Journal of Molecular Biology
Discoidin I from Dictyostelium discoideum and Interactions with Oligosaccharides: Specificity, Affinity, Crystal Structures, and Comparison with Discoidin II
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.
Section snippets
Specificity of D. discoideum lectins with glycan microarray resource
Recombinant DiscI was obtained with a yield of 20 mg l− 1, and its molecular weight, checked by mass spectrometry, corresponded to the expected value. Recombinant DiscII was produced as previously described.29 Both lectins were labeled with Alexa 488 and assayed on Glycan Microarray available at the Consortium for Functional Glycomics, using concentrations varying from 0.01 to 200 μg ml− 1. DiscI gave excellent signals at very low concentrations, and the analysis displayed in Fig. 1a has been
Discussion
DiscI and DiscII are two domain proteins assembled as trimers. We reported previously the trimeric structure of DiscII, while the present work focuses on isoform A of DiscI. The structures of isoforms C and D of DiscI should be almost identical with this one, since they only differ by 13 and 9 amino acids, respectively. These mutations are mainly located at the protein surface, and only one is not found in the DS domain. It is very likely that heterotrimers combining the different isoforms are
Construction of plasmid for the expression and purification of recombinant DiscI
The discI gene of isoform A (P02886) was cloned in-frame with an N-terminal hexahistidine tag (His-tag) and the tobacco etch virus protease cleavage site of the prokaryotic expression vector pProEXHTb (Invitrogen) using polymerase chain reaction and standard molecular biology techniques. The primers used are 5′-GTG CCA TGG CTA CCC AAG GTT TAG TTC-3′ and 5′-GAC TCG AGT TAT TCC AAA GCG GTA GC-3′. Sequence analysis was in accordance with the one deposited.
The protein was overexpressed in
Acknowledgements
The glycan microarray analysis was provided by the Consortium for Functional Glycomics funded by National Institute of General Medical Sciences grant GM62116. We acknowledge the European Synchrotron Radiation Facility for provision of synchrotron radiation facilities. We thank Michel Satre and Christopher West for helpful discussions on D. discoideum. This work was supported by Centre National de la Recherche Scientifique and the French Ministry of Research. K.S.A. was the recipient of a CNPq
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2020, International Journal of Biological MacromoleculesCitation Excerpt :The dissociation constant (Kd) for HPA-GalNAc complex is 0.13 mM [10], whereas the affinity of DiscII to GalNAc is almost ten times lower (Kd = 1.11 mM) [16] and the affinity does not change significantly for oligosaccharides containing galactose instead of GalNAc. Interestingly, DiscI has a relatively high affinity to GalNAc (Kd = 0.30 mM), only slightly lower in comparison to HPA, but DiscI affinity to galactose is significantly lower (Kd = 3.8 mM) [15]. In DiscI, N236 interacts with oxygen O7 of the N-acetyl group of GalNAc (Fig. 2F, H) and this gives specificity to GalNAc.
Discoidin domain receptors: Micro insights into macro assemblies
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2016, Journal of Molecular BiologyCitation Excerpt :Overall, trimerization was principally mediated by the extensive hydrophobic interactions among domains 2 and 3. Structural comparisons using the MATRAS server revealed marked structural and topological similarity of domain 3 to that of the C-terminal domain of discoidin I from Dictyostelium discoideum (PDB code 2WN3) with a Z-score of 23.28, despite a sequence identity of only 6.9% (Fig. 3c) [32,33]. This structure represents the immunoglobulin-like β-sandwich fold typical of H-type lectins and first observed in Helix pomatia agglutinin (HPA) [34].
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S.V.M. and K.S.A. contributed equally to this work.
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Present address: K. S. Aragão, Laboratorio de Moléculas Biologicamente Ativas, Universidade Federal do Ceará, Caixa Postal 6043, Campus do Pici, 60455-970 Fortaleza CE, Brazil.