Toxoplasma gondii micronemal protein MIC1 is a lactose-binding lectin

Eimeria tenella, in common with other parasitic protozoa of the phylum Apicomplexa, invades host cells using an actinomyosin-powered "glideosome" complex and requires the secretion of adhesive proteins from the microneme organelles onto the parasite surface. Microneme proteins of E. tenella include EtMIC4, a transmembrane protein that has multiple thrombospondin type I domains and calcium-binding epidermal growth factor-like domains in its extracellular domain, and EtMIC5, a soluble protein composed of 11 tandemly repeated domains that belong to the plasminogen-apple-nematode superfamily. We show here that EtMIC4 and EtMIC5 interact to form an oligomeric, ultrahigh molecular mass protein complex. The complex was purified from lysed parasites by non-denaturing techniques, and the stoichiometry was shown to be [EtMIC4] 2 :[EtMIC5] 1 , with an octamer of EtMIC4 bound noncovalently to a tetramer of EtMIC5. The complex is formed within the parasite secretory pathway and is maintained after secretion onto the surface of the parasite. The purified complex binds to a number of epithelial cell lines in culture. Identification and characterization of this complex contributes to an overall understanding of the role of multimolecular protein complexes in specific interactions between pathogens and their hosts during infection.

Section: Discussionmentioning

confidence: 98%

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

Periz¹,

Gill

Hunt

et al. 2007

“…4E). In the light of literature reporting a lactose lectin activity of TgMIC1 (23) and the similarity of TgMIC1-CT with the galectin family, the lactose binding activity of TgMIC1-CT was monitored using NMR chemical shift mapping experiments. No detectable binding could be observed up to several hundred molar equivalents of lactose (100 mM), which substantiates the observation of an altered binding site.…”

Section: Discussionmentioning

confidence: 99%

A Novel Galectin-like Domain from Toxoplasma gondii Micronemal Protein 1 Assists the Folding, Assembly, and Transport of a Cell Adhesion Complex

Saouros

Edwards-Jones

Reiss

et al. 2005

Immediately prior to invasion Toxoplasma gondii tachyzoites release a large number of micronemal proteins (TgMICs) that participate in host cell attachment and penetration. The TgMIC4-MIC1-MIC6 complex was the first to be identified in T. gondii and has been recently shown to be critical in invasion. This study establishes that the N-terminal throm-bospondin type I repeat-like domains (TSR1-like) from TgMIC1 function as an independent adhesin as well as promoting association with TgMIC4. Using the newly solved three-dimensional structure of the C-terminal domain of TgMIC1 we have identified a novel Galectin-like fold that does not possess carbohydrate binding properties and redefines the architecture of TgMIC1. Instead, the TgMIC1 Galectin-like domain interacts and stabilizes TgMIC6, which provides the basis for a highly specific quality control mechanism for successful exit from the early secretory compartments and for subsequent trafficking of the complex to the micronemes.Toxoplasma gondii is a protozoan parasite of the phylum Apicomplexa, which infects virtually all warm-blooded animals and invades almost any cell type. Host cell invasion by this obligate intracellular parasite is an active process initiated by the formation of a tight association/junction with the host cell plasma membrane and leading to the creation of a parasitophorous vacuole. Contact with the host cell results in an increase in parasite intracellular calcium ions, which trigger apical organelles called micronemes to discharge their contents (1). Several micronemal proteins act as ligands for host cell receptors (2), while TgMIC2 and other transmembrane proteins establish a connection with the parasite actinomyosin system via their cytoplasmic tail (3), thus providing the motive force for penetration (4). It is becoming increasingly apparent that many microneme proteins are found in stable adhesive complexes, which are formed in the endoplasmic reticulum, and normally comprise an escorter protein, which is responsible for correct micronemal targeting, and one or more soluble effector proteins. The first such complex to be discovered in T. gondii was TgMIC4-MIC1-MIC6, in which TgMIC6 fulfils the role of the escorter protein, whereas TgMIC1 and TgMIC4 function as adhesins (5). Although TgMIC4-MIC1-MIC6 and the recently identified micronemal complex, TgMIC3-MIC8 (5, 6), are individually dispensable, the generation of double knock-outs for TgMIC1 and TgMIC3 renders the parasites avirulent in vivo, demonstrating functional synergy between these complexes (7). Deletion of the mic1 gene in T. gondii also confirmed the specific and critical role played by TgMIC1 in host cell attachment and invasion in vitro.Micronemal proteins have a modular structure with common themes in domain organization, for example many possess thrombospondin type-1 repeat domains (TSR1), 4 apple (or PAN) domains, and epidermal growth factor-like (EGF) domains (8). A schematic representation of the organization within the TgMIC4-MIC1-MIC6 complex is depicted in Fig. 1. Tg...

“…Blue Native PAGE and SDS-PAGE of Native TgMIC1-4 Complex-Native TgMIC1-4 complex from tachyzoites of the virulent RH strain of T. gondii was purified as described previously (18). BN-PAGE experiments were performed with the Native PAGE Novex BisTris Gel System (Invitrogen) according to the instructions, using a 4 -16% gradient gel.…”

Section: Cloning Expression and Purification From Escherichia Coli-mentioning

confidence: 99%

“…S1) (16). Although lectin activity has not yet been reported for TgMIC4, speculation has recently arisen due to an earlier report showing that a TgMIC1-4 subcomplex could be recovered from a lactose-affinity column (18) and our previous studies revealing TgMIC1 specificity for sialylated oligosaccharides only (13). Here, we combine atomic resolution studies with data from carbohydrate microarrays to reveal the basis of the interaction between TgMIC4 and a variety of galactose (Gal)-terminating oligosaccharides, and further define the interaction between TgMIC4 and TgMIC1.…”

mentioning

confidence: 99%

Galactose Recognition by the Apicomplexan Parasite Toxoplasma gondii

Marchant

Cowper

Liu

et al. 2012

Background: TgMIC4 is an important microneme effector protein from Toxoplasma gondii. Results: The structure of TgMIC4 together with carbohydrate microarray analyses reveal a broad specificity for galactoseterminating sequences. Conclusion: Lectin activity within the fifth apple domain of TgMIC4 is reminiscent of the mammalian galectin family. Significance: TgMIC4 may contribute to parasite dissemination within the host or down-regulation of the immune response.