• cholesterol-dependent cytolysins;
  • receptor recognition;
  • Streptococcus mitis-derived human platelet aggregation factor;
  • vaginolysin


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  9. Supporting Information

Cholesterol-dependent cytolysins (CDCs) are bacterial pore-forming toxins secreted mainly by pathogenic Gram-positive bacteria. CDCs generally recognize and bind to membrane cholesterol to create pores and lyse target cells. However, in contrast to typical CDCs such as streptolysin O, several atypical CDCs have been reported. The first of these was intermedilysin, which is secreted by Streptococcus intermedius and has human cell-specificity, human CD59 (huCD59) being its receptor. In the study reported here, the diversity of receptor recognition among CDCs was investigated and multi-receptor recognition characteristics were identified within this toxin family. Streptococcus mitis-derived human platelet aggregation factor (Sm-hPAF) secreted by S. mitis strain Nm-65 isolated from a patient with Kawasaki disease was previously shown to hemolyze erythrocytes in a species-dependent manner, its maximum activity being in human cells. In the present study, it was found that Sm-hPAF recognizes both membrane cholesterol and huCD59 as receptors for triggering pore-formation. Moreover, vaginolysin (VLY) of Gardnerella vaginalis showed similar characteristics to Sm-hPAF regarding receptor recognition. On the basis of the results presented here, the mode of receptor recognition of CDCs can be categorized into the following three groups: (i) Group I, comprising typical CDCs with high affinity to cholesterol and no or very little affinity to huCD59; (ii) Group II, including atypical CDCs such as ILY, with no or very little affinity to cholesterol and high affinity to huCD59; and (iii) Group III, which contains atypical CDCs such as Sm-hPAF and VLY with affinity to both cholesterol and huCD59.

List of Abbreviations

Coomassie brilliant blue


cholesterol-dependent cytolysin


cholesterol recognition/binding motif


domain 1


domain 2


domain 3


domain 4


Dulbecco's modified Eagle's medium


extra domain




recombinant CDC with N-terminal hexa-His-tag


pore-formation restricted type of His-CDC


His-tagged D4 of CDC


human CD59




Lewis b


Lewis y


listeriolysin O








perfringolysin O






response unit


streptolysin O




S. mitis-derived human platelet aggregation factor


surface plasmon resonance


transmembrane β-hairpin



Cholesterol-dependent cytolysins, bacterial pore-forming toxins secreted by several pathogenic Gram-positive bacteria, such as Streptococcus spp., Clostridium spp. and Listeria spp., are considered to be important virulence factors. SLO, secreted by Streptococcus pyogenes, has been shown to be important for pathogenicity in a mouse infection model [1], to accelerate caspase-dependent apoptosis in macrophages [2], and to induce various cellular immune responses [3-5]. PLY, from Streptococcus pneumoniae, is reported to be associated with pneumococcal pneumonia in mice [6] and to induce immune responses ([7-12], review in [13]). PFO, from Clostridium perfringens, contributes to cytotoxicity against macrophages and escape from macrophage phagosomes [14]. LLO, from Listeria monocytogenes, facilitates escape of the bacterium from phagosomes [15], resulting in cellular internalization of bacteria [16]; it also causes some immune responses in target cells ([17-20], review in [21]).

Within the CDC family, the overall molecular structure is highly conserved. CDCs are typically composed of four domains (D1–D4) of molecular weight approximately 50–60 kDa. Domains D1, D2 and D3 contribute to creation of membrane pores by penetration of part of D3 into target cell membranes, whereas domain D4 is associated with receptor(s) recognition on target cell membranes [22]. The exact mechanisms of membrane recognition and pore-formation by CDCs have been elucidated in detail [23]. Briefly, within a CDC molecule (monomer), a loop structure comprising β-strand 5 (β5) and α-strand 1 (α1) in domain D3 is rotated away from β-strand 4 (β4), enabling the exposed edge of the latter to pair with structure β-strand 1 (β1) of another monomer. This facilitates and, through repetition, extends CDC monomer–monomer contacts, resulting in formation of a large, ring-shaped structure or “prepore” [23, 24]. Interaction of D4 and the target cell membrane causes extensive structural change, namely, unfolding in TMH 1 and TMH2 of D3 [24]. These pore-forming mechanisms are thought to be highly conserved in CDCs.

Unlike the secretion mechanism observed with typical CDCs, until recently secretion of PLY had been thought to depend on autolysis induced by autolysin LytA encoded by the lytA gene [25]. However, research findings have suggested that LytA is not responsible for release of PLY [26]; rather, PLY is exported from the cytoplasm in a PLY D2-dependent manner and localized in the bacterial cell wall [27]. Similarly an N-terminal secretion signal sequence is absent in MLY, a homologue of PLY expressed by some S. mitis strains that is secreted into culture supernatants [28]. It is therefore of interest to investigate associations between the secretion mechanisms of CDCs that lack an important unit for secretion and their pathogenicity. Furthermore, a new type of CDC named LLY, which is a homologue of Sm-hPAF and has an additional-domain in the N-terminal, has been reported from S. mitis strain SK597 [29]. This additional domain has amino-acid sequence similarity with both Anguilla anguilla agglutinin and the glycan binding domain of the family 98 glycoside hydrolases from S. pneumoniae, and functions as an Ley and Leb specific lectin domain [29]. In summary, variations in molecular structure and mode of secretion of CDCs have been elucidated by several recent studies.

There are also variations in CDC receptor recognition. It has been generally accepted that the receptor for CDCs is membrane cholesterol. However, after discovery of the receptor for the human-specific CDC ILY secreted by human oral commensal S. intermedius, this generalized perception had to be changed. The receptor for ILY is the GPI-anchored glycoprotein, huCD59 [30]; the interaction between ILY and huCD59 forms the basis for its human specificity. Furthermore, it was recently reported that the hemolytic activity of LLY against human erythrocytes is inhibited by anti-huCD59 antibody and also by prepore-locked ILY [31]. In addition, VLY secreted by Gardnerella vaginalis reportedly recognizes huCD59 as its receptor and shows human-specific activity [32]. Regarding the mode of receptor recognition, it has become apparent that there are at least two groups of CDCs: typical CDCs recognizing and binding to membrane cholesterol and atypical CDCs that recognize huCD59.

Previously, Ohkuni et al. reported Sm-hPAF secreted from S. mitis strain Nm-65 isolated from a patient with Kawasaki disease [33]. According to the full-length amino acid sequence encoded by the sm-hpaf gene (GenBank ID: AB051299), Sm-hPAF is the most homologous to LLY, differing from it by only 12 amino acid residues. Interestingly, Sm-hPAF has unique, species-dependent hemolytic activity, humans being the most susceptible species followed by horses, rabbits, rats, sheep and chickens in descending order of susceptibility [34]. Therefore, the demonstration of an additional category of CDCs displaying features intermediate between cholesterol binding (“typical”) CDCs and CDCs recognizing huCD59 suggests that the mode of receptor recognition in CDCs is more diverse than hitherto recognized.

In the present study, we investigated the diversity in mode of receptor recognition of CDCs, focusing on comparisons between Sm-hPAF, which has species-dependent activity but relatively relaxed human-specificity, the more stringently human-specific ILY, and the non-species-specific typical CDCs. We also re-evaluated the human-specificity of VLY.


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  9. Supporting Information

Purification of native ILY from culture supernatant of S. intermedius

Native ILY was purified from the culture supernatant of S. intermedius UNS46 based on a method previously reported [35] with some modifications. The purification procedure is described in the Supporting Information. Purified native ILY was stored at −80 °C until use.

Expression and purification of recombinant CDCs and their derivatives

The recombinant CDCs used in this study were prepared by an Escherichia coli expression system as N-terminal hexa-His tagged proteins; the construction of expression vectors is described in detail in the Supporting Information. The expression systems of His-Sm-hPAF and of His-SLY have been reported previously [34]. Combinations of expression vector and host strain were tested and the clone showing the most efficient expression of the recombinant CDC was selected for preparation of CDCs. All primer sequences used in this study are listed in Table S1 in the Supporting Information. The purity of each recombinant protein was checked by CBB-R250 staining after SDS–PAGE. The purified proteins were kept at −80 °C until use.

Measurement of hemolytic activity

Human blood was obtained with informed consent from a healthy volunteer and stored in sterilized Alsever solution at 4 °C until use. Horse, rabbit and sheep blood was purchased from Nippon Bio-Supply Center (Tokyo, Japan). For hemolysis assay, stored blood was centrifuged (800 g, 5 min) to remove the supernatant and buffy-coat. Erythrocytes were then washed three times in PBS (137 mM NaCl, 1.47 mM KH2PO4, 8.10 mM Na2HPO4 ∙ 12H2O, 2.68 mM KCl) with centrifugation. The washed erythrocytes were added at a final concentration of 0.5% (v/v) to the CDCs serially diluted with PBS. De-ionized water instead of the CDCs solution was used for 100% hemolysis controls and PBS without CDC for 0% hemolysis controls. After incubation at 37 °C for 1 hr, each mixture was centrifuged (800 g, 5 min) and the absorbance of the supernatants at 540 nm determined on a microplate reader (Model 550, Bio-Rad, Hercules, CA, USA). Hemolytic activity was calculated as previously described [35].

Competition of ILYD4 for hemolysis of Sm-hPAF

Competition between His-Sm-hPAFD4 or His-ILYD4 and His-Sm-hPAF was investigated based on the method for the measurement of hemolytic activity described above except for pre-incubation of PBS-washed human erythrocytes for 30 min at 37 °C with the competitors, His-Sm-hPAFD4 or His-ILYD4, respectively.

Evaluation of the binding affinity of CDCs to membrane cholesterol using surface plasmon resonance

Liposomes made with egg york lecithin (Wako Pure Chemical Industries, Osaka, Japan) containing cholesterol were prepared as follows. Fifty microliters chloroform solutions of both 100 mM lecithin (as dipalmitoylphosphatidylcholine) and 100 mM cholesterol were mixed with 5 mL of chloroform in a round-bottom flask and the solvent evaporated. A sample without cholesterol was also prepared for assaying the background. After desication for 2 hr under reduced pressure, 0.5 mL of pre-warmed (50 °C) running buffer (10 mM 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid [pH 7.4] containing 150 mM NaCl and 1 mM EDTA) was added and the lipid-film layer suspended to form liposomes. Uni-lamellar liposomes were prepared using a Mini-Extruder (Avanti Polar Lipids, Alabaster, AL, USA) with a 100 nm filter (Whatman, Tokyo, Japan) according to the manufacturer's instructions and used in the analyses.

The association of His-CDCs with liposomes was investigated using Biacore 1000 (GE Healthcare, Tokyo, Japan). The programs for the assay were as used in a report describing binding of LLO, a CDC produced by L. monocytogenes [36]. Briefly, 25 μL of liposome solution diluted with running buffer was injected onto an L1 sensor chip (GE Healthcare) at a flow rate of 5 μL/min to cover the surface of the sensor chip, followed by injection of 5 μL of 50 mM NaOH at the same flow rate. After liposome-coating, the exposed surface of the sensor chip was blocked with 30 μL of 0.1 mg/mL BSA. The association of His-CDCD4s was investigated at a flow rate of 30 μL/min. The analyte diluted with the running buffer was injected for 1 min and the dissociation monitored for 3 min in the running buffer. The results obtained were analyzed using BIAevaluation software.

Absorption of His-CDCs by cholesterol-embedded lecithin liposomes

Reaction mixtures consisting of PBS containing 0.8–1.0% (v/v) of 50 mol% cholesterol-embedded egg yolk-lecithin liposomes and 10 nM His-CDCs were incubated at 37 °C for 30 min. After incubation, the reaction mixtures were centrifuged (20,600 g, 5 min, 4 °C) and each supernatant incubated with a final concentration of 0.5% (v/v) PBS-washed human erythrocytes at 37 °C for 1 hr. Each reaction mixture was centrifuged (800 g, 5 min) and the absorbance of the resulting supernatants measured at 540 nm.

Construction of the huCD59 transformant of rat hepatic cell BRL3A

The procedure for constructing the huCD59 transformant of rat hepatic cell BRL3A is described in the Supporting Information. Cell-surface expression of huCD59 was confirmed by immunofluorescent staining using anti-huCD59 antibody (11–234-M001; EXBIO Diagnostics, Vestec, Czech Republic) as the first antibody and Alexa Fluor 488-labeled F(ab’)2 anti-mouse IgG (H + L) (Invitrogen, Carlsbad, CA, USA) as the second antibody, and observed by fluorescent microscopy (ECLIPSE TE2000; Nikon, Tokyo, Japan).

Analysis of the binding characteristics of fluorescent-labeled His-CDC(ss)s and His-ILYD4 to human cells

His-CDC(ss)s, namely His-ILY(ss), His-SLY(ss) and His-Sm-hPAF(ss), carrying N-terminal Cys (1.0–1.2 mg of protein) were reacted with 0.1 mM of Alexa Fluor 532 C5 maleimide (Invitrogen) for 2 hr at room temperature. The remaining Alexa fluorophore was removed by gel filtration chromatography using a HiLoad 200 prepgrade column (GE Healthcare) with PBS as the elution buffer (flow rate: 0.3 mL/min), or Hitrap Desalting column (GE Healthcare) equilibrated with PBS. For fluorescent-labeling of His-ILYD4 with N-terminal Cys, 0.26 mg of protein was reacted with 0.1 mM of Alexa Fluor 532 C5 maleimide (Invitrogen) for 2 hr at room temperature and any remaining Alexa fluorophore removed using a Hitrap Desalting column (GE Healthcare) equilibrated with PBS. The purity of the fluorescent-labeled His-ILYD4 fraction and efficiency of fluorescent-labeling were checked as described above.

To deplete cholesterol from the cell membranes of human promyelocytic leukemia cells (U-937 DE-4 [huCD59-unexpressed cell] [37] and HL60 [huCD59-expressed cell]), the cells were treated with a final concentration of 5 mM MβCD at 37 °C for 30 min. For preparation of a sample of adherent cells, that is, BRL3A and their huCD59-expressing transformant, cells were detached by incubation in PBS containing 1 mM EDTA at 37 °C for 10 min. In order to deplete cell membrane cholesterol, cells were incubated in culture medium containing 5 mM MβCD without FBS at 37 °C for total 30 min (10 min, three times). The cells for analysis were washed once and re-suspended to adjust the cell density to 1.0–2.0 × 105 cells in culture medium without FBS, then incubated with each fluorescent-labeled His-CDC(ss) at 10-fold concentrations of LD50, namely final concentrations of 750 ng/mL for His-ILY(ss), 742 ng/mL for His-SLY(ss) and 960 ng/mL for His-Sm-hPAF(ss), for 30 min at 30 °C under dark conditions. His-ILYD4 was also reacted with the cells at the same molar concentration as His-ILY(ss) (final concentration of 198 ng/mL for His-ILYD4). After incubation, the cells were 10-fold diluted with PBS then analyzed by cell analyzer Guava PCA (EMD Millipore, Billerica, MA, USA). Data analysis was performed with Summit version 5.1 (Beckman Coulter, Fullerton, CA, USA).

Measurement of cytotoxicity of CDCs in culture cells

Human promyelocytic leukemia cell lines (U-937 DE-4 and HL60) were cultured in RPMI1640 with 10% (v/v) FBS and used for this assay. The rat hepatic cell line BRL3A, its huCD59-expressed transformant and human hepatoma cell HepG2 were also cultured in DMEM with 10% (v/v) FBS and used. Expression of huCD59 was confirmed by standard immunoblotting using anti-huCD59 antibody (EXBIO Diagnostics) as the first antibody and peroxidase-conjugated goat-anti mouse IgG (MP Biomedicals, Santa Ana, CA, USA) as the second antibody. The cells were diluted to 1.0 × 105 cells/well in culture medium (RPMI1640 without FBS for suspension cells, DMEM with 10% [v/v] FBS for adherent cells) in 96-well plates. For adherent cells, incubation was conducted overnight to allow cells to attach to the assay plates; the culture medium was changed to DMEM without FBS immediately prior to measurement. To prepare MβCD-treated cells, the cells were incubated with culture medium containing 5 mM MβCD without FBS at 37 °C for 30 min, washed once with the medium without FBS to remove MβCD and re-suspended in the same medium without FBS. After this treatment, CDCs serially diluted with PBS were added and incubated for 1 hr at 37 °C in 5% CO2. The viability of the CDC-treated cells was assayed using a reagent for cell proliferation assays, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-1; Dojindo, Kumamoto, Japan), according to the manufacturer's instructions.

Amino-acid sequence of domain 4 of CDCs

A phylogenetic tree of CDCD4s was constructed by Njplot [38]. Amino acid sequences of CDCD4s used for phylogenetic analysis were as follows: pyolysin (AAC45754), arcanolysin (ACV96715), inerolysin (ZP05744302, registered as PFO in the database), SLY (CAA85378), PLY (AAK75991), MLY (ABK58690), ILY (BAA89790), VLY (ACD39459), LLY (ACE79194), Sm-hPAF (BAE72438), SLO (AAZ50760), alveolysin (AAA22224), PFO (BAB79869), sphaericolysin (ACA41562), cereolysin O (AAX88798), anthrolysin O (AAP27127), PFO from Bacillus thuringiensis (AAT63862), tetanolysin O (AAO36403), Hly from Listeria ivanovii subsp. ivanovii (AAR97343), seeligeriolysin O (AAR97361) and LLO (ADX21052). Prior to Njplot analysis, the amino acid sequences described above were aligned by Clustal X [39]. Amino acid sequence identity was calculated with GENETYX software.

Molecular modeling of Sm-hPAF

The molecular model of Sm-hPAFD1–4 was constructed by insightII-discover with homology module (Accelrys, San Diego, CA, USA), using information about the molecular structure of ILY (PDB ID: 1S3R) as a reference, and displayed by Swiss PDB Viewer ver. 4.01.


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  9. Supporting Information

Species-dependent hemolytic activity of Sm-hPAF does not rely on an N-terminal extra domain

Recently, it was reported that His-Sm-hPAF has species-dependent hemolytic activity (34). Unlike typical CDCs, Sm-hPAF has an extra N-terminal domain designated here as “ExD”. For LLY, the CDC with the strongest amino acid sequence identity to Sm-hPAF, this domain (designated as “lectin domain” in LLY) is reportedly specific for difucosylated glycans within Leb and Ley antigens; the pore-forming activity of LLY is significantly increased in a glycan-dependent manner [29]. However, it is commonly recognized that the domain contributing to receptor recognition (membrane cholesterol for typical CDCs and huCD59 for atypical CDCs) is C-terminal D4. Therefore the N-terminal ExD of Sm-hPAF may not affect species-dependent hemolysis. In order to confirm this, hemolytic activity of an ExD deficient mutant of Sm-hPAF (designated here as His-Sm-hPAFD1–4) against several different animal erythrocytes was investigated and species-dependent hemolysis similar to that observed for full-length His-Sm-hPAF (insert of Fig. 1a) shown (Fig. 1a). This finding indicates that the species-dependent hemolytic activity of Sm-hPAF does not rely on the presence of ExD. Further confirmation of the relevance of ExD of Sm-hPAF to species-dependent hemolytic activity was obtained by using ExD-connected chimeras of ILY (His-ILYExD) and SLY (His-SLYExD). As shown in Figure 1b, His-ILYExD showed human erythrocyte-specific hemolysis similar to that of His-ILY (insert of Fig. 1b). Additionally, His-SLYExD showed non species-dependent hemolysis (Fig. 1c) similar to that of His-SLY (insert of Fig. 1c). These findings demonstrate that ExD is not the determinant of species-dependency in hemolysis of Sm-hPAF and also that the ExD of Sm-hPAF does not contribute to the strength of hemolytic activity.


Figure 1. Species-dependent hemolytic activity of Sm-hPAF does not rely on the ExD.

(a) His-tagged recombinants of ExD-deleted Sm-hPAF, His-Sm-hPAFD1–4. (b) His-tagged ILY with Sm-hPAFExD between His-tag and ILY, His-ILYExD. (c) His-tagged SLY with Sm-hPAFExD between His-tag and SLY, His-SLYExD. Inserts in each graph show the hemolytic activity of the relevant wild type; namely, non-mutated recombinant protein, His-Sm-hPAF (insert a), His-ILY (insert b) and His-SLY (insert c). Each assay was performed in duplicate and the results are shown as mean values with differences. Filled circles, human; filled squares, horse; filled triangles, rabbit; filled diamonds, sheep.

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Species-dependent hemolysis of Sm-hPAF relies on possession of domain 4.

Next, the hemolytic activities of D4 mutants of Sm-hPAF were investigated. Substitution of D4 from Sm-hPAFD4 to ILYD4 confers human-specificity on this chimera Sm-hPAF (Fig. 2a). In contrast, the chimera Sm-hPAF with SLYD4 completely lost species-dependent hemolysis (Fig. 2b). Furthermore, substitution of D4 from ILYD4 or SLYD4 to Sm-hPAFD4 eliminated their original hemolytic properties. In the case of His-ILY with Sm-hPAFD4 (His-ILYP4D), the human-specific hemolysis attributable to ILY was eliminated whereas hemolytic activity to animal erythrocytes other than those of humans was enhanced (Fig. 2c). In the case of His-SLY with Sm-hPAFD4 (His-SLYP4D), the non-species-specific hemolysis attributable to SLY was also eliminated; species-dependent hemolysis with some variations in the animal species spectrum compared with that of the original Sm-hPAF remained (Fig. 2d). These findings suggest that species-dependent activity of Sm-hPAF depends on its D4 structure. Judging from a report that the lectin domain of LLY (corresponding to ExD of Sm-hPAF) modulates pore-forming activity “after” binding to the cell membrane [29] and the present findings showing no contribution of the domain to receptor recognition, the species-dependency of Sm-hPAF depends solely on receptor recognition by D4.


Figure 2. The domains from ExD to D3 of Sm-hPAF do not participate in species-dependent hemolytic activity but D4 does contribute.

(a) His-tagged Sm-hPAF recombinant with substituted D4 to ILYD4, His-Sm-hPAFID4 and (b) SLYD4, His-Sm-hPAFSD4. (c) His-tagged ILY recombinant with substituted D4 to Sm-hPAFD4, His-ILYPD4 and (d) His-tagged SLY recombinant with substituted D4 to Sm-hPAFD4, His-SLYPD4 were also investigated. Each assay was performed in duplicate and the results are shown as mean values with differences. Filled circles, human; filled squares, horse; filled triangles, rabbit; filled diamonds, sheep.

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Receptor competition between Sm-hPAF and ILY

Because D4 interacts with the target membrane [40], receptor competition between Sm-hPAF and ILY was investigated using D4 of Sm-hPAF (His-Sm-hPAFD4) and of ILY (His-ILYD4) as the competitors. According to the findings shown in Figure 3, the hemolytic activity of His-Sm-hPAF is masked by pre-incubation with both His-Sm-hPAFD4 and His-ILYD4, suggesting that Sm-hPAF recognizes the same receptor as ILY, that is, huCD59.


Figure 3. Receptor competition by CDCD4s in hemolytic activity of His-Sm-hPAF.

Human erythrocytes were pre-incubated with His-Sm-hPAFD4 or His-ILYD4 at 37 °C for 30 min, then hemolytic activity of His-Sm-hPAF was determined as described in “Materials and Methods” under the heading “Measurement of hemolytic activity”. “N” indicates background hemolysis (pre-incubation with competitor only). Each assay was performed in duplicate and the results are shown as mean values with differences. Filled circles, human erythrocytes pre-incubated with His-Sm-hPAFD4, filled squares, human erythrocytes pre-incubated with His-ILYD4, open circles, pre-incubated with no competitor.

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Association of Sm-hPAF with membrane cholesterol.

The association of Sm-hPAF with membrane cholesterol was assessed using a Biacore 1000 (GE Healthcare). Representative sensorgrams of His-Sm-hPAFD4, His-ILYD4 and His-SLYD4 are shown in Figure 4a–c, respectively. Both analytes, His-Sm-hPAFD4 and His-SLYD4, interacted with cholesterol embedded in lecithin-liposome (Fig. 4a,c, respectively), the intensity of the response units increasing with increasing analyte concentration. No significant binding was observed for His-ILYD4 (Fig. 4b). No His-CDCD4s showed interaction with lecithin liposomes without cholesterol (data not shown). The kinetic parameters for His-Sm-hPAFD4 and His-SLYD4 were calculated using Biacore 1000 BIAevaluation software. The results, shown in Table 1, indicate that the affinity of His-Sm-hPAFD4 to cholesterol embedded in lecithin liposomes is about 60% of that of His-SLYD4.


Figure 4. Surface plasmon resonance sensorgrams of His-tagged recombinant CDCD4s associated with cholesterol-embedded lecithin liposomes.

Analytes were injected for 1 min at 30 μL/min, then dissociation monitored for 3 min. (a) His-Sm-hPAFD4, (b) His-ILYD4, and (c) His-SLYD4. Representative results are shown. Solid line, 5.0 μg/mL; broken line, 2.5 μg/mL; dotted line, 1.0 μg/mL.

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Table 1. Kinetic parameters for the association between His-Sm-hPAFD4 and His-SLYD4 and cholesterol embedded in lecithin-liposomes
CDCska (×105 M−1 s−1)kd (×10−3 s−1)KA (×107 M−1)KD (×10−8 M)
His-Sm-hPAFD42.35 ± 0.464.06 ± 0.765.79 ± 0.661.74 ± 0.20
His-SLYD41.34 ± 1.561.24 ± 1.299.36 ± 2.141.10 ± 0.25
Ratio (vs. His-SLYD4)1.753.270.621.58

Absorption of His-CDCs with cholesterol-embedded lecithin liposomes

In order to evaluate the reactivity of CDCs with cholesterol in the membranes, the reactivity of full-length recombinant CDCs with cholesterol-embedded lecithin liposomes was further investigated. The results, shown in Table 2, correlated well with the data from the SPR assay by BIAcore (Fig. 4). When a typical CDC (SLY) was absorbed by incubation with cholesterol-embedded lecithin liposomes, residual hemolytic activity was not detected. However, ILY, which does not interact with membrane cholesterol (Fig. 4b), showed no reactivity towards cholesterol-embedded lecithin liposomes: significant hemolysis was observed even after absorption treatment. As with SLY, Sm-hPAF also bound to cholesterol-embedded lecithin liposomes and little residual hemolytic activity was observed in the reaction supernatant. Interestingly, VLY also showed reactivity with cholesterol-embedded lecithin liposomes and reduction of hemolytic activity in the supernatant.

Table 2. Residual hemolytic activity of CDCs after absorption with cholesterol-embedded lecithin liposome
CDCMean hemolysis ± difference (%)a
  1. a

    Each assay was performed in duplicate and the results are shown as mean values with differences. N.D, Not detectable.

His-ILY94.7 ± 0.8
His-VLY30.8 ± 6.0
His-Sm-hPAF0.3 ± 0.9

Analysis of binding characteristics of fluorescent-labeled His-CDC(ss)s and His-ILYD4 to human cells

In order to further characterize the mode of receptor recognition of CDCs, binding of CDCs (ILY, SLY and Sm-hPAF) to human promyelocytic leukemia cells was also investigated using a cell analyzer. Each N-terminal fluorescent-labeled, pore-formation restricted CDC, namely, ILY(ss), SLY(ss) or Sm-hPAF(ss), was incubated with huCD59-expressing HL60 and huCD59-nonexpressing U-937 DE-4 with or without MβCD treatment. Significant binding of fluorescent-labeled ILY(ss) was observed in huCD59-expressing HL60 whereas this was not observed for huCD59-nonexpressing U-937 DE-4 (Fig. 5a,b; light gray zone). Significant binding of fluorescent-labeled SLY(ss) was observed in both HL60 and U-937 DE-4 (Fig. 5c,d; light gray zone). Binding of fluorescent-labeled Sm-hPAF(ss) was also observed in both HL60 and U-937 DE-4, the binding being stronger to HL60 than to U-937 DE-4 (Fig. 5e,f; light gray zone). With regard to the effects of cholesterol-depletion treatment by MβCD on CDCs binding to human promyelocytic leukemia cells, binding of fluorescent-labeled SLY(ss) was significantly decreased both in HL60 and U-937 DE-4 (Fig. 5c,d; compare dark gray and light gray zones). Similarly, a decrease in fluorescent-labeled Sm-hPAF(ss) binding to MβCD-treated cells was also observed both in HL60 and U-937 DE-4 (Fig. 5e,f; compare dark gray and light gray zones). Expression of huCD59 for U-937 DE-4 and HL60 was determined by immunoblotting; the results are shown in Figure 5g. In contrast, despite a slight decrease binding of fluorescent-labeled ILY(ss) to HL60 was maintained after MβCD treatment (Fig. 5a; compare dark gray and light gray zones). However, binding of fluorescent-labeled His-ILYD4 to HL60 was significantly reduced to the level of U-937 DE-4 by MβCD treatment (inserts of Fig. 5a,b); however, no reduction in huCD59 was observed even after MβCD-treatment (Fig. 5h). Moreover, the amount of fluorescent-labeled ILY(ss) bound to HL60 cells after washing three times with PBS was significantly less than in cells not subjected to MβCD-treatment (Fig. 5i; compare dark gray and light gray zones), indicating that the stability of ILY binding is weakened by depletion of cholesterol from the membranes.


Figure 5. Contribution of huCD59 and membrane cholesterol to binding of CDCs to human promyelocytic leukemia cell lines.

(a, b, and i) N-terminal Alexa Fluor 532-labeled ILY(ss), (c and d) SLY(ss), or (e and f) Sm-hPAF(ss) were incubated with human promyelocytic leukemia cell lines, (a, c, e and i) huCD59 expressing HL60 or (b, d and f) non-huCD59 expressing U-937 DE-4 and with or without MβCD treatment. Domain 4 of ILY labeled with the same fluorophore, Alexa Fluor 532-labeled ILYD4, was also reacted with the cells (inserts a, b). The cell surface-bound fluorescent-labeled CDCs were analyzed with a cell analyzer Guava PCA (EMD Millipore). Light gray, normal cells reacted with fluorescent-labeled CDCs; dark gray, MβCD-treated cells reacted with fluorescent-labeled CDCs; fine broken lines, normal cells only; bold broken lines, MβCD-treated cells only. (g) SDS–PAGE patterns of all cellular proteins stained by CBB and endogenous huCD59 detected by immunoblotting in U-937 DE-4 and HL60. Lane 1, U-937 DE-4; lane 2, HL60; Lane M, molecular weight marker. (h) SDS–PAGE patterns of all cellular proteins stained by CBB and huCD59 detected by immunoblotting in HL60 with or without MβCD treatment. Lane 1, HL60; lane 2, MβCD-treated HL60; Lane M, molecular weight marker. (i) The amount of Alexa Fluor 532-labeled ILY(ss) bound to HL60 cells after washing three times with PBS was also investigated in MβCD-treated (dark gray) and non-treated HL60 (light gray). Bold broken lines, MβCD-treated HL60 cells; fine broken lines, non-treated HL60 cells. The data shown are typical of the results of three independent experiments.

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Cytotoxicity of Sm-hPAF enhanced by huCD59

In order to further investigate the mode of receptor recognition of Sm-hPAF, its cytotoxicity against culture cell lines with or without expression of huCD59 was assessed. Figure 6 shows the cytotoxicity of CDCs against human promyelocytic leukemia cell lines U-937 DE-4 and HL60 (Fig. 6a for His-Sm-hPAF, Fig. 6b for ILY, and Fig. 6c for His-SLY). ILY showed no cytotoxicity against U-937 DE-4 because of the absence of receptor huCD59 (Fig. 6b, the absence of expression of huCD59 in U-937 DE-4 is shown in Fig. 5g). On the other hand, His-SLY reacted with cholesterol in the target cell membrane and showed similar cytotoxic activity against U-937 DE-4 and HL60 (Fig. 6c). Unlike the above findings, His-Sm-hPAF had higher activity against HL60 (huCD59 expression of HL60 is shown in Fig. 5g) than against U-937 DE-4 (Fig. 6a). The cytotoxicity of His-Sm-hPAF against HL60 was about 30-fold higher than that against U-937 DE-4.


Figure 6. Cytotoxicity of His-Sm-hPAF, native ILY and His-SLY against culture cell lines with or without expression of huCD59.

Cytotoxicity of (a) His-Sm-hPAF, (b) native ILY and (c) His-SLY against a huCD59-negative cell line, U-937 DE-4 and a huCD59-positive cell line, HL60. Cells were incubated with serially diluted CDCs for 1 hr at 37 °C in 5% (v/v) CO2, then the viability determined as described in “Materials and Methods” under the heading “Measurement of cytotoxicity of CDCs in culture cells”. Open circles, U-937 DE-4; closed circles, HL60. Cytotoxicity of (d) His-Sm-hPAF, (e) native ILY and (f) His-SLY against a human hepatoma cell line, HepG2; a rat hepatic cell line, BRL3A; and a huCD59-expressing transformant of BRL3A. Cells were incubated with serially diluted CDCs for 1 hr at 37 °C under 5% (v/v) CO2 condition and then the viability was determined. Each assay was performed in duplicate and the results are shown as mean values with differences. Closed squares, HepG2; open circles, rat hepatic cell line, BRL3A; closed circles, huCD59-expressing transformant of BRL3A. Expression of huCD59 on the cell surface of (g) HepG2, (h) BRL3A and (i) BRL3A-transformant expressing huCD59 was detected by immunofluorescent staining. Scale bars represent 20 μm. (j) SDS–PAGE pattern of total cellular proteins stained by CBB (7.5 × 104 cells/lane) and huCD59 detected by immunoblotting (2.5 × 104 cells/lane). Lane 1, U-937 DE-4; lane 2, HL60; lane 3, BRL3A-transformant expressing huCD59; lane 4, BRL3A; lane 5, HepG2; Lane M, molecular weight marker. Cytotoxicity of His-Sm-hPAF against huCD59-null cell lines (k) U-937 DE-4 and (l) BRL3A with or without MβCD treatment. Each assay was performed in duplicate and the results are shown as mean values with differences. Open symbols, with MβCD; closed symbols, without MβCD.

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Using a human hepatoma HepG2 as a reference, a non-human cell line (rat hepatic cell BRL3A) with or without expression of huCD59 was also investigated (Fig. 6d, His-Sm-hPAF; Fig. 6e, ILY; Fig. 6f, His-SLY). As with the results obtained in human promyelocytic leukemia cell lines, ILY showed specificity towards huCD59-expressing BRL3A and HepG2 with endogenous huCD59, and His-SLY displayed similar levels of cytotoxicity against all cell lines tested (Fig. 6e,f). Interestingly, His-Sm-hPAF also displayed a similar increase in cytotoxicity to that against HepG2 when huCD59 was expressed in BRL3A. Moreover, cytotoxicity was still observable for BRL3A (no huCD59 expression) in the presence of higher concentrations of His-Sm-hPAF (Fig. 6d). The susceptibility of huCD59-expressing cells to His-Sm-hPAF was also more than 10-fold higher than that of BRL3A with no huCD59. Expression of huCD59 in these cell lines was assessed by immunofluorescent staining for huCD59 (Fig. 6g–i) and the amount of expression of huCD59 in the tested cell lines was compared by immonoblotting (Fig. 6j).

To determine the dependency of cytotoxicity of His-Sm-hPAF on membrane cholesterol, the cytotoxic properties of His-Sm-hPAF were further investigated in the huCD59-null cells, U-937 DE-4 (Fig. 6k) and BRL3A (Fig. 6l), after treatment with or without MβCD. As shown in Figure 6k,l, the cytotoxicity of His-Sm-hPAF against membrane cholesterol-depleted cells (open symbols) was significantly decreased compared to that against normal cells (closed symbols). These results clearly show that His-Sm-hPAF can trigger pore-formation without huCD59, indicating that cytotoxicity against huCD59-null cells depends on the presence of membrane cholesterol.

Binding of fluorescent-labeled ILY(ss) (Fig. 7a,b), fluorescent-labeled SLY(ss) (Fig. 7c,d) and fluorescent-labeled Sm-hPAF(ss) (Fig. 7e,f) to huCD59-expressing transformant of rat hepatic cell BRL3A (Fig. 7a,c,e) and their parent huCD59-null BRL3A (Fig. 7b,d,f) was also investigated. Binding of fluorescent-labeled CDC(ss)s showed similar tendencies to those shown in Figure 5a–f. Binding of fluorescent-labeled ILY(ss) was observed on huCD59-expressing transformant of BRL3A and this binding was less influenced by MβCD-treatment (Fig. 7a). On the other hand, because of the absence of huCD59 expression, binding of fluorescent-labeled ILY(ss) was not, or to a lesser extent, on parent BRL3A (Fig. 7b). Binding of fluorescent-labeled ILY(ss) was observed in both cell lines; binding in both cases being significantly reduced by MβCD-treatment (Fig. 7c,d; compare light gray dark gray zones). Again, binding of His-Sm-hPAF(ss) was detected on both cell lines (Fig. 7e,f; light gray zone) but this binding was decreased by cholesterol-depletion using MβCD, especially in huCD59-null BRL3A (Fig. 7e,f; dark gray zone).


Figure 7. Contribution of huCD59 and membrane cholesterol to binding of CDCs to non-human adherent cells.

N-terminal Alexa Fluor 532-labeled (a and b) ILY(ss), (c and d) SLY(ss) or (e and f) Sm-hPAF(ss) was incubated with (a, c and e) huCD59-expressing transformant of BRL3A or (b, d and f) its parent BRL3A. Cell surface-bound fluorescent-labeled CDCs were analyzed with the cell analyzer Guava PCA. Light gray, normal cells reacted with fluorescent-labeled CDCs; dark gray, MβCD-treated cells reacted with fluorescent-labeled CDCs; fine broken lines, normal cells only; bold broken lines, MβCD-treated cells only.

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VLY is a species-dependent CDC like Sm-hPAF

Vaginolysin, a CDC secreted by G. vaginalis, is reportedly a “human-specific cytolysin” (32). However, VLYD4 had greater amino acid identity with Sm-hPAFD4 (68%) than ILYD4 (53%) (Table 3). Because this observation suggests that the hemolytic properties of VLY are similar to those of Sm-hPAF (that is, it exhibits species-dependent activity), the hemolytic activities of His-VLY against various animal erythrocytes were investigated. As shown in Figure 8a, the highest activity was observed against human erythrocytes. Moreover, hemolytic activity of His-VLY against non-human erythrocytes (horse and rabbit) was also observed and, as expected, the species-dependent affinity of His-VLY to erythrocytes followed a similar pattern to that of His-Sm-hPAF [34] (Fig. 8a). The direct association of His-VLY with cholesterol was also investigated by surface plasmon resonance. Figure 8b shows that obvious affinity of His-VLYD4 to cholesterol embedded in lecithin liposomes, this affinity being intermediate between that of His-SLYD4 and His-ILYD4 (Fig. 8b). These results indicate that VLY is not a human-specific cytolysin but rather a human-preferential, species-dependent cytolysin, as is true of Sm-hPAF.

Table 3. Amino acid sequence identity between Sm-hPAF (BAE72438) and related CDCs
CDCsAmino acid sequence identityaGenBank ID
  1. a

    The numbers in parentheses are the number of amino acids for analysis (number of identical amino acids vs. total compared).

Lectinolysin (LLY)98% (653/665)97% (462/472)99% (110/111)ACE79194
Vaginolysin (VLY)56% (276/486)58% (275/470)68% (76/111)ACD39459
Intermedilysin (ILY)55% (257/467)55% (257/467)53% (59/111)BAA89790
Mitilysin (MLY)50% (238/467)50% (238/467)55% (61/109)ABK58690
Suilysin (SLY)44% (184/417)45% (216/473)51% (55/108)CAA85378

Figure 8. Characteristics of VLY similar to Sm-hPAF.

(a) Species-dependent hemolytic activity of His-tagged VLY to erythrocytes of human, horse, rabbit and sheep. Each assay was performed in duplicate and the results are shown as mean values with differences. Filled circles, human; filled squares, horse; filled triangles, rabbit; filled diamonds, sheep. (b) SPR sensorgram of His-tagged VLYD4 associated with cholesterol-embedded liposomes (broken line). Each 500 nM of analyte (His-VLYD4, 7.3 μg/mL; His-ILYD4, 7.3 μg/mL; His-SLYD4, 7.0 μg/mL) was injected for 1 min at 30 μL/min, then dissociation was monitored for 2 min. For comparison, sensorgrams of His-ILYD4 (dotted line) and His-SLYD4 (solid line) are also shown.

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Amino acid sequence of domain 4 of CDCs

The structural diversity (amino acid sequence) of CDCD4s was investigated. Only one residue difference in amino acid sequence (99% identity) was found between Sm-hPAFD4 and LLYD4 (Table 3). D4 of huCD59-recognizing VLY had the next closest similarity to Sm-hPAFD4 (68% sequence identity). Unexpectedly, there was relatively little similarity between Sm-hPAFD4 and ILYD4 (53% sequence identity); this is close to that found with typical, cholesterol-binding CDCs such as MLYD4 and SLYD4 (Table 3).

Figure 9a shows a model structure of Sm-hPAFD1–4 (a close-up view of D4 is also shown in Fig. 9b) and the amino acid sequence alignments for Sm-hPAFD4 and other CDCD4s (Fig. 9c). A signature motif for huCD59-binding CDCs (Tyr-X-Tyr-X14-Arg-Ser) (31) was completely conserved among Sm-hPAF, LLY, VLY and ILY, but not conserved among other cholesterol-binding CDCs (Fig. 9c). On the other hand, as reported previously (41), the amino acids that reportedly insert into the membrane surface, the CRM in L1, were completely conserved. The amino acids in L2 and L3 were also highly conserved among CDCs; however, some variation was observed in the amino acids of L3 (42–44). Moreover, for LLY, an additional variation was observed in L2, in which the amino acid is Ile. In the conserved 11-mer region of CDCs, the amino acids were completely conserved among Sm-hPAF, LLY and VLY (EKTGLVWEPWR); three amino acid substitutions being observed only in ILY (GATGLAWEPWR). As shown in the phylogenetic tree of CDCD4s, huCD59-recognizing CDCs (ILY, VLY, LLY and Sm-hPAF) were mapped to the same branch and were separate from other clusters of cholesterol-binding CDCs (Fig. 10).


Figure 9. Model of structure of Sm-hPAFD1–4 and alignment of amino acid sequences of CDCD4s.

(a) Molecular model of Sm-hPAFD1–4 constructed by insightII-discover with homology module (Accelrys) based on information about the molecular structure of ILY (PDB ID: 1S3R) as a reference. (b) A close-up view of region D4 of modeled Sm-hPAF showing the region of amino acids composing the 11mer conserved region, reported as a signature motif for huCD59-recognizing CDCs (Tyr-X-Tyr-X14-Arg-Ser) (31); the region that inserts into the membrane surface (CRM, L2 and L3) (41, 42) is indicated. (c) The alignment of amino acid sequences of CDCD4s is shown with CRM (white with black shading), L2 and L3 (white with gray shading). Signature motifs for huCD59-recognizing CDCs are indicated by arrowheads. Roman numerals indicate the classification of the mode of receptor recognition of CDCs (refer to the legend of Fig. 10). The symbols indicating identity/homology of primary sequences beneath the figure are as follows: “*”, amino acid conserved; “:”, amino acid highly conserved; “.”, amino acid moderately conserved.

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Figure 10. Phylogenetic tree of CDCD4s.

The amino acid sequences of CDCD4s were aligned by Clustal X and the phylogenetic tree constructed by Njplot. CDCs that recognize both cholesterol and huCD59 as their receptor (Sm-hPAF, LLY, and VLY) are indicated in bold face. The mode of receptor recognition is classified into three groups: Group I are cholesterol-binding CDCs with high affinity to cholesterol and no or very little affinity to huCD59; Group II are huCD59-recognizing CDCs with no or very little affinity to cholesterol and high affinity to huCD59 (only one member so far); and Group III are cholesterol-binding/huCD59-recognizing CDCs with intermediate affinity to both cholesterol and huCD59.

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  9. Supporting Information

Cholesterol-dependent cytolysins are bacterial protein toxins secreted particularly from several species of pathogenic Gram-positive bacteria. This toxin family was formerly called “thiol-activated cytolysins” or “cholesterol-binding cytolysins.” However, because the discovery of ILY introduced a new sub-group into the gene family (huCD59-recognizing cytolysins with dependency on the presence of cholesterol in the target membrane for pore-forming activity), all these cytolysins were termed CDCs. CD59 is a GPI-anchored glycoprotein that helps to confer self-protection from the cytotoxic effect of the membrane attack complex induced by activation of the complement system [45]. Since the demonstration of ILY and its receptor huCD59, several studies on this atypical type of CDC have shown no or very low cholesterol binding [30, 31, 35, 42, 43, 46-51]. A second huCD59-recognizing CDC, named VLY, was subsequently discovered [32], followed by a report of the recognition of huCD59 by LLY [31]. Consequently a novel CDCs sub-family recognizing huCD59 as the receptor is now established.

To date, two modes of receptor recognition by CDCs have been reported: (i) the more typical cholesterol-binding mode; and (ii) an atypical huCD59-recognizing mode. The latter mode constitutes a sub-family of CDCs that includes ILY, VLY and LLY. As shown in the present study, Sm-hPAF, a homologue of LLY, also belongs to the sub-family with an atypical huCD59-recognizing mode (Fig. 3). This property of Sm-hPAF seems to be responsible for the observed human-preferential activity. However, unlike the more strictly human-specific activity of ILY, Sm-hPAF displays hemolytic activity/cytotoxicity against non-human erythrocytes (34), human cells not expressing huCD59 (Fig. 6a), and rat hepatic cells with no huCD59 (Fig. 6d). This property of the hemolytic activity/cytotoxicity of Sm-hPAF may be partly attributable to their ability to recognize membrane cholesterol, as do the typical CDCs (Figs. 4a, 6k,l and Tables 1, 2). These findings enabled recognition of a new group of Sm-hPAF that possesses receptor recognition properties intermediate between cholesterol-binding typical CDCs and huCD59-recognizing atypical CDCs. Furthermore, the results of the present study suggest that Sm-hPAF preferentially recognizes huCD59 over cholesterol because of (i) the greater susceptibility to Sm-hPAF of huCD59-expressing BRL3A cells compared with the parent, non-human BRL3A cells (Fig. 6d), and (ii) the lesser susceptibility to Sm-hPAF of huCD59-negative U-937 DE-4 compared with huCD59-positive HL60 (Fig. 6a). Quantitative comparison of the affinity of Sm-hPAF to huCD59 with that to cholesterol has not yet been accomplished because of the difficulties in efficient purification of native huCD59 and in reconstitution of the physiological state of huCD59, that is, huCD59 in lipid raft, without cholesterol condition. However, we speculate that the choice by Sm-hPAF between huCD59 and cholesterol receptors might depend on differences in affinity to these molecules on biological membranes. Thus, huCD59-preferential recognition by Sm-hPAF would contribute to their cytotoxicity because of the characteristic of preferentially targeting human cells. Though the precise mechanism of human-preferential, species-dependent hemolytic activity/cytotoxicity of Sm-hPAF is so far unclear, we speculate that, because the primary- and the secondary-structures of the region in huCD59 reported to interact with ILY (30) is not conserved in other CD59 of animal origin, the factor most likely to be responsible for this remarkable property of Sm-hPAF is the difference in affinity between Sm-hPAF and CD59 of various animal origins. Unlike ILY, it seems that the CD59-binding sites of Sm-hPAF and the homologues belonging to this CDC type are not best fit for huCD59; rather, they are somewhat loose, allowing interactions with not only huCD59 but also with other animal CD59s. Therefore Sm-hPAF can also bind to animal CD59s with individual affinity. Attempts at constructing transformants of U-937 DE-4 (huCD59 null cells) with CD59 genes from other animal species in order to further understand the basis for Sm-hPAF species-specificity have so far remained unsuccessful because there has been no detectable expression of CD59 (data not shown). Thus, further study is necessary in order to understand the species-specificity shown by Sm-hPAF.

As to the association of ILY with cholesterol, Dowd et al. have reported that ILY binds to cholesterol-rich POPC liposomes [51]. However, we found no significant association between ILY and cholesterol-containing membranes (Fig. 4b and Table 2); our data strongly suggest that ILY does not specifically associate/interact with membrane cholesterol. Therefore, Dowd et al.'s finding of relatively high concentrations of ILY on cholesterol-rich POPC liposomes may have been attributable to non-specific adsorption rather than reflecting an interaction between ILY and human cells under physiological conditions. Such non-specific adsorption may be facilitated by ILY's highly positive charges (calculated pI: 9.87), which cause non-specific binding as we found during purification of ILY by ion-exchange chromatography [35].

Recently, Johnson et al. reported that the crystal structure of the ILY-huCD59 complex has two interfaces on huCD59 coordinate ILY monomers [52]. The feature of this interaction may indicate that clustering of huCD59 facilitates binding and ring oligomer (known as “prepore”) formation of ILY. As shown in a previous study, after incubation with human erythrocyte ghost membrane, ILY forms membrane pores that are irreversibly embedded in membranes with SDS-resistance [22]. However, binding of ILY is thought to induce reversible oligomerization that forms “prepores”, the stage prior to formation of membrane-embedded pores of CDCs. We speculate that, supported by the avidity with which ILY molecules associate, ILY binds to huCD59 clusters in lipid rafts with high affinity. However, the binding affinity of ILY to dispersed huCD59 would be significantly weakened by MβCD treatment, which disrupts the lipid raft structure and thus the support for association avidity. This explains why PBS washing only easily washed out ILY bound to MβCD-treated HL60 cells, whereas very little bound ILY was washed out of normal HL60 cells (Fig. 5i). Johnson et al. reported that some amino acid residues involve primary- and secondary-binding sites of ILY for interactions with huCD59 [52]. Among the residues involved in ILY-huCD59 interactions, the conserved residues in huCD59-binding CDC are Y436 and R480. In order to describe the determinant structure/residue for receptor recognition of ILY (Group II) and huCD59-binding CDC (Group III), further information is necessary.

In this study, we suggest a new categorization of the modes of receptor recognition in CDCs (Figs. 9, 10). Group I comprises typical CDCs with high affinity to cholesterol and no or very little affinity to huCD59: most CDCs so far described belong to this group. Group II, ILY being the only group member so far, comprises atypical CDCs with no or only very little affinity to cholesterol and high affinity to huCD59. Group III, a novel group of atypical CDCs with affinity to both cholesterol and huCD59, includes Sm-hPAF, VLY and possibly LLY. The amino acid sequence data for CDCD4s also support this categorization; the amino acid sequence of a signature motif for huCD59-recognizing CDCs (Tyr-X-Tyr-X14-Arg-Ser) [31] and 11mer region was completely conserved amongst the members of Group III (Fig. 9c). Two of the five amino acids in the CRM (L1), L2 and L3 that are inserted into the membrane surface, are not completely conserved (Fig. 9c). We also observed one of the amino acid variations in L3 in CDCs belonging to Group I (Fig. 9c). However, previously reported data showing that the A464D mutation in ILY does not affect oligomer assembly while blocking the conversion required for pore formation [43] suggest that this amino acid makes only a small contribution to the interaction with membrane cholesterol and that the amino acid in L3 is not very important in the cholesterol-binding property of CDCs. Another variation has been observed only in LLY; namely, the amino acid Ile in L2 replacing the Val that is present in other CDCs. L3 is located immediately behind this residue [42], variation in which may also be relatively unimportant for cholesterol binding. Furthermore, it has been reported that only two amino acids of CRM (L1) are essential for cholesterol recognition [41]. Thus, it seems that all CDCs potentially possess the property of cholesterol binding.

These observations invite speculation as to why there is diversity in receptor recognition. Interestingly, previously reported data shows that substitution of the 11mer region in ILYD4 with that of the consensus 11mer of CDCs, ECTGLAWEWWR, makes ILY non-human specific and susceptible to cholesterol inhibition, as seen with typical (i.e., Group I) CDCs [22]. On the other hand, in a review of the interaction between ILY and huCD59, two important functions were suggested [23]: one being initiation of structural change for pore formation and the other positioning of CRM in order to bind to membrane cholesterol and initiate further insertion of short hydrophobic loops into the membrane. Based on these reported findings, although the above consensus sequence in typical CDCs does not affect the interaction between CRM and cholesterol, this region in ILY may inhibit the interaction of CRM on the surface of D4 with the target membrane because the 11mer region is a structure that loops out from the rigid body of D4. It is possible that the interaction of ILY with huCD59 induces conformational changes in the 11mer region that unmask steric hindrance and allow interaction between CRM and membrane cholesterol. In this context, a clear classification of the 11mer region into three patterns representing Groups I–III is desirable (Fig. 9c). In the case of Group III CDCs with 11mer region sequence consensus, CDCs which can directly bind to cholesterol but with an affinity lowered by incomplete hindrance of the region, the inhibitory or masking effect in the interaction of CRM with membrane cholesterol may be less marked than with ILY. However, how this region of Groups II and III participates in the selective interaction with huCD59 is so far unclear. Further research is necessary to identify the basis for the observed species-dependency of Sm-hPAF. This would have to take into account the variations in affinity to CD59 of humans and other animals seen in CDCs of Groups II and III, defined by the complex interactions between the 11mer region and the signature motifs for cholesterol binding and huCD59-recognition. The answer to this question may be found in future studies using in silico analysis, such as molecular modeling of CDCs and their receptors, cholesterol and CD59.


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  9. Supporting Information

This research was partially supported by Grants-in-Aids for Young Scientists (Start-up) No. 18890127, for Scientific Research (C) Nos. 24592769 and 24590221, and the Program for the Strategic Research Foundation at Private Universities (2012–2016) from the Ministry of Education, Culture, Sports, Science, and Technology of the Japanese Government. We are grateful to S. Saito, E. Sakakura, Y. Shinohara, K. Suzuki, M. Aoyagi, N. Ozaki, M. Hasegawa, M. Ishikawa and Y. Nakamura for technical assistance.


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  9. Supporting Information

The authors have no conflicts of interest associated with this study.


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mim12131-sm-0001-SuppData-S1.pdf365KTable S1. Primers used in the study

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