Detailed insights from microarray and crystallographic studies into carbohydrate recognition by microneme protein 1 (MIC1) of Toxoplasma gondii

Abstract

The intracellular protozoan Toxoplasma gondii is among the most widespread parasites. The broad host cell range of the parasite can be explained by carbohydrate microarray screening analyses that have demonstrated the ability of the T. gondii adhesive protein, TgMIC1, to bind to a wide spectrum of sialyl oligosaccharide ligands. Here, we investigate by further microarray analyses in a dose-response format the differential binding of TgMIC1 to 2-3-and 2-6-linked sialyl carbohydrates. Interestingly, two novel synthetic fluorinated analogs of 3 0 SiaLacNAc 1-4 and 3 0 SiaLacNAc 1-3 were identified as highly potent ligands. To understand the structural basis of the carbohydrate binding specificity of TgMIC1, we have determined the crystal structures of TgMIC1 micronemal adhesive repeat (MAR)-region (TgMIC1-MARR) in complex with five sialyl-Nacetyllactosamine analogs. These crystal structures have revealed a specific, water-mediated hydrogen bond network that accounts for the preferential binding of TgMIC1-MARR to arrayed 2-3-linked sialyl oligosaccharides and the high potency of the fluorinated analogs. Furthermore, we provide strong evidence for the first observation of a CAFÁÁÁHAO hydrogen bond within a lectin-carbohydrate complex. Finally, detailed comparison with other oligosaccharide-protein complexes in the Protein Data Bank (PDB) reveals a new family of sialic-acid binding sites from lectins in parasites, bacteria, and viruses.

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

“…Binding was predominantly observed to ␣2-3-linked sialyl probes with little or no binding to the ␣2-6-linked sialyl probes. This is in agreement with a recent study revealing the importance of a glutamic acid residue (Glu-222) in TgMIC1 for this preference (29), which is conserved in TgMIC13 and NcMIC1 (supplemental Fig. S2).…”

“…Analyses-To better understand the basis of the binding preference revealed by the microarray studies, we compared the crystal structure of TgMIC1-MARR in complex with 3ЈSiaLacNAc 1-3 (Protein Data Bank code 3F5A) (29) to the TgMIC13 MAR domains. Although the first three MAR domains of TgMIC13 possess the Sia-binding signature, we have assumed that the type II domains (MAR2) make the dominant contribution to Sia recognition as observed for TgMIC1-MARR (1); therefore, MAR2 of TgMIC1 and TgMIC13, in particular, was considered.…”

Sialic acids: Key determinants for invasion by the Apicomplexa

“…This increased affinity is due to additional contacts to the galactose moiety observed in the crystal structure of TgMIC1 in complex with 3 0 SiaLacNAc (Garnett et al, 2009), and is likely involved in defining the preference for a2-3 linked sialylated glycoconjugates.…”

“…More recently, the atomic structure of TgMIC1 uncovered a novel cell-binding motif called microneme adhesive repeat (MAR), which provides a highly specialised structure for glycan discrimination (Blumenschein et al, 2007). Using carbohydrate microarrays, this adhesin was shown to interact selectively with sialylated oligosaccharides and further structural studies of various complexes between TgMIC1 and sialylated oligosaccharides provided high-resolution insights into the mechanism of recognition (Garnett et al, 2009).…”

Galactose Recognition by the Apicomplexan Parasite Toxoplasma gondii

“…Some of the microneme protein complexes contribute to host cell attachment and provide a link between host cell receptors and the parasite actomyosin motor and hence provides the motive force necessary for host cell invasion (9,10) Among the microneme protein complexes operating in invasion, the TgMIC1-4-6 complex is important for the efficiency of this process and has been shown to contribute to the virulence of the parasite in mice (11,12). The structure of the N-terminal region of TgMIC1 revealed a pair of novel domains termed the microneme adhesive repeat region (MARR) 4 (13), in complex with a wide range of sialylated glycans (14). TgMIC1 not only interacts with a range of sialylated glycans on host cell receptors, but also recruits TgMIC4, which is anticipated to exert adhesive function during invasion (15).…”