Biochimica et Biophysica Acta (BBA) - General Subjects
Endocytosis of pulchellin and its recombinant B-chain into K-562 cells: Binding and uptake studies
Introduction
Pulchellin is a type 2 ribosome-inactivating protein (RIP) found in the seeds of Abrus pulchellus tenuiflorus. Members of this group of proteins play a remarkable role in the history of clinical medicine and biomedical research (reviewed in [1]) and include abrin, volkesin, viscumin, ebulin [2] and the most extensively studied among all RIPs, ricin. Type 2 RIPs are structurally heterodimeric proteins composed of an enzymatic A-chain covalently linked by a single disulfide bond to a B-chain. The first bears the toxic rRNA-specific N-glycosidase activity. The B-chain has lectin activity towards specific carbohydrate moieties present in mammal cell outer surface [3]. Ricin irreversibly removes an adenine residue from the conserved sarcin/ricin loop of the 28 S rRNA inhibiting the EF 1 and 2 binding to the ribosome and arresting protein synthesis ultimately resulting in cell death [4].
Ricin is synthesized as a single polypeptide precursor that must be cleaved yielding the independent chains of the mature protein. The uncleaved precursor is enzymatically inactive [5], which could be a means to prevent self-intoxication. The B-chain folds into two globular domains, each one with a carbohydrate-binding site, responsible for the agglutinating activity of the toxins. The B-chain has possibly evolved from a series of gene duplications as each domain seems to be derived from three ancestral galactose binding peptides named α, β and γ. Only two of these, 1α and 2γ, are able to bind galactose [6]. Cell-bound RIP is endocytosed by both clathrin-dependent and clathrin-independent processes operating in the same cell [7]. Additionally, an alternative mechanism of binding and uptake was described for ricin, which involves recognition of the native toxin glycosyl residues by the mannose receptors present in the macrophage and liver endothelial cells [8], [9], [10]. Regardless of their form of up take, RIPs were shown to be transported to endosomes. From there, a fraction is recycled back to the cell surface, another part degraded in the lysosomes and only a small fraction (only 5% for ricin) is translocated to the cytosol. This small amount seems to use a retrograde transport from endosomes to the Golgi apparatus, to the ER and finally to the cytosol, exploring the cellular sorting mechanism for misfolded proteins [11], [12].
Pulchellin B-chain exhibits affinity for β-d-galactose substrate. Recombinant pulchellin B-chain (rPBC) was produced as inclusion bodies in E. coli that were successfully refolded recovering biological activity [13], [14]. New approaches for using this kind of protein as a biotechnological tool demand a better understanding of cell targeting, binding, uptake, intracellular sorting and delivery [2], [12]. In this work, cell adhesion experiments were used to determine rPBC interaction with mammalian cells, fluorescence microscopy to verify its endocytosis and confocal laser-scanning microscopy to probe its intracellular localization. Native pulchellin subcellular sorting could also be determined. The results are consistent with the hypothesis that the endosomal internalization path and retrograde transport through Golgi apparatus might be used by both the native and the recombinant pulchellin B-chain.
Section snippets
Materials and methods
Mammalian cell lines were from the American Type Culture Collection (ATCC—www.atcc.org): TK cells, Mus musculus fibroblast (ATCC number CCL-1.3); HeLa, Homo sapiens cervix adenocarcinoma epithelial cells (ATCC number CCL-2); MDA-MB-231 cells, H. sapiens mammal gland breast cancer epithelial cells (ATCC number HTB-26); K-562 cells, human bone marrow leukemia lymphoblasts (ATCC number CCL-243). K-562α2β1, K-562 cells transfected with α2β1 integrin were a gift from Dr. M.E. Hemler (Dana Farber,
Results and discussion
RIPs were shown to trigger diverse cell responses, most of them related to the toxic A-chain catalytic activity on ribosomes, DNA or apoptotic response [15], [16], [17]. Promotion of cell adhesion is a useful way to probe for protein interaction with living cells, and the interacting cell line should be a good candidate to investigate the endocytosis pathway. Recombinant pulchellin B-chain supported adhesion of MDA-MB-231 and K-562 cancer cell lines in a protein concentration-dependent way but
Acknowledgments
This work was supported by grants from CAPES and FAPESP. The authors are thankful to Dr. Sara Teresinha Ollala Saad, who kindly gave support in confocal laser-scanning experiments.
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The role of the C-terminal region of pulchellin A-chain in the interaction with membrane model systems
2012, Biochimica et Biophysica Acta - BiomembranesCitation Excerpt :PAC is a highly specific RNA N-glycosylase that inhibits protein synthesis by irreversible depurination of 28S RNA of the 60S ribosome subunit (both in vivo and in vitro), while PBC is a galactose-specific lectin [2]. Analogously to ricin and abrin, pulchellin displays strong cytotoxic effects on mammalian cells [3,4] at low protein concentrations. Pulchellin enters cells through lectin–carbohydrate interactions at the cell surface followed by endocytic uptake and subsequent retrograde transport [3].
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