Gut commensal bacteria are known to have a significant role in regulating the innate and adaptive immune homeostasis. Alterations in the intestinal microbial composition have been associated with several disease states, including autoimmune and inflammatory conditions. However, it is not entirely clear how commensal gut microbiota modulate and contribute to the systemic immunity, and whether circulating elements of the host immune system could regulate the microbiome. Thus, we have studied the diversity and abundance of specific taxons in the gut microbiota of inbred GalT-KO mice during 7 months of animal life by metagenetic high-throughput sequencing (16S rRNA gene, variable regions V3–V5). The repertoire of glycan-specific natural antibodies, obtained by printed glycan array technology, was then associated with the microbial diversity for each animal by metagenome-wide association studies (MWAS). Our data show that the orders clostridiales (most abundant), bacteriodales, lactobacillales, and deferribacterales may be associated with the development of the final repertoire of natural anti-glycan antibodies in GalT-KO mice. The main changes in microbiota diversity (month-2 and month-3) were related to important changes in levels and repertoire of natural anti-glycan antibodies in these mice. Additionally, significant positive and negative associations were found between the gut microbiota and the pattern of specific anti-glycan antibodies. Regarding individual features, the gut microbiota and the corresponding repertoire of natural anti-glycan antibodies showed differences among the examined animals. We also found redundancy in different taxa associated with the development of specific anti-glycan antibodies. Differences in microbial diversity did not, therefore, necessarily influence the overall functional output of the gut microbiome of GalT-KO mice. In summary, the repertoire of natural anti-carbohydrate antibodies may be partially determined by the continuous antigenic stimulation produced by the gut bacterial population of each GalT-KO mouse. Small differences in gut microbiota diversity could determine different repertoire and levels of natural anti-glycan antibodies and consequently might induce different immune responses to pathogens or other potential threats.

Background: Endogenous lectins are multifunctional effectors in cell physiology. Adding the sixth member of the galectin family in chicken, a model organism for systematic profiling of these adhesion/growth-regulatory proteins, is a step toward comprehensive network monitoring. Methods: Database mining and computational data processing are applied for gene detection, chromosomal location and sequence alignments. Cloning, recombinant production and fusion-protein technology gain access to the protein, mass spectrometry and gel electrophoresis/filtration provide analytical data. Haemagglutination, glycan microarray and cell assays assess binding capacity, and crystallography of a shortened variant (also analyzed by ultracentrifugation and small angle X-ray scattering) determines its structure. Results: The gene for the galectin-related protein (GRP) is present exclusively in vertebrates with high-level sequence conservation and similar chromosomal positioning. The chicken protein is monomeric and has lost the canonical galectin property of binding lactose. The crystal structure of the variant without the 36-amino-acid extension at the start provides explanations for this lack of binding. Conclusions: Chicken GRP is special within this family of six proteins by being unable to bind lactose. The documented high degree of sequence conservation among vertebrate orthologues confers the status of a model for delineating an assumedly shared functionality to this GRP. General significance: Biochemical characterization of a product of a gene under strong positive selection is a prerequisite for functional characterization. It is also essential for network monitoring by adding a new member to this lectin family.

Galectins are multifunctional effectors, for example acting as regulators of cell growth via protein-glycan interactions. The observation of capacity to kill bacteria for two tandem-repeat-type galectins, which target histo-blood epitopes toward this end (Stowell et al. Nat. Med. 16:295-301, 2010), prompted us to establish an array with bacterial polysaccharides. We addressed the question whether sugar determinants other than β-galactosides may be docking sites, using human galectins-4, -8, and -9. Positive controls with histo-blood group ABH-epitopes and the E. coli 086 polysaccharide ascertained the suitability of the set-up. Significant signal generation, depending on type of galectin and polysacchride, was obtained. Presence of cognate β-galactoside-related epitopes within a polysaccharide chain or its branch will not automatically establish binding properties, and structural constellations lacking galactosides, like rhamnan, were found to be active. These data establish the array as valuable screening tool, giving direction to further functional and structural studies.

It is generally accepted that the generation of antibodies proceeds due to immunization of an organism by alien antigens, and the level and affinity of antibodies are directly correlated to the presence of immunogen. At the same time, vast experimental material has been obtained providing evidence of antibodies whose level remains unchanged and affinity is constant during a lifetime. In contrast to the first, adaptive immunoglobulins, the latter are named natural antibodies (nAbs). The nAbs are produced by B1 cells, whereas adaptive Abs are produced by B2. This review summarizes general data on nAbs and presents in more detail data on antigens of carbohydrate origin. Hypotheses on the origin of nAbs and their activation mechanisms are discussed. We present our thoughts on this matter supported by our experimental data on nAbs to glycans.

Natural antibodies, part of the innate immunity system, are produced at strictly regulated levels in normal sera without immunization and thus are part of the innate immune system. The best studied natural antibodies are those directed against blood group antigens A and B and xeno-antigens including glycolylneuraminic acid containing Hanganutziu-Deicher (HD) glycolipid. Abnormal levels of anti-glycan antibodies were found in a number of pathologies. In many cases pathological antibodies are known to bind gangliosides. The genesis of anti-glycan antibodies in healthy humans and the reasons for their changes in pathologies are poorly understood. With a growing interest in their diagnostic applications, it is important to determine the carbohydrate structures that are recognized by antibodies present in the circulation of healthy individuals. We tested a large number of healthy donors using a printed glycan array (PGA) in a microchip format. The PGA contained ~300 glycans, representing mostly normal mammalian structures of glycoproteins and glycolipids, and many of the structures presented are biologically relevant sialylated motifs. As revealed by PGA, the sera interacted with at least 70 normal human glycans. With only few exceptions, antibodies recognizing sialosides have not been identified. Moderate levels of antibodies and moderate variability were observed in the case of SiaT n and its glycolyl variant. Unexpectedly, we found minimal antibody titer directed against Neu5Gcα and the trisaccharide Neu5Gcα2-6Galβ1-4GlcNAc, although this form of neuraminic acid does not occur naturally in humans. Antibodies recognizing sialosides in unnatural β-configuration have been detected and confirmed Springer's paradigm that circulating antibodies represent a reaction against bacteria. Gram-negative bacteria contain LPS with βKDN and/or βKDO which are very close analogs of Neu5Ac that are found in β-connected form. Antibodies against the biantennary N-glycan chain, (Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα)2-3,6-Manβ1-4GlcNAcβ1-4GlcNAc were never observed and similarly we never saw antibodies directed against the SiaLe(a)/SiaLe (x) motifs. Anti-sialoglycan antibodies can be masked with gangliosides: for example, we observe about a five times higher level of anti-GD3 in purified total IgG compared to the same concentration of total Ig in the composition of native serum. For several antibodies we observed anomalous binding in diluted sera, namely, the signals towards sialylated glycans were increased in the PGA if diluted sera were used.