Discoidin I from Dictyostelium discoideum and Interactions with Oligosaccharides: Specificity, Affinity, Crystal Structures, and Comparison with Discoidin II

Abstract

Discoidin I (DiscI) and discoidin II (DiscII) are N-acetylgalactosamine (GalNAc)-binding proteins from Dictyostelium discoideum. They consist of two domains: an N-terminal discoidin domain and a C-terminal H-type lectin domain. They were cloned and expressed in high yield in recombinant form in Escherichia coli. Although both lectins bind galactose (Gal) and GalNAc, glycan array experiments performed on the recombinant proteins displayed strong differences in their specificity for oligosaccharides. DiscI and DiscII bind preferentially to Gal/GalNAcβ1–3Gal/GalNAc-containing and Gal/GalNAcβ1–4GlcNAcβ1–6Gal/GalNAc-containing glycans, respectively. The affinity of the interaction of DiscI with monosaccharides and disaccharides was evaluated using isothermal titration calorimetry experiments. The three-dimensional structures of native DiscI and its complexes with GalNAc, GalNAcβ1–3Gal, and Galβ1–3GalNAc were solved by X-ray crystallography. DiscI forms trimers with involvement of calcium at the monomer interface. The N-terminal discoidin domain presents a structural similarity to F-type lectins such as the eel agglutinin, where an amphiphilic binding pocket suggests possible carbohydrate-binding activity. In the C-terminal H-type lectin domain, the GalNAc residue establishes specific hydrogen bonds that explain the observed affinity (K_d = 3 × 10^− 4 M). The different specificities of DiscI and DiscII for oligosaccharides were rationalized from the different structures obtained by either X-ray crystallography or molecular modeling.

Introduction

Lectins are ubiquitous proteins of nonimmune origin that interact reversibly and specifically with carbohydrates without modifying them. A large panel of lectins has been identified in invertebrates, and considerable attention is focused on their roles in biological recognition. Invertebrate lectins are often involved in specific binding to bacterial polysaccharides, playing a role in the establishment of symbiosis or in innate immunity.1, 2, 3 In other cases, they are involved in self-recognition and cell–cell aggregation, which are required for the building of multicellular organisms such as corals and sponges.⁴ They can be useful tools in cancer diagnosis/prognosis as histochemical markers or in cancer therapy, thanks to antitumoral activities.5, 6, 7

The slime mold Dictyostelium discoideum grows as free-living amoebae in the soil, feeding on bacteria. Upon starvation, it undergoes a complex developmental cycle in which about 10,000–50,000 individual amoebae aggregate to form a multicellular fruiting body that is able to produce spores. The aggregation of individual amoeba occurs by chemotaxis to periodic cAMP signals (reviewed by Kessin⁸). During differentiation, numerous new proteins are synthesized, and some of the most abundant of these new products are the two N-acetylgalactosamine (GalNAc)-binding proteins discoidin I (DiscI) and discoidin II (DiscII).9, 10 They are virtually undetectable in cells growing on bacteria, but constitute over 1% of cellular proteins in aggregated cells.⁹ They present overlapping but distinct sugar specificities.¹⁰ The production of DiscI is prominent in aggregating cells, accumulating intracellularly and upon externalization in multilamellar bodies, while DiscII is prominent during fruiting body formation and is localized in pre-spore vesicules.11, 12 The precise function of these lectins, as well as their biological ligands, remains unknown.

DiscI is polymorphic, with at least three isoforms per strain coded by genes, forming a small, coordinately regulated multigene family.13, 14 The three genes dscA, dscC, and dscD are found on chromosome 2 and are duplicated on the same chromosome in some laboratory strains (AX3 and AX4). They are often used as markers of early development in D. discoideum.15, 16 DiscI expression is developmentally regulated at the transcriptional level by several signal transduction pathways involving cAMP, prestarvation factor, and conditioned medium factor.17, 18, 19 Several mutants and antisense transformant analysis showed that DiscI was apparently not necessary for aggregation, since mutants plated at high density can aggregate and form fruiting bodies.¹⁷ The lack of gene product impairs the process of cell streaming in early development, where it seems important in the formation of head-to-tail streams by aggregating cells.²⁰ DiscI was also shown to be involved in cell substratum attachment and ordered cell migration during aggregation by a fibronectin-like mechanism.²¹ It contains the RGD motif, which is the cell attachment site found in a large collection of adhesive proteins.22, 23 The proposed receptor for this RGD sequence is a 67-kDa developmentally regulated cell surface glycoprotein.²⁴ DiscI also recognizes glycoconjugates that contain GalNAc in the slime coat around aggregates and in multilamellar bodies, since the interaction is completely blocked in the presence of GalNAc.25, 26, 27 The sugar binding site was shown to be different from the cell adhesion site and requires a divalent cation.24, 28

DiscI is a two-domain protein of 253 amino acids with a higher affinity for GalNAc than for galactose (Gal).¹⁰ It displays a 48% sequence identity with DiscII, whose trimeric structure has been recently solved in unliganded state and in complex with Gal and GalNAc.²⁹ The N-terminal domain, referred to as the discoidin domain (DS domain), is a structural and functional motif found in various proteins from both eukaryotic and prokaryotic origins. This domain shows considerable functional diversity via interactions with a wide range of molecules and is mainly involved in cell-surface-mediated regulatory events and glycoconjugates binding.30, 31, 32 The C-terminal domain belongs to the H-type lectin family recently identified in invertebrates such as snails and corals.³³

We present here the specificity and affinity of DiscI and DiscII for a variety of carbohydrate ligands, as well as the crystal structures of DiscI in unliganded form and in complex with GalNAc, GalNAcβ1–3Gal, and Galβ1–3GalNAc. The specificities, affinities, structures, and binding sites of both discoidins are compared and discussed. DiscI and DiscII present interesting differences in terms of oligosaccharide binding, which may be correlated to their differences in localization and their role in the slime mold.