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ORIGINAL RESEARCH ARTICLE

Identification of Staphylococcal Enterotoxin B Domains Involved in Binding to Cultured Human Kidney Proximal Tubular Cells: Imparting Proliferation and Death

Subroto Chatterjee^*,1, Roger Neill, Jeffrey W. Shupp, Rasha Hammamieh, Boris Ionin and Marti Jett

^* Department of Pediatrics, Johns Hopkins University, Baltimore, Maryland 21205; and Division of Pathology, Department of Molecular Pathology, Walter Reed Army Institute of Research, Silver Spring, Maryland 20910

To whom requests for reprints should be addressed at ¹ Department of Pediatrics, Johns Hopkins University, 550 North Broadway, Suite 312, Baltimore, MD 21205. E-mail: schatte2{at}jhmi.edu

Studies suggest that staphylococcal enterotoxin B (SEB) is initially harbored in the kidney by binding to digalactosylceramide molecules in the proximal tubular cells. However, little is known in regard to the peptide motif within SEB that binds to these cells and imparts toxic effects. Herein, using human kidney proximal tubular cells (PTs) we have performed a systematic study on the binding of various peptides and peptide analogs of SEB and demonstrate a structure-functional relationship. Using [¹²⁵I]labeled SEB peptides, we show a high affinity and displaceable binding of SEB 191–220 to human PT cells. Binding was mitigated by the use of antibody against SEB, by digalactosylceramide (the putative receptor), and by the use of endoglycoceramidase, which selectively removes the oligosaccharide backbones from glycosphingolipids. Our structure/ functional studies revealed that peptide 130–160 induces a concentration-dependent increase in programmed cell death/ apoptosis in human proximal tubular cells. Mechanistic studies further suggest that SEB/SEB peptide (130–160) impart apoptosis via the activation of neutral sphingomyelinase, which hydrolizes sphingomyelin to ceramide and phosphocholine. SEB 130–160 mediated apoptosis was mitigated by preincubation of cells with antibody against SEB and an SEB 130–160 antibody.

Key Words: enterotoxin • SEB • renal proximal tubule cells • sphingolipids