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Serum amyloid P component is the Shiga toxin 2-neutralizing factor in human blood
T Kimura1, S Tani, Y Matsumoto Yi
1Teijin Institute for Biomedical Research, Teijin Ltd., 4-3-2, Asahigaoka, Hino City, Tokyo 191-8512, Japan. t.kimura@teijin.co.jp
Abstract:
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.
Insights
A human plasma factor neutralizes Shiga toxin 2 (Stx2). Researchers purified and identified this factor as human serum amyloid P component (HuSAP), crucial for understanding Shiga toxin-producing E. coli infections.
Area of Science:
- Microbiology
- Immunology
- Biochemistry
Background:
- A factor in human plasma neutralizes Shiga toxin 2 (Stx2) in vitro.
- This Stx2-neutralizing factor is specific to humans and absent in other species.
- Understanding this factor is vital for studying Shiga toxin-producing Escherichia coli pathogenesis.
Purpose of the Study:
- To purify the Stx2-neutralizing factor from human plasma.
- To identify the molecular identity of this human-specific factor.
Main Methods:
- Multi-step purification of human plasma using Con A-Sepharose, DEAE-Sepharose, hydroxyapatite, and HPLC.
- Assay of Stx2-neutralizing activity to guide purification.
- SDS-polyacrylamide gel electrophoresis and N-terminal amino acid sequencing for identification.
Main Results:
- A single protein band was isolated via gel-filtration HPLC.
- N-terminal sequencing revealed the protein to be human serum amyloid P component (HuSAP).
- Commercially available HuSAP demonstrated Stx2 binding and neutralizing activity.
Conclusions:
- Human serum amyloid P component (HuSAP) is the factor in human plasma that neutralizes Shiga toxin 2 (Stx2).
- This identification provides a key insight into the host's defense against Stx2 and the pathology of Stx-producing E. coli infections.