Influence of protein (human galectin-3) design on aspects of lectin activity

The concept of biomedical significance of the functional pairing between tissue lectins and their glycoconjugate counterreceptors has reached the mainstream of research on the flow of biological information. A major challenge now is to identify the principles of structure–activity relationships that underlie specificity of recognition and the ensuing post-binding processes. Toward this end, we focus on a distinct feature on the side of the lectin, i.e. its architecture to present the carbohydrate recognition domain (CRD). Working with a multifunctional human lectin, i.e. galectin-3, as model, its CRD is used in protein engineering to build variants with different modular assembly. Hereby, it becomes possible to compare activity features of the natural design, i.e. CRD attached to an N-terminal tail, with those of homo- and heterodimers and the tail-free protein. Thermodynamics of binding disaccharides proved full activity of all proteins at very similar affinity. The following glycan array testing revealed maintained preferential contact formation with N-acetyllactosamine oligomers and histo-blood group ABH epitopes irrespective of variant design. The study of carbohydrate-inhibitable binding of the test panel disclosed up to qualitative cell-type-dependent differences in sections of fixed murine epididymis and especially jejunum. By probing topological aspects of binding, the susceptibility to inhibition by a tetravalent glycocluster was markedly different for the wild-type vs the homodimeric variant proteins. The results teach the salient lesson that protein design matters: the type of CRD presentation can have a profound bearing on whether basically suited oligosaccharides, which for example tested positively in an array, will become binding partners in situ. When lectin-glycoconjugate aggregates (lattices) are formed, their structural organization will depend on this parameter. Further testing (ga)lectin variants will thus be instrumental (i) to define the full range of impact of altering protein assembly and (ii) to explain why certain types of design have been favored during the course of evolution, besides opening biomedical perspectives for potential applications of the novel galectin forms

Removal of natural anti-αGal antibodies elicits protective immunity against Gram-negative bacterial infections

Antibody-dependent enhancement (ADE) of bacterial infections occurs when blocking or inhibitory antibodies facilitate the infectivity of pathogens. In humans, antibodies involved in ADE of bacterial infections may include those naturally produced against Galα1-3Galβ1-4GlcNAcβ (αGal). Here, we investigate whether eliminating circulating anti-αGal antibodies using a soluble αGal glycopolymer confers protection against Gram-negative bacterial infections. We demonstrated that the in vivo intra-corporeal removal of anti-αGal antibodies in α1,3-galactosyltransferase knockout (GalT-KO) mice was associated with protection against mortality from Gram-negative sepsis after cecal ligation and puncture (CLP). The improved survival of GalT-KO mice was associated with an increased killing capacity of serum against Escherichia coli isolated after CLP and reduced binding of IgG1 and IgG3 to the bacteria. Additionally, inhibition of anti-αGal antibodies from human serum in vitro increases the bactericidal killing of E. coli O86:B7 and multidrug-resistant Klebsiella pneumoniae and Pseudomonas aeruginosa. In the case of E. coli O86:B7, there was also an improvement in bacteria opsonophagocytosis by macrophages. Both lytic mechanisms were related to a decreased binding of IgG2 to the bacteria. Our results show that protective immunity against Gram-negative bacterial pathogens can be elicited, and infectious diseases caused by these bacteria can be prevented by removing natural anti-αGal antibodies

Allergic reactions to tick saliva components in zebrafish model

[Background]: Alpha-Gal syndrome (AGS) is a tick-borne food allergy caused by IgE antibodies against the glycan galactose-alpha-1,3-galactose (α-Gal) present in glycoproteins and glycolipids from mammalian meat. To advance in the diagnosis and treatment of AGS, further research is needed to unravel the molecular and immune mechanisms underlying this syndrome. The objective of this study is the characterization of tick salivary components and proteins with and without α-Gal modifications involved in modulating human immune response against this carbohydrate.[Methods]: Protein and α-Gal content were determined in tick saliva components, and proteins were identified by proteomics analysis of tick saliva fractions. Pathophysiological changes were recorded in the zebrafish (Danio rerio) model after exposure to distinct Ixodes ricinus tick salivary components. Serum samples were collected from zebrafish at day 8 of exposure to determine anti-α-Gal, anti-glycan, and anti-tick saliva protein IgM antibody titers by enzyme-linked immunosorbent assay (ELISA).[Results]: Zebrafish treated with tick saliva and saliva protein fractions combined with non-protein fractions demonstrated significantly higher incidence of hemorrhagic type allergic reactions, abnormal behavioral patterns, or mortality when compared to the phosphate-buffered saline (PBS)-treated control group. The main tick salivary proteins identified in these fractions with possible functional implication in AGS were the secreted protein B7P208-salivary antigen p23 and metalloproteases. Anti-α-Gal and anti-tick salivary gland IgM antibody titers were significantly higher in distinct saliva protein fractions and deglycosylated saliva group when compared with PBS-treated controls. Anti-glycan antibodies showed group-related profiles.[Conclusions]: Results support the hypothesis that tick salivary biomolecules with and without α-Gal modifications are involved in modulating immune response against this carbohydrate.This study was supported by Ministerio de Ciencia e Innovación/Agencia Estatal de Investigación MCIN/AEI/10.13039/501100011033, Spain and EU-FEDER (Grant BIOGAL PID2020-116761GB-I00). M. Contreras was funded by the Ministerio de Ciencia, Innovación y Universidades, Spain, grant IJC2020-042710-I. R. Vaz-Rodrigues was supported by a doctoral contract (2022/20675), from Universidad de Castilla-La Mancha (UCLM), Spain, co-financed by the European Social Fund (ESF). L. Mazuecos was supported by UCLM co-financed by ESF, post-doctoral grant 2021/32002). E. Ferreras-Colino was funded by UCLM co-financed by ESF, doctoral grant 2020/3836.Peer reviewe

Structural Characterization of Rat Galectin-5, an N-Tailed Monomeric Proto-Type-like Galectin

Galectins are multi-purpose effectors acting via interactions with distinct counterreceptors based on protein-glycan/protein recognition. These processes are emerging to involve several regions on the protein so that the availability of a detailed structural characterization of a full-length galectin is essential. We report here the first crystallographic information on the N-terminal extension of the carbohydrate recognition domain of rat galectin-5, which is precisely described as an N-tailed proto-type-like galectin. In the ligand-free protein, the three amino-acid stretch from Ser2 to Ser5 is revealed to form an extra β-strand (F0), and the residues from Thr6 to Asn12 are part of a loop protruding from strands S1 and F0. In the ligand-bound structure, amino acids Ser2–Tyr10 switch position and are aligned to the edge of the β-sandwich. Interestingly, the signal profile in our glycan array screening shows the sugar-binding site to preferentially accommodate the histo-blood-group B (type 2) tetrasaccharide and N-acetyllactosamine-based di- and oligomers. The crystal structures revealed the characteristically preformed structural organization around the central Trp77 of the CRD with involvement of the sequence signature’s amino acids in binding. Ligand binding was also characterized calorimetrically. The presented data shows that the N-terminal extension can adopt an ordered structure and shapes the hypothesis that a ligand-induced shift in the equilibrium between flexible and ordered conformers potentially acts as a molecular switch, enabling new contacts in this region

Supplementary table3 -Supplemental material for Repertoire of Abs primed by bacteria in gnotobiotic mice

<p>Supplementary tables</p

Supplementary table4 -Supplemental material for Repertoire of Abs primed by bacteria in gnotobiotic mice

<p>Supplementary tables</p