Carbohydrates are the most abundant organic compound on Earth. They are easily identified by the fact that they all have two H atoms for every O and C atom, giving them the general molecular formula of (CH2O)n.
Beyond nucleic acids and proteins, the family of complex carbohydrates (a.k.a. polysaccharides or glycans) constitutes an enormous biomass, cellulose and chitin being the two most profuse elements in the world (Sharon, 1975; Merzendorfer, 2009).
But besides the polysaccharides, conjugates of proteins (or glycoproteins) are present in organisms of all branches of the evolutionary tree including virus, fungi, Metazoa, Protozoa, and planta. In fact, more than half of all proteins are glycosylated and they form a major part of the human proteome (1, 2).
This broad occurrence of cellular glycoconjugates strongly argues against eukaryotic glycosylation being a “whim of nature”, but more a “multipurpose tool” (3).
Glycan, a fabulous multipurpose tool
In fact, glycans should be considered as Swiss army knife information-storage molecules, and are engaged in many processes including:
- maintenance of the conformational stability and protection from proteolysis (Huber et al., 1989; Rupley and Careri, 1991; Martins and Santos, 1995; Martins et al., 1997; Ramakrishnan et al., 1997),
- antigenic presentation
o triggering phagocytosis via C-type lectins on antigen presenting cells (4, 5),
o triggering CD1d-restricted and TCR-mediated activation of NKT cells (6)
- immunogenicity of glycosidic epitopes
o increasing the immune response to glycoproteins (7)
o generating xeno auto-antibodies (8)
o creating allergenic reactions to xylose and fucose residues (9)
- control of intracellular transport with the presence of glycan-recognizing proteins in the ER-Golgi pathway, involved in trafficking and chaperone functions (10)
- intracellular adhesion with the particular role of selectins in cancer progression (11)
- cell age encryption since glycans are a biomarker of chronological and biological ages (12).
But such a diversity of function at the cellular level should not be unrelated to how diseases spread in our bodies.
The special case of sialic acid
As early as 1979, the development of hybridoma technology made it possible to identify the first tumor antigens of which carcinoembryonic antigen (CEA). CEA - monomeric glycoprotein of 180 kDa - rapidly became a preferred marker of the diagnosis of colorectal cancer in blood test (13). Since then, new glycomarkers have been identified, including carbohydrate sialyl Lewis a CA19-9 (14), carbohydrate antigen 50 (CA50) (15) and especially sialic acid-containing carbohydrate antigen (CA242) for pancreatic cancer (16).
But it must be said that sialic acid (SA) is not only involved in cancer.
Sialic acids are a broad family of 2-keto-3-deoxy-nononic acids and the three major players are N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc), and 3-deoxy-D-glycero-D-galacto-2-nonulosonic acid (KDN).
They interact with surface proteins « Sialic acid-binding immunoglobulin-type lectins » (Siglecs), on macrophages, NK cells, eosinophils, B cells and dendritic cells, playing a major role in immunity.
The covalent addition of sialic acid to the terminal end of glycoproteins is named sialylation, and this process is regulated by the activity of sialyltransferases or sialidase.
As sialidases are ubiquitous in the reign of the living, the most famous sialidase has been found in a virus.
The role of sialidases in diseases
In 1958, Alfred Gottschalk discovered that Influenza virus has two major surface proteins, hemagglutinin and neuraminidase (17). Hemagglutinin initiates infection by binding to cell-surface SAs, whereas neuraminidase disrupts the interaction between hemagglutinin and the SA receptors to promote the release of viral particles out of infected cells.
Since then, sialidase and neuraminidase are synonymous. In mammlian, they are members of the N-acylneuraminosyl glycohydrolase family and referred as Neu.
Neuraminidase increase in-vitro adherence of Staphylococcus aureus to pharynx cells (18) and mutations in the NEU1 gene result in sialidase Neu-1 (the most common Neu in humans) deficiency and lead to sialidosis, one type of lysosomal storage and orphan diseases.
Human immunodeficiency virus type 1 (HIV-1) envelope protein gp120 is heavily sialylated. Desialylation of this glycoprotein greatly increases virus–cell interaction, leading to enhanced viral replication and cytopathogenicity (19).
In Chagas disease, the desialylation of host lysosomal-associated membrane proteins (LAMPs) facilitates Trypanosoma cruzi entry into the host cell cytoplasm (20).
More recently, variations in glycosite location arising from antigenic drift can be expected to have a profound effect on SARS-CoV-2 S protein antigenicity and potentially COVID-19 vaccine efficacy (21).
This is just a few examples since the involvement of glycans in immune mechanisms is pretty extensive. And with so many applications, there’s a growing need for antibody highly specific to carbohydrate structures.
But generating monoclonal antibodies against glycans is not so simple.
Why it is so difficult to generate high specifity and high affinity monoclonals against glycans
Each time an antigen is presented to the CD4 T cells of the adaptive immune system, it must first be internalized and digested by antigen-presenting cells (APCs) before being presented on the surface of macrophages, dendritic cells and B cells as simple peptides by a MHC class II molecule (Watts and Powis, 1999).
In case of polysaccharides antigens, the answer of the immune system to infection (capsule from bacteria or cell wall from fungi) is a T cell-independent mechanism.
Glycans don’t undergo endocytosis in APCs, and the IgM become the first soldier of the adaptive immunity using its multivalency to compensate for the low affinity and low specificity of binding to complex carbohydrates. The IgG class switching is very uncommon and no immunologic memory has been demonstrated (Abbas et al., 2000).
Combining the appropriate antigen synthesis, the design of particular carrier linked at the perfect attachment site, its proprietary adjuvant and the access to lymph nodes, SYnabs has developed an expertise in the generation of high affinity IgG monoclonal antibodies against glycans.
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