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J. Biol. Chem., Vol. 276, Issue 45, 41576-41579, November 9, 2001

This Article Abstract Full Text (PDF) All Versions of this Article: 276/45/41576    most recent M107819200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kimura, T. Articles by Takeda, T. Search for Related Content PubMed PubMed Citation Articles by Kimura, T. Articles by Takeda, T. Social Bookmarking                      What's this?

Serum Amyloid P Component Is the Shiga Toxin 2-neutralizing Factor in Human Blood^*

Tsuyoshi Kimura^§, Shinobu Tani, Yoh-ichi Matsumoto^¶, and Tae Takeda

From the  Teijin Institute for Biomedical Research, Teijin Ltd., 4-3-2, Asahigaoka, Hino City, Tokyo 191-8512, Japan, ^¶ Pharmaceutical Development Department I, Teijin Ltd., 2-1-1, Uchisaiwaicho, Chiyoda-ku, Tokyo 100-8585, Japan, and  Department of Infectious Diseases Research, National Children's Medical Research Center, 3-35-31, Taishido, Setagaya-ku, Tokyo 154-8509, Japan

Received for publication, August 14, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

It has been suggested that some factor present in human plasma binds to Shiga toxin 2 (Stx2) and neutralizes it in vitro (Bitzan, M., Klemt, M., Steffens, R., and Muller-Wiefel, D. E. (1993) Infection 21, 140-145). This factor does not exist in other species (Caprioli, A., Luzzi, I., Seganti, L., Marchetti, M., Karmali, M., Clarke, I., and Boyd, B. (1994) Recent Adv. VTEC Infect. 353-356). Because analysis of this factor is important to understanding the pathology induced by Shiga toxin-producing Escherichia coli, we purified this factor from human plasma and identified it. Purification was carried out by serially subjecting human plasma to Con A-Sepharose, DEAE-Sepharose, hydroxyapatite, and gel-filtration high performance liquid chromatography (HPLC), using Stx2-neutralizing activity as the indicator. The gel-filtration HPLC fraction yielded a single band on SDS-polyacrylamide gel electrophoresis. Twenty N-terminal amino acid residues of this fraction were analyzed and found to correspond perfectly to human serum amyloid P component (HuSAP). Because commercially available HuSAP also showed Stx2 binding and neutralizing activity, we identified this factor as HuSAP.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Shiga toxin (Stx)¹-producing Escherichia coli (STEC) has been recognized as a pathogen that causes bloody diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS) mostly in developed countries (3). STEC produces Stxs, which mediate STEC virulence (4). Stxs consist of an A-subunit monomer, which has RNA N-glycosidase activity, and a B-subunit pentamer, which is involved in receptor binding (5-7). Stxs can be divided broadly into two groups, Stx1 and Stx2. Stx2, but not Stx1, is thought to be a major cause of toxicity that induces HUS in humans based on epidemiological analysis and studies on STEC-infected animals (8-10).

Bitzan et al. (1) have shown that some factor present in human plasma binds to Stx2 and neutralizes it in vitro, and Caprioli et al. (2) have shown that this activity does not exist in other species. It is unclear why Stx2 induces HUS in humans despite the presence of this factor. To assess the role and function of this factor in STEC infection in humans, we purified this factor from human plasma and identified it.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials

Con A-Sepharose, DEAE-Sepharose, PD-10, NAP-5, and gel-filtration HPLC (Superose 6 HR 10/30) were from Amersham Pharmacia Biotech. Hydroxyapatite (Gigapite, grade K-100S) was from Seikagaku Corp. D-Mannose was from Wako Pure Chemicals. -Methyl-D-mannoside was from Nacalai Tesque. Polyvinylidene difluoride membrane (ProSpin) was from Applied Biosystems. Human serum amyloid P component (HuSAP) was from Alexis.

Human renal adenocarcinoma ACHN (ATCC CRL1611) was obtained from the American Type Culture Collection. Eagle's minimum essential medium was purchased from Nissui Pharmaceutical. Fetal calf serum was from Life Technologies, Inc. and bovine serum albumin (BSA) was from Roche Molecular Biochemicals.

Stx1 and Stx2 were prepared from recombinant E. coli VS-1 and E. coli MC1061 (pITY1) (11, 12), respectively. Each recombinant E. coli was cultured in Luria-Bertani (LB) broth with 100 µg/ml ampicillin (for Stx1) or Mueller-Hinton broth with 5 µg/ml trimethoprim (for Stx2) at 37 °C for 2 days with vigorous shaking. Stx1 and Stx2 were purified by DEAE-Sephacel (for Stx1) or DEAE-Sepharose (for Stx2) column chromatography, PBE94 chromatofocusing chromatography (Amersham Pharmacia Biotech), and TSK-gel G-2000 SW (Tohso) as described previously (13, 14).

Stxs Binding Assay

A 1 µg/ml volume of Stx1 or Stx2 in PBS was inoculated on MaxiSorp enzyme-linked immunosorbent assay plates (Nunc) and incubated at 4 °C for 1 day. The wells were then washed with 1% BSA-PBS and blocked with 3% BSA-PBS at 37 °C for 1 h. After removing the blocking buffer, HuSAP serially diluted with 3% BSA-PBS was added to the wells, and the plates were incubated at 37 °C for 1 h. After washing with 1% BSA-PBS, the wells were incubated at 37 °C for 1 h with rabbit anti-HuSAP serum (Biogenesis) diluted 4000-fold with 3% BSA-PBS. The wells were then washed with 1% BSA-PBS and incubated at 37 °C for 1 h with peroxidase-conjugated goat anti-rabbit immunoglobulin (Kirkegaard & Perry Laboratories) diluted 1:2000 with 3% BSA-PBS. After washing with 1% BSA-PBS, peroxidase activity was detected colorimetrically by adding TMB (Kirkegaard & Perry Laboratories).

Stx2 Neutralizing Assay

A 100-µl volume of a 1 × 10⁵ cells/ml suspension of human renal adenocarcinoma ACHN cells in growth medium (Eagle's minimum essential medium-10% fetal calf serum) was inoculated into 96-well flat-bottomed plates and incubated at 37 °C with 5% CO₂ for 1 day. After removing the growth medium and adding 50 µl of fresh growth medium to the wells, 50 µl of the pre-incubated mixture of test sample and Stx2 (5-10 CD₅₀) was added to each well. The neutralizing assay for the fractions from each purification step was performed according to method 1 below and for commercial HuSAP by methods 1 and 2 below.

Method 1-- After incubation at 37 °C with 5% CO₂ for 1 h, the mixture was removed, and the wells were washed once with growth medium. Next, 100 µl of growth medium was added, and the plates were incubated at 37 °C with 5% CO₂ for 4 days. A 100-µl volume of 0.028% (w/v) neutral red (Merck) in growth medium was added, and incubation at 37 °C with 5% CO₂ was continued for 1 h to stain the living cells. After washing the wells with PBS, 100 µl of 50% ethanol, 1% acetic acid solution was added to extract the dye, and the absorbance at 550 nm was measured.

Method 2-- After incubation at 37 °C with 5% CO₂ for 4 days, living cells were stained with neutral red and quantified as described above. Neutralizing activity was calculated according to the following formula: neutralization (%) = (A_{550(Stx2 + test sample)}  A_550(Stx2))/(A_550(medium)  A_550(Stx2)) × 100.

Other Procedures

SDS-PAGE was performed according to Laemmli, using 4-20% polyacrylamide gradient slab gel (Daiichi Pure Chemicals), and the separated proteins were silver-stained (Wako Pure Chemicals). Throughout the purification procedures, the protein concentration was estimated by BCA protein assay or micro BCA protein assay (Pierce) with BSA as a standard. Spectrophotometric determinations were made in a Shimadzu UV-160A spectrophotometer. Protein sequencing was performed using an Applied Biosystems 494 protein sequencer.

	RESULTS

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Purification of Stx2-neutralizing Factor from Human Blood

Bitzan et al. (1) have reported the presence of a non-immunoglobulin factor in human plasma that neutralizes Stx2 in vitro. Caprioli et al. (2) have shown that this activity does not exist in other species and that the human high-density lipoprotein fraction neutralizes Stx2 in vitro. However, we were unable to detect any Stx2-neutralizing activity by human HDL (data not shown). Because analysis of this Stx2-neutralizing factor is important to understanding the pathology of STEC, we purified this factor from human plasma and identified it. Plasma obtained from one healthy male donor was used as the starting material. Purification was serially carried out by Con A-Sepharose, DEAE-Sepharose, hydroxyapatite, and gel-filtration HPLC.

Step 1-- Chromatography on Con A-SepharoseThe human plasma was applied to a Con A-Sepharose column (column bed volume, 40 ml) equilibrated with PBS, and the column was thoroughly washed with PBS and then serially eluted with 0.1 M D-mannose in PBS and 0.5 M -methyl-D-mannoside in PBS. As shown in Fig. 1a, Stx2-neutralizing activity was observed in the eluate from 0.1 M D-mannose in PBS. This eluate was applied to a PD-10 column equilibrated with 10 mM Tris-HCl (pH 8.0) to equilibrate the same buffer.

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Column chromatography of each purification step. Each column chromatography experiment was carried out as described under "Results." a, Con A-Sepharose column chromatography. Fractions of 10 ml were collected. The arrow indicates the fractions containing Stx2-neutralizing activity. b, DEAE-Sepharose column chromatography. Fractions of 5 ml were collected. c, hydroxyapatite column chromatography. Fractions of 6 ml were collected. d, gel-filtration HPLC. The flow rate was 0.5 ml/min. Fractions of 0.5 ml were collected.

Step 2-- Chromatography on DEAE-SepharoseThe eluate from the PD-10 column was applied to a DEAE-Sepharose column (column bed volume, 5 ml) equilibrated with 10 mM Tris-HCl (pH 8.0), and the column was serially washed with 10 mM Tris-HCl (pH 8.0) and 0.1 M NaCl in 10 mM Tris-HCl (pH 8.0), then developed with a linear salt gradient from 0.1 to 0.3 M NaCl in 10 mM Tris-HCl (pH 8.0), and finally washed with 2 M NaCl in 10 mM Tris-HCl (pH 8.0). As shown in Fig. 1b, Stx2-neutralizing activity was observed in the eluate at about 0.2-0.24 M NaCl in 10 mM Tris-HCl (pH 8.0). These fractions were collected and applied to a PD-10 column equilibrated with 50 mM sodium phosphate (pH 6.8) to equilibrate the same buffer.

Step 3-- Chromatography on HydroxyapatiteThe eluate was applied to a hydroxyapatite column (column bed volume, 3 ml) equilibrated with 50 mM sodium phosphate (pH 6.8). The column was washed with 50 mM sodium phosphate (pH 6.8), then developed with a linear gradient of 50-250 mM sodium phosphate (pH 6.8), and finally washed with 250 mM sodium phosphate (pH 6.8). As shown in Fig. 1c, Stx2-neutralizing activity was observed mainly in the eluate at ~125-250 mM sodium phosphate (pH 6.8). Fractions of the eluate at ~200-250 mM sodium phosphate (pH 6.8) were collected, frozen, and dried in a rotary evaporator. The dried sample was dissolved in distilled water.

Step 4-- Chromatography by Gel-filtration HPLCThe pool of the eluate from Step 3 was applied to a gel-filtration HPLC (Superose 6 HR 10/30) using PBS as the elution buffer. As shown in Fig. 1d, Stx2-neutralizing activity was observed in the peak fractions, and the peak fractions (fractions 27-29) were pooled.

SDS-PAGE analysis of the peak fractions obtained by gel-filtration HPLC indicated a single staining band of 25 kDa (Fig. 2). This Stx2-neutralizing factor was purified about 1270-fold and the yield of the purified protein was 2.5% (Table I).

View larger version (43K): [in this window] [in a new window]   Fig. 2.   SDS-PAGE analysis of fractions from each purification step. SDS-PAGE was performed under reducing conditions as described under "Experimental Procedures." The arrow indicates the purified protein. Lane 1, molecular size marker; lane 2, human plasma; lane 3, Con A-Sepharose fraction; lane 4, DEAE-Sepharose fraction; lane 5, hydroxyapatite fraction; lane 6, gel-filtration fraction.

Identification of the Purified Protein

The purified protein was applied to a polyvinylidene difluoride membrane, and the N-terminal amino acid sequence of the protein was analyzed with an Applied Biosystems 494 protein sequencer. Twenty N-terminal amino acid residues could be analyzed, and the sequence obtained (HTDLSGKVFVFPRESVTDHV) corresponded completely to the human serum amyloid P component, which is a member of the human plasma pentraxin family consisting of a complex of 10 identical 25-kDa subunits non-covalently associated in two pentameric rings interacting face-to-face (15).

Characterization of HuSAP

To confirm that the Stx2-neutralizing factor in human blood is HuSAP, we tested the Stx2-binding activity and the Stx2-neutralizing activity of commercially available HuSAP in vitro. The binding capacity of HuSAP to Stxs was estimated by enzyme-linked immunosorbent assay. As shown in Fig. 3, HuSAP bound to Stx2 but not to Stx1. The Stx2-neutralizing activity of HuSAP was also tested by a cell cytotoxicity assay. As shown in Fig. 4, HuSAP was found to neutralize Stx2 cytotoxicity. Because commercial HuSAP showed Stx2 binding and neutralizing activity, we ultimately concluded that the Stx2-neutralizing factor in human blood was HuSAP.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Stxs binding activity of commercial HuSAP. The Stxs binding assay was performed as described under "Experimental Procedures." Black circles and black squares represent the results of the binding activity of HuSAP with Stx1 and Stx2, respectively.

View larger version (13K): [in this window] [in a new window]   Fig. 4.   In vitro Stx2-neutralizing activity of commercial HuSAP. The in vitro neutralizing assay was performed as described under "Experimental Procedures." The black circles represent the results for incubation of HuSAP and Stx2 with the target cells for 1 h. The black squares represent the results for incubation of HuSAP and Stx2 with the target cells for 4 days.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

HuSAP is synthesized by the liver and circulates in human blood at 30-45 µg/ml (16). It is found in all types of amyloid deposits (17), including in plaque from Alzheimer's disease (18), in glomerular basement membrane, and in elastic fibers in blood vessels (19, 20). It also binds in a calcium-dependent manner to a variety of ligands, including DNA and chromatin (21, 22), fibronectin (23), C4-binding protein (23, 24), glycosaminoglycans (25, 26), collagen (27), and laminin (28). HuSAP shows no polymorphism or heterogeneity, and no deficiency of HuSAP has been reported, suggesting that it has important functions. However, there has been no report on the function of HuSAP in STEC infections.

Surveillance studies have shown a strong statistical association between the Stx2 gene and severity of disease (8, 9). In addition, when mice were challenged with STEC co-producing Stx1 and Stx2, anti-Stx2 monoclonal antibodies rescued the mice from death. On the other hand, anti-Stx1 monoclonal antibodies could not rescue them (10). These studies suggest that Stx2 is a more significant factor than Stx1 in the progression of the disease in STEC infections. It is unclear why only some STEC-infected patients develop HUS despite the presence of Stx2-neutralizing factor (HuSAP) in their blood. This phenomenon can be explained by hypothesizing that HuSAP acts as a carrier protein of Stx2 as well as a Stx2-neutralizing factor in humans as described below.

Caprioli et al. (2) have reported that the Stx2-neutralizing activity in serum is unique to humans. We tested mouse, rat, hamster, guinea pig, rabbit, chicken, dog, pig, sheep, deer, goat, horse, bovine, and monkey sera and confirmed that they were negative for in vitro Stx2-neutralizing activity (data not shown). Mice challenged with STEC had a higher mortality rate (29, 30), whereas the rate of occurrence of HUS in STEC-infected patients is 8-14% (31, 32), and the mortality rate is much lower. HuSAP probably neutralizes Stx2 cytotoxicity in human blood, thus decreasing the frequency of progression to HUS.

As shown in Fig. 4, the in vitro Stx2-neutralizing activity of HuSAP was much lower when the incubation time of HuSAP and Stx2 with the target cells was changed to 4 days instead of 1 h, suggesting that the binding between HuSAP and Stx2 is relatively weak and that Stx2 was gradually released from HuSAP day after day and finally killed the target cells. HuSAP may act as a carrier protein of Stx2 to the target cells.

Another possibility is that HuSAP is a factor that reduces the immunogenicity of Stx2 by binding to the antigenic site of Stx2, for the following reasons. First, no Stx2-neutralizing antibodies were produced in humans infected with STEC (33). Second, in vitro neutralizing activity against Stx1, but not Stx2, has been detected in intravenous immunoglobulins (34, 35). Third, studies on serum amyloid P component knockout mice have suggested that serum amyloid P component has an important physiological role in inhibiting the formation of pathogenic autoantibodies against chromatin and DNA (36).

In summary, this study has confirmed that HuSAP is the factor in human plasma that binds to Stx2 and neutralizes it in vitro. HuSAP may function not only as a Stx2-neutralizing factor in STEC infections but as a carrier of Stx2 as well. However, further studies will be necessary to clarify more precisely the physiological role of HuSAP in STEC pathology.

	FOOTNOTES

^* This work was supported by the Program for Promotion of Fundamental Studies of the Organization for Pharmaceutical Safety and Research of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ To whom correspondence should be addressed. Tel.: +81-42-586-8137; Fax: +81-42-587-5515; E-mail: t.kimura@teijin.co.jp.

Published, JBC Papers in Press, August 31, 2001, DOI 10.1074/jbc.M107819200

	ABBREVIATIONS

The abbreviations used are: Stx, Shiga toxin; HPLC, high-performance liquid chromatography; HuSAP, human serum amyloid P component; STEC, Shiga toxin-producing Escherichia coli; HUS, hemolytic uremic syndrome; BSA, bovine serum albumin; PBS, phosphate-buffered saline.

	REFERENCES

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