Identification of glycosphingolipid receptors for pierisin-1, a guanine-specific ADP-ribosylating toxin from the cabbage butterfly

J Biol Chem. 2003 Mar 14;278(11):9972-8. doi: 10.1074/jbc.m212114200.

Abstract

Pierisin-1, a cytotoxic protein found naturally in the cabbage butterfly, induces apoptosis of mammalian cells. Our recent studies suggest that pierisin-1 consists of an N-terminal ADP-ribosyltransferase domain, and a C-terminal region that binds to receptors on the surfaces of target cells and incorporates the protein into cells. The present study was undertaken to identify receptors for pierisin-1. The cross-linking and cloning experiments suggested that the proteins on cell membrane had no binding ability to pierisin-1. Inhibitory assays of fractionated lipids from human cervical carcinoma HeLa cells, which are highly sensitive to pierisin-1, indicated neutral glycosphingolipids on the cell surface to show receptor activity. Inhibitory assays and TLC immunostaining using anti-pierisin-1 antibodies demonstrated two neutral glycosphingolipids as active components. Analysis of their structures with glycosphingolipid-specific antibodies and negative secondary ion mass spectrometry identified them as globotriaosylceramide (Gb3) and globotetraosylceramide (Gb4). The receptor activities of Gb3 and Gb4 for pierisin-1 were also confirmed with these authentic compounds. Pierisin-1-insensitive mouse melanoma MEB4 cells were found to lack pierisin-1 receptors, including Gb3 and Gb4, but pretreatment of the cells with glycosphingolipid Gb3 or Gb4 enhanced their sensitivity to pierisin-1. Thus, Gb3 and Gb4 were proven to serve as pierisin-1 receptors. The C-terminal region of pierisin-1 consists of possible lectin domains of a ricin B-chain, containing QXW sequences, which are essential for its structural organization. Alteration of QXW by site-directed mutagenesis caused marked reduction of pierisin-1 cytotoxicity. Thus, our results suggest that pierisin-1 binds to Gb3 and Gb4 receptors at the C-terminal region, in a manner similar to ricin, and then exhibits cytotoxicity after incorporation into the cell.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ADP Ribose Transferases
  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Butterflies
  • Chromatography, Thin Layer
  • Cross-Linking Reagents / pharmacology
  • Dose-Response Relationship, Drug
  • Glycosphingolipids / metabolism*
  • HeLa Cells
  • Humans
  • Inhibitory Concentration 50
  • Insect Proteins / chemistry*
  • Insect Proteins / metabolism*
  • Lectins
  • Mass Spectrometry
  • Mice
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Phospholipids / metabolism
  • Protein Binding
  • Protein Structure, Tertiary
  • Receptors, Cell Surface / metabolism*
  • Sequence Homology, Amino Acid

Substances

  • Cross-Linking Reagents
  • Glycosphingolipids
  • Insect Proteins
  • Lectins
  • Phospholipids
  • Receptors, Cell Surface
  • pierisin protein, insect
  • ADP Ribose Transferases