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

Abstract

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...

Members of a Novel Protein Family Containing Microneme Adhesive Repeat Domains Act as Sialic Acid-binding Lectins during Host Cell Invasion by Apicomplexan Parasites

“…Correct trafficking of the complex to the micronemes depends not only on a sorting determinant in the C-terminal tail of TgMIC6 but also on the interaction between the galectin-like domain of TgMIC1 (TgMIC1-GLD) with the third membrane-proximal EGF-like domain of TgMIC6 (TgMIC6-EGF3). This interaction is crucial for transport of the entire complex through the early secretory pathway as it assists proper folding of TgMIC6-EGF3, providing a quality control checkpoint (12,15,17).…”

“…Each of the remaining two EGF-like domains is able to recruit one molecule of TgMIC1 via interaction with its C-terminal galectin-like domain (for a schematic see Fig. 1A) (12,15,16). Correct trafficking of the complex to the micronemes depends not only on a sorting determinant in the C-terminal tail of TgMIC6 but also on the interaction between the galectin-like domain of TgMIC1 (TgMIC1-GLD) with the third membrane-proximal EGF-like domain of TgMIC6 (TgMIC6-EGF3).…”

Cryptosporidium p30, a Galactose/N-Acetylgalactosamine-specific Lectin, Mediates Infection in Vitro

“…For example, the membrane-bound micronemal protein TgMIC 6 functions as an escorter for the soluble adhesive proteins TgMIC 1 and TgMIC 4 (47-50). TgMIC 1, which forms the core of the complex, simultaneously interacts with TgMIC 4 and TgMIC 6 as well as with host cells, thus forming a "bridge" between parasite and host cells (48,49). In addition to functioning as an escorter, TgMIC 6 is also thought to have adhesive functions (49).…”

“…RNA was extracted from uninfected and infected Caco-2A monolayers at different time points (24,48, and 72 h) using an RNeasy kit (Qiagen). Contaminating DNA was removed by RNase-free DNase treatment using a DNA-free TM kit (Ambion).…”

Sialic acids: Key determinants for invasion by the Apicomplexa

“…TgMIC1 interacts with an appledomain-containing protein, TgMIC4 (Reiss et al, 2001;Saouros et al, 2005). The apple domain is another potential carbohydrate-binding fold and several apple-domain-containing proteins are present in the T. gondii genome.…”