Background: Apoptotic cells release vesicles, which expose "eat-me" signals. Results: Vesicles originated from endoplasmic reticulum expose immature glycoepitopes and are preferentially phagocytosed by macrophages. Conclusion: Immature surface glycoepitopes serve as "eat-me" signals for the clearance of apoptotic vesicles originated from endoplasmic reticulum. Significance: Understanding the distinction by macrophages of apoptotic blebs may provide new insights into clearance-related diseases.
Recently, we found increased levels of alpha-d-mannose- and beta-d-galactose-containing glycoproteins in plasma membrane of the apoptotic murine leukemia L1210 cells (Bilyy & Stoika 2003). That indicator was suggested to be a novel marker of apoptosis in L1210 cells. The aim of our present work was to reveal if these changes in glycoprotein expression can be common for apoptotic cells of different origin and for various ways of apoptosis induction. It was demonstrated that an elevated expression of plasma membrane glycoproteins rich in alpha-d-mannose and beta-d-galactose did not depend on type of cell line and its tissue origin as well as on nature of apoptosis-inducing agent. We also found that an increase in membrane glycoprotein expression was dependent on concentration of apoptosis-inducing agent and was time-dependent. Changes in glycoproteins' expression were detected as early as 9-12 hours after apoptosis induction. Two hours pretreatment of cells with non-labeled lectin decreased plasma membrane staining with corresponding peroxidase-labeled lectin, probably because of lectin-induced internalization of specific membrane glycoproteins. PSL-lectin-affinity procedure was developed for isolation of apoptotic cells from their mixed population with normal cells. Lectin-dependent agglutination analysis showed that this process occurs at much lower lectin dilutions in the apoptotic cells than in the non-apoptotic cells. Thus, we found that alpha-d-mannose- and beta-d-galactose-containing glycoproteins can be used for lectinocytochemical detection, study and isolation of apoptotic cells.
A new highly cytotoxic protein, toxophallin, was recently isolated from the fruit body of the death cap Amanita phalloides mushroom [Stasyk et al. (2008) Studia Biologica 2, 21–32]. The physico‐chemical, chemical and biological characteristics of toxophallin differ distinctly from those of another death cap toxic protein, namely phallolysin. The interaction of toxophallin with target cells is not mediated by a specific cell surface receptor. It induces chromatin condensation, as well as DNA and nucleus fragmentation, which are typical for apoptosis. However, caspase III inhibitor [benzyloxycarbonyl‐Asp(OMe)‐fluoromethylketone] did not stop toxophallin‐induced DNA fragmentation. Thus, toxophallin uses a caspase‐independent pathway of apoptosis induction. In the present study, we applied a complementary approach based on a combination of proteomics and molecular biology tools for the protein identification of toxophallin. The primary structure of toxophallin was partially studied via direct sequencing of its tryptic peptides, followed by PCR‐based cloning of the corresponding cDNA. A subsequent bioinformatic search revealed a structural homology of toxophallin with the l‐amino acid oxidase of the Laccaria bicolor mushroom. This demonstrates the usefulness of our approach for the identification of proteins in organisms with unknown genomes. We also found a broad substrate specificity of toxophallin with respect to oxidizing selected amino acids. Ascorbic acid inhibited the cytotoxic effect of toxophallin, most likely as a result of scavenging hydrogen peroxide, which is the product of oxidase catalysis. Thus, in addition to highly toxic cyclopeptides and toxic lectin phallolysin, the death cap fruit body contains another cytotoxic protein in the form of an enzyme, namely l‐amino acid oxidase.
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