Calcium binding activity of the epidermal growth factor-like domains of the apicomplexan microneme protein EtMIC4

Abstract

Microneme proteins are secreted from apicomplexan parasites during invasion of host cells and they play crucial roles in parasite–host cell adhesion. EtMIC4 is a 240 kDa transmembrane protein from Eimeria tenella that contains 31 tandemly arranged epidermal growth factor (EGF), like repeats within its extracellular domain. The majority of these repeats have calcium binding (cb) consensus sequences. Little is known about cbEGFs in apicomplexan parasites but their presence in microneme proteins suggests that they may contribute to parasite–host interactions. To investigate the potential role of cbEGFs we have expressed and correctly refolded a cbEGF triplet from EtMIC4 (cbEGF7–9) and demonstrated that this triplet binds calcium. Circular dichroism spectroscopic analysis of cbEGF7–9 demonstrates that the molecule undergoes a gradual change in conformation with increasing levels of calcium. In the presence of calcium, the triplet becomes resistant to proteolytic degradation by a variety of proteases, a characteristic feature of cbEGF repeats from higher eukaryotic proteins, such as fibrillin, suggesting that calcium binding induces the formation of a rigid conformation. Moreover, mass spectrometric mapping of the cleavage sites that are protected by calcium shows that these sites are located both close to and distant from the calcium binding sites, indicating that protection is not due to steric hindrance by calcium ions, but rather due to the overall conformation adopted by the triplet in the presence of calcium. Thus, the tandemly-arranged cbEGF repeats within EtMIC4 provide a mechanism whereby, in the calcium-rich extracellular environment, the molecule could adopt a protease-resistant, rigid structure that could favour its interaction with host cell ligands

The Microneme Proteins EtMIC4 and EtMIC5 of Eimeria tenella Form a Novel, Ultra-high Molecular Mass Protein Complex That Binds Target Host Cells

“…This form of selective sorting, mediated by aggregation, occurs upon exposure to the mildly acidic and high calcium environments encountered in the trans-Golgi network (41) and within immature secretory granules (42). EtMIC4 is an acidic protein (pI 3.9) that has 31 EGF-like domains, most of which can bind calcium (26). Oligomerization and complex formation of EtMIC4 with EtMIC5 (pI 6.3) in the parasite late secretory pathway may modify its aggregation/ multimerization/folding state.…”

“…In all experiments, whether using lysates of sporozoites alone or lysates of sporozoiteinfected MDBK epithelial cells, two independent polypeptide species were precipitated. One, which migrated under reducing SDS-PAGE conditions as a single ϳ100-kDa band, was identified by Western blotting as EtMIC5, and the other, a single ϳ240-kDa band, was identified as EtMIC4, a multimodular, transmembrane MIC protein also under study in our laboratory (25,26). Silver staining of the precipitated material did not identify any additional proteins pulled down specifically by the antibodies to EtMIC5, indicating that EtMIC4 and EtMIC5 most likely form a two-partner protein complex.…”

“…Moreover, complementation studies in T. gondii show that expression of EtMIC1-2 permits the knock-out of the essential endogenous gene, TgMIC2 (24). Studies to characterize additional apicomplexan MIC complexes await identification of novel MICs, and among those already described in E. tenella are EtMIC4, a 240-kDa TM MIC with 16 thrombospondin type I (TSR) domains and 31 epidermal growth factor-like (EGF) domains (25,26), and EtMIC5, a 100-kDa soluble MIC with 11 domains that belong to the plasminogen-apple-nematode (PAN) superfamily (27,28). We show now that these MICs associate to form an ultrahigh molecular mass oligomeric complex.…”

Molecular characterization, tissue-specific expression and RNAi knockdown of the first vitellogenin receptor from a tick

“…As in other VgRs and LDLRs, the EGF-like domain (approximately 50 amino acids with three internal disulfide bridges) serves to bind calcium with high affinity to specific cell-surface receptors (Selander-Sunnerhagen et al, 1992;Rao et al, 1995). Calcium binding is believed to induce a rigid conformation of the EGF-like domain that makes it highly resistant to proteolytic degradation by various proteases, a characteristic feature of EGF repeats from higher eukaryotic proteins, such as fibrillin (Periz et al, 2005). The O-linked sugar domain has been proposed as a region important for maintaining the stable expression of the receptor on cell surfaces; its absence has been found to result in release of the receptor from the cell into the extracellular space (Magrane et al, 1999).…”

Aplysia temptin − the ‘glue’ in the water‐borne attractin pheromone complex

“…Alternatively, complexation with temptin may be necessary for attractin to bind to the host cell. For example, the parasitic protozoan, Eimeria tenella , synthesizes a microneme protein (EtMIC4) containing multiple extracellular Ca 2+ ‐binding EGF‐like repeats [29] that forms a high molecular mass ([EtMIC4] 8 [EtMIC5] 4 ; > 2 MDa) complex with a soluble protein EtMIC5. This complex, secreted to the protozoan surface, mediates the binding of the parasite to mammalian cells [30].…”