical to the first one except for the addition of 2 × 1011 plaque-forming units ( pfu) from previously amplified phages. The third round of biopanning was performed once again with 2 × 1011 pfu of second-round amplified phages. The third-round eluted phages were titered on LB/IPTG/X-Gal plates and selected for ELISA.

Identification and sequencing of positive phage clones The ELISA plate was coated with 10 µg/mL of protein in 0.1 M NaHCO3 ( pH 8.6) buffer for 2 h at RT and blocked with blocking buffer at 4°C overnight. The diluted phages were incubated with coated plates for 1 h at RT. After washing, the bound phages were probed with horseradish peroxidase (HRP)-conjugated mouse anti-M13 monoclonal antibody (mAb) (GE Healthcare Biosciences, Piscataway, NJ). The positive phage clones were further sequenced with the −96 primer 5′-CCCTCATAGTTAGCGTAACG-3′, which corresponded to the pIII gene sequence of M13 phage. The phage-displayed peptide sequences were translated with ExPASy Proteomics Server.

Peptide synthesis The GM1-replica peptides P1–P6 were synthesized using PEPTIDE 2.0 (http://www.peptide2.com). The GD3-like peptide, PGD3 (RHAYRSMAEWGF), was similarly synthesized.

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GM1-replica peptides block cholera toxin binding

CTB-binding assay for GM1 and other ligands

Adsorption treatment of CTB using peptide matrices

We first evaluated the inhibition avidity and specificity of GM1-replica peptides for GM1 binding to CTB on a 96-well flat-bottom polystyrene microtiter plate (Immulon 1B; Thermo Lab Systems, Franklin, MA). Each well of the ELISA plate was coated with 25 ng of GM1 in absolute ethanol. After the solvent was evaporated in an incubator at 37°C, nonspecific binding sites in the wells were blocked using 100 µL of washing buffer (1% BSA in PBS) for 30 min at RT. After decanting the blocking solution, the plate was washed five times with 300 µL of the washing buffer using the EL × 50 microplate strip washer (BioTek Instruments, Inc., Winooski, VT). After further washing, the plate was filled with a reaction solution of CTB, HRP-conjugated cholera toxin B subunit (HRP-CTB) 1:20,000, containing a GM1-replica peptide (10, 50, 100, 200, 300 or 500 pmol/ mL) for 2 h at RT. The plate was then washed with washing buffer as described above, followed by addition of a chromogenic reagent [100 µL of o-phenylenediamine dihydrochloride (OPD) peroxidase substrate in PBS; Sigma-Aldrich]. The plate was incubated for 2 min in the dark and then the reaction was terminated by addition of 50 µL of 3 N sulfuric acid. The absorbance of each well was measured at 492 nm with a microplate spectrophotometer (Bio-Rad, Hemel Hempstead, UK). The dose–response relationship of peptides was expressed numerically as IC 50 value using data points from each peptide treatment and was analyzed by a curve-fitting method. In addition to GM1, several other analogs that are known to be ligands for CTB including F-GM1, GD1b, LOSGM1 and Lipid A-F were tested for their avidity for binding. The structures of GM1, F-GM1, LOSGM1, GD1b and Lipid A-F are shown in Figure 3. Based on the IC50 concentrations of peptides for GM1 (Figure 2), each peptide was tested for CTB-binding inhibition by an appropriate ligand using the ELISA assay. Each well of the ELISA plate was coated with 25 ng of F-GM1, LOSGM1, GD1b or Lipid A-F and tested as described above for GM1 binding.

GM1-replica peptide matrices were prepared using synthetic peptides P1–P6 (2 mg) with 2 mL of a coupling resin (AminoLink Coupling Resin, Pierce, Rockford, IL). Two hundred milliliter of a peptide matrix slurry was dispensed to a Spin column (Pierce Spin Cups/ Columns, column volume 600 µL, Pierce). Excess solvents were removed by centrifugation at 1000 rpm for 15 s. After the aliquot in the collection tube was discarded, 400 µL of b-CTB (Sigma-Aldrich), at 1:5000 dilution, was applied to each of the cups containing the GM1-replica peptide matrices. The applied sample was passed through the spin column by centrifugation at 1000 rpm for 15 s. The processed samples were obtained from the collection tubes and labeled as “passed” samples. Subsequently, the spin column was washed with 400 µL of 1% BSA in PBS and centrifuged at 5000 rpm for 5 min. The amount of b-CTB in the passed sample vs. the amount in the applied sample was then determined using the ABC reagent (ABC peroxidase staining kit, Thermo Scientific Pierce). An ELISA plate was developed using 100 µL of OPD Peroxidase Substrate solution in each well (OPD EASY-tablet for ELISA, Acros Organics, NJ). The absorbance of each well was then measured at 492 nm with a microplate spectrophotometer (Bio-Rad, Hercules, CA). Percent binding was determined by the following equation:

Anti-GM1 binding assay for GM1 The effect of the peptides on anti-GM1 antibody binding to GM1 was compared for five different preparations of rabbit anti-GM1 antibodies (Ab1, Ab2, Ab3, Ab4 and Ab5) by ELISA. Each well on the plate was coated with 25 ng of GM1. After blocking the nonspecific sites on the plate as described above and washing five times with 1% BSA, each well on the plate was filled with a solution of anti-GM1 (diluted to 1:500) containing GM1-replica peptides (10, 50, 100, 200, 300 or 500 pmol/mL) for 2 h at RT. The plate was then incubated for 15–16 h at 4°C. After further washing of the plate, a secondary antibody, HRP-conjugated goat anti-rabbit IgG at 1:100,000 dilution (Sigma-Aldrich) was added. After incubation for 2 h at RT, the wells were washed, followed by coloring reaction using OPD as described above.

Other anti-glycolipid antibody-binding assays The peptide mimics were tested for interfering with other glycolipid antibody–antigen interactions. The antibodies tested included anti-GM2, -GM4, -GD1b, -GD3, -F-GM1; and -SGPG (Ab GM2 , AbGM4 , AbGD1a, Ab GD1b , Ab GD3 , Ab F-GM1 and AbSGPG , respectively) at the IC 50 concentrations of peptides for anti-GM1 (Ab GM1 ) by use of the ELISA plate coated with 25 ng of each of the glycolipid antigens. The plate was treated in the same manner as described above except for the use of an appropriate secondary antibody.

Binding ð%Þ ¼

Absapplied  Abspassed × 100: Absapplied

In this formula, Absapplied refers to ELISA absorbance of the applied CTB concentration. Abspassed corresponds to ELISA absorbance of passed sample passed through P1–P6 matrix.

Cell culture The Caco-2 human intestinal epithelial cell line was established from a human colorectal adenocarcinoma (Fogh and Trempe 1975) and was obtained from American Type Culture Collection (ATCC #HTB-37, Manassas, VA). Cells were grown in Dulbecco’s Modified Eagle Medium (DMEM, Life Technologies) containing 10% fetal bovine serum supplemented with penicillin (100 units/mL) and streptomycin (100 µg/mL) on a dish that had been coated with poly-L-ornithine (0.1%, Sigma-Aldrich). Cells were incubated in a humidified atmosphere containing 5% CO2 at 37°C.

cAMP assay Caco-2 cells (7.0 × 105 cells/2 mL medium) were seeded in 6-well plastic plates. One week later, the cells were washed twice with PBS and exposed for 30 min–2.5 mM HEPES buffer containing 0.5 µg/mL of GM1 or in the absence of GM1. Subsequently, the cells were washed twice, and then CT and 1 mM IBMX were added to the cultured cells in 2 mL of 0.01% BSA in 2.5 mM HEPES buffer containing peptide P3 (50 nM/mL or 50 µM) or in the absence of P3. The cells were incubated at 37°C for 6 h, followed by washing twice. The cells were harvested by centrifugation and sonicated briefly in 1 mL of 0.1 N HCl, and the supernatants collected into micro-centrifuge tubes. For protein assay, the supernatants were diluted and adjusted to 1.0 mg/ mL (10 µL of the supernatant). The amount of cAMP in the supernatant was quantified using a cAMP EIA kit (Cayman Chemical Company, Ann Arbor, MI). A standard curve was plotted for the absorbance ratio % B/B0 (standard bound/maximum bound) for logarithm of the standard cAMP concentrations. The data points from the standard cAMP concentrations were simulated by the following

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mathematical formula of the Hill equation: Y¼

Funding This work was supported in part by a VA Merit Award (1 IO1BX001388 to R.K.Y.) and NIH grants (RO1 NS26994 and RO1 NS11853 to R.K.Y.).

100 1 þ ð50%B=B0 =XÞH

where Y is % B/B0 and H is the trend of the slope, described as steep or shallow. The curve-fitting resulted in convergent parameters: 50% B/ B0 = 7.383, and H = 0.6595. The cAMP concentrations ( pmol/mg protein) in the samples were determined from the standard curve.

Blot Fully confluent Caco-2 cells were incubated with 100 nM b-CTB (Sigma-Aldrich) with or without peptides (0, 1, 10, 100 and 1000 µM) for 1 h at RT. After incubation, cells were lysed and the protein concentration was measured using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Waltham, MA). Proteins (40 µg) were separated by SDS-PAGE under reducing conditions and transferred to polyvinylidene difluoride (PVDF; Bio-Rad) membranes. The membranes were probed with peroxidase-conjugated streptavidin (Jackson Laboratory, Bar Harbor, ME) and visualized with Western Lightning Chemiluminescence reagent (PerkinElmer Life Sciences, Waltham, MA). The developed bands were quantified using NIH ImageJ 1.46r image processing program (rsb.info.nih.gov).

Microscopy Cells cultured on glass cover slips were incubated with 1 µg/mL of CTB-488 and 1 mM of P3 peptide for 30 min on ice. Cells were fixed in 4% formaldehyde and then incubated with 4,6-diamidino-2-phenylindole (DAPI, Life Technologies) for 5 min to stain the nuclei. Cells were observed by LSM 510 confocal microscopy (Carl Zeiss GmbH, Jena, Germany).

Statistical analysis and curve fitting Statistical analyses were performed using the GraphPad Prism 5.0 software package (GraphPad, San Diego, CA). Statistical differences of the means were tested by one-way ANOVA, followed by Tukey’s multiple comparison test and Dunnet’s test. The data points from peptide dose–response and standard cAMP samples were curve-fitted using the following logarithmic concentration equation to obtain best-fitted parameters by nonlinear regression analysis without weighting. The data points were simulated by the following mathematical formula, given as a Hill-type equation (Hill 1910; Wagner 1968). A competitive inhibition is defined by negative Hill slope values, with IC50 given as: Y ¼ bottom þ

ðtop  bottomÞ 1 þ ðIC50 =XÞhill slope

;

where Y is the observed absorbance value, X is a logarithmic concentration of additives; Bottom is the lowest observed absorbance value, Top is the highest observed absorbance value and the Hill Slope is the trend of the slope described as steep or shallow.

Acknowledgements We thank Ms Dawn O’Brien for excellent technical assistance.

Conflict of interest statement None declared.

Abbreviations Ab, antibody; b-CTB, biotinylated-cholera toxin B; BSA, bovine serum albumin; Caco-2 cells, human epithelial colorectal adenocarcinoma cell line; cAMP, cyclic adenosine monophosphate; CIDP, chronic inflammatory demyelinating neuropathies; CT, cholera toxin; CTA, cholera toxin A subunit; CTB, cholera toxin B subunit; CFTR, cystic fibrosis transmembrane conductance regulator; DAPI, 4,6-diamidino-2-phenylindole; ELISA, enzyme-linked immunosorbent assay; HRP, horseradish peroxidase; HRP-CTB, HRP-conjugated CTB; IBMX, 3-isobutyl-1-methylxanthine; LOS, lipooligosaccharide; mAb; monoclonal antibody; NMIgG, normal mouse IgG; OPD, o-phenylenediamine dihydrochloride; PBS, phosphate-buffered saline; PFU, plaque-forming units; PVDF, polyvinylidene difluoride; RT, room temperature; SGPG, sulfoglucuronosyl paragloboside.

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