Studies on Lectin Mediated Agglutination Reaction on Red Blood Cell Surface Antigens Using Hot and Cold Water Plant Extracts

Lectin has various physiological roles in cell agglutination, based on their carbohydrate-binding properties, plant lectins are widely used for the detection, segregation, and characterization of glycoconjugates. Rhesus (Rh) factor is a protein that is inherited and found on the surface of red blood cells. If the surface protein is present, the RBC is Rh positive; otherwise, it is Rh-negative in nature. In this paper, we use agglutination reactions to investigate the effect of different cold and hot water extracted plants on RBC antigens as an alternative to commercial monoclonal antibodies. Extensive research on the sequence homology and 3-D structure of various plant lectins suggests that they have been conserved throughout evolution and may play important physiological roles that are still unknown.

A Neoglycoprotein-Immobilized Fluorescent Magnetic Bead Suspension Multiplex Array for Galectin-Binding Studies

Carbohydrate-protein conjugates have diverse applications. They have been used clinically as vaccines against bacterial infection and have been developed for high-throughput assays to elucidate the ligand specificities of glycan-binding proteins (GBPs) and antibodies. Here, we report an effective process that combines highly efficient chemoenzymatic synthesis of carbohydrates, production of carbohydrate-bovine serum albumin (glycan-BSA) conjugates using a squarate linker, and convenient immobilization of the resulting neoglycoproteins on carboxylate-coated fluorescent magnetic beads for the development of a suspension multiplex array platform. A glycan-BSA-bead array containing BSA and 50 glycan-BSA conjugates with tuned glycan valency was generated. The binding profiles of six plant lectins with binding preference towards Gal and/or GalNAc, as well as human galectin-3 and galectin-8, were readily obtained. Our results provide useful information to understand the multivalent glycan-binding properties of human galectins. The neoglycoprotein-immobilized fluorescent magnetic bead suspension multiplex array is a robust and flexible platform for rapid analysis of glycan and GBP interactions and will find broad applications.

Glycoproteins Presenting Galactose and N-Acetylgalactosamine in Human Seminal Plasma as Potential Players Involved in Immune Modulation in the Fertilization Process

In light of recent research, there is increasing evidence showing that extracellular semen components have a significant impact on the immune reaction of the female partner, leading to the tolerogenic response enabling the embryo development and implantation as well as further progress of healthy pregnancy. Seminal plasma glycoproteins are rich in the unique immunomodulatory glycoepitopes that may serve as ligands for endogenous lectins that decorate the surface of immune cells. Such interaction may be involved in modulation of the maternal immune response. Among immunomodulatory glycans, Lewis type antigens have been of interest for at least two decades, while the importance of T/Tn antigens and related structures is still far from understanding. In the current work, we applied two plant lectins capable of distinguishing glycoepitopes with terminal GalNAc and Gal to identify glycoproteins that are their efficient carriers. By means of lectin blotting and lectin affinity chromatography followed by LC-MS, we identified lactotransferrin, prolactin inducible protein as well as fibronectin and semenogelins 1 and 2 as lectin-reactive. Net-O-glycosylation analysis results indicated that the latter three may actually carry T and/or Tn antigens, while in the case of prolactin inducible protein and lactotransferrin LacdiNAc and lactosamine glycoepitopes were more probable. STRING bioinformatics analysis linked the identified glycoproteins in the close network, indicating their involvement in immune (partially innate) processes. Overall, our research revealed potential seminal plasma ligands for endogenous Gal/GalNAc specific lectins with a possible role in modulation of maternal immune response during fertilization.

Chemoenzymatic modular assembly of O-GalNAc glycans for functional glycomics

O-GalNAc glycans (or mucin O-glycans) play pivotal roles in diverse biological and pathological processes, including tumor growth and progression. Structurally defined O-GalNAc glycans are essential for functional studies but synthetic challenges and their inherent structural diversity and complexity have limited access to these compounds. Herein, we report an efficient and robust chemoenzymatic modular assembly (CEMA) strategy to construct structurally diverse O-GalNAc glycans. The key to this strategy is the convergent assembly of O-GalNAc cores 1–4 and 6 from three chemical building blocks, followed by enzymatic diversification of the cores by 13 well-tailored enzyme modules. A total of 83 O-GalNAc glycans presenting various natural glycan epitopes are obtained and used to generate a unique synthetic mucin O-glycan microarray. Binding specificities of glycan-binding proteins (GBPs) including plant lectins and selected anti-glycan antibodies towards these O-GalNAc glycans are revealed by this microarray, promoting their applicability in functional O-glycomics. Serum samples from colorectal cancer patients and healthy controls are assayed using the array reveal higher bindings towards less common cores 3, 4, and 6 than abundant cores 1 and 2, providing insights into O-GalNAc glycan structure-activity relationships.

Development and applications of sialoglycan-recognizing probes (SGRPs) with defined specificities: exploring the dynamic mammalian sialoglycome

Glycans that are abundantly displayed on vertebrate cell surface and secreted molecules are often capped with terminal sialic acids (Sias). These diverse 9-carbon-backbone monosaccharides are involved in numerous intrinsic biological processes. They also interact with commensals and pathogens, while undergoing dynamic changes in time and space, often influenced by environmental conditions. However, most of this sialoglycan complexity and variation remains poorly characterized by conventional techniques, which often tend to destroy or overlook crucial aspects of Sia diversity and/or fail to elucidate native structures in biological systems i.e., in the intact sialome. To date, in situ detection and analysis of sialoglycans has largely relied on the use of plant lectins, sialidases or antibodies, whose preferences (with certain exceptions) are limited and/or uncertain. We took advantage of naturally-evolved microbial molecules (bacterial adhesins, toxin subunits and viral hemagglutinin-esterases) that recognize sialoglycans with defined specificity to delineate 9 classes of Sialoglycan Recognizing Probes (SGRPs: SGRP1SGRP9) that can be used to explore mammalian sialome changes in a simple and systematic manner, using techniques common in most laboratories. SGRP candidates with specificity defined by sialoglycan microarray studies were engineered as tagged probes, each with a corresponding non-binding mutant probe as a simple and reliable negative control. The optimized panel of SGRPs can be used in methods commonly available in most bioscience labs, such as ELISA, Western Blot, flow cytometry and histochemistry. To demonstrate the utility of this approach, we provide examples of sialoglycome differences in tissues from C57BL/6 wild type mice and human-like Cmah-/- mice.

Can Plant Lectins Help to Elucidate Insect Lectin-Mediated Immune Response?

Lectins are carbohydrate-binding proteins that recognize and selectively bind to specific sugar structures. This group of proteins is widespread in plants, animals, and microorganisms, and exerts a broad range of functions. Many plant lectins were identified as exogenous stimuli of vertebrate immunity. Despite being the largest and most diverse taxon on earth, the study of lectins and their functions in insects is lagging behind. In insects, research on lectins and their biological importance has mainly focused on the C-type lectin (CTL) family, limiting our global understanding of the function of insect lectins and their role in insect immunity. In contrast, plant lectins have been well characterized and the immunomodulatory effects of several plant lectins have been documented extensively in vertebrates. This information could complement the missing knowledge on endogenous insect lectins and contribute to understanding of the processes and mechanisms by which lectins participate in insect immunity. This review summarizes existing studies of immune responses stimulated by endogenous or exogenous lectins. Understanding how lectins modulate insect immune responses can provide insight which, in turn, can help to elaborate novel ideas applicable for the protection of beneficial insects and the development of novel pest control strategies.