Glycans in the immune system and The Altered Glycan Theory of Autoimmunity: a critical review

Abstract

Herein we will review the role of glycans in the immune system. Specific topics covered include: the glycosylation sites of IgE, IgM, IgD, IgE, IgA, and IgG; how glycans can encode "self" identity by functioning as either danger associated molecular patterns (DAMPs) or self-associated molecular patterns (SAMPs); the role of glycans as markers of protein integrity and age; how the glycocalyx can dictate the migration pattern of immune cells; and how the combination of Fc N-glycans and Ig isotype dictate the effector function of immunoglobulins. We speculate that the latter may be responsible for the well-documented association between alterations of the serum glycome and autoimmunity. Due to technological limitations, the extent of these autoimmune-associated glycan alterations and their role in disease pathophysiology has not been fully elucidated. Thus, we also review the current technologies available for glycan analysis, placing an emphasis on Multiple Reaction Monitoring (MRM), a rapid high-throughput technology that has great potential for glycan biomarker research. Finally, we put forth The Altered Glycan Theory of Autoimmunity, which states that each autoimmune disease will have a unique glycan signature characterized by the site-specific relative abundances of individual glycan structures on immune cells and extracellular proteins, especially the site-specific glycosylation patterns of the different immunoglobulin(Ig) classes and subclasses.

Keywords: Autoimmunity; Glycan; Glycome; Glycosylation; Immunoglobulin.

Figure 1. Immunoglobulin Isotypes and their sites of glycosylation

Depicted here are the antibody structures including their sites of glycosylation: IgM [(N-glycans at Asn-46, 209, 272, 279, and 439 (UNIPROT), or CH1-45, CH2-120, CH3-81, CH3-84.4, and CHS-7 (IMGT)]; IgA1 [(N-glycans at Asn-144 and 352 (UNIPROT) or CH2-20, CHS-7 (IMGT)]; IgA2 [(N-glycans at Asn-47, 92, 131, 205, 327 (UNIPROT) or CH1-45.2, CH1-114, CH2-20, CH2-120 and CHS-7 (IMGT)]; IgG [(N-glycans at Asn-180 (IgG1), Asn-176 (IgG2), Asn-227 (IgG3), Asn-177 (IgG4), and Asn-322 (IgG3) (UNIPROT) or CH2-84.4 (IgG1-4) and CH3- 79 (IgG3) (IMGT)]; IgD [(N-glycans at Asn-225, 316, and 367 (UNIPROT) or CH2-84.4, CH3-45.4, CH3- 116 (IMGT)], and IgE [(N-glycans at Asn-21, 49, 99, 146, 252, 264, 275 (UNIPROT) or CH1-15.2, CH1-45.2, CH1-118, CH2-38, CH3-38, CH3-77, and CH3-84.4 (IMGT)]. Each immunoglobulin is comprised of two heavy chains (blue) and two light chains (purple) that are linked together by disulfide bonds (black lines). IgA, IgD, and IgG have a flexible hinge region that link the Igs’ antigen-binding Fab region to their Fc receptor-binding region. O-glycosylation sites are depicted in yellow and N-glycosylation sites are depicted in brown. The depicted glycans are important for the structural integrity of the antibodies and their effector function [2].

Figure 2. A limited number of sugar monomers can create thousands of complex glycans

Post-translational glycan modifications are generally thought to be important for protein folding; steric protection from proteolytic degradation; and regulation of protein-protein interactions. It is estimated that up to 70% of mammalian proteins are glycosylated. The glycans are attached to proteins via “N” or “O” linkages, with N-glycosylations being more common. N-glycans are attached to asparagine (Asn) residues, whereas O-glycans are attached to amino acids serine (Ser) or threonine (Thr). Depicted here is the process of N-glycosylation, which begins in the endoplasmic reticulum (ER) and ends in the Golgi. N-glycans are attached to proteins at specific motifs; Asparagine-X-Serine or Asparagine-X-threonine, where X can be any amino acid except proline. During the process of N-glycosylation, monosaccharides (often donated by UDP or GDP-sugars) are sequentially added to the glycan structure. Initially, two N-Acetylglucosamine residues are added consecutively to Dol in the cytosol. This is followed by the addition of several mannose (Man) residues. After formation of the intermediate (Man5HexNAc2-PP-Dol), the complex is flipped into the ER-lumen. Then, four additional Man residues are added. This is followed by the addition of 3 glucose (Glc) residues, donated by Glc-P-dolichol, to form the Glc3Man9GlcNAc2-PP-dolichol precursor glycan, which is then transferred to an Asn residue on a newly synthesized protein. Glycosidases and glycosyltransferases then modify the precursor glycan to potentially generate over 10 thousand unique structures, which can be separated into three very broad structural categories (High Mannose, Hybrid, and Complex).

Figure 3. Asialoglycoprotein receptors

Sialylated serum proteins and cells are not recognized by asialoglycoprotein receptors in the liver and are thus protected from uptake and degradation. However, as an initially sialylated molecule ages it progressively loses its sialic acid moieties making it a target for asialoglycoprotein receptors. In the liver these receptors identify desialylated proteins, targeting them for uptake and degradation.

Figure 4. Cutaneous Leukocyte Antigen (CLA) is a glycovariant of P-selectin glycoprotein ligand-1 (PSGL-1)

A) PSGL-1, present on the surface of T cells, binds to P-selectin, which is upregulated on endothelial cells in the setting of inflammation. PSGL-1 binding to P-selectin helps initiate leukocyte rolling. B) In contrast, PSGL-1 does not bind to E-selectin, which is present on endothelial cells within the skin. C) Skin-homing T cells up-regulate the glycosylation enzyme FucT-VII, which leads to an increase in the number of sialyl-Lewis X moieties on PSGL-1, bestowing it with the capacity to bind to E-selectin. The sialyl-Lewis X-decorated E-selectin-binding glycoform of PSGL-1 is called CLA, which also differs from PSGL-1 in that it occurs as a monomer.

Figure 5. Fc receptors can have unique Ig specificities

FcγRI, FcγRIIA, FcγRIIIA are activating Fc receptors that differ in their affinities for the individual IgG subclasses; the only commonality being that each of them binds best to IgG1. Of the activating receptors, FcγRI is unique in that it can bind with high affinity to monomeric IgG antibodies. In contrast, FcγRIIA and FcγRIIIA are low affinity Fc receptors that bind only to antigen-antibody complexes. Activating signals originating from FcR-Ig interactions can be initiated by intracellular immunoreceptor tyrosine-based activation motifs (ITAMs), either within the Fc receptor or as part of the Fc receptor complex. In contrast, FcγRIIB is a low affinity inhibitory Fc receptor, which has an immunoreceptor tyrosine-based inhibition motif (ITIM) as part of its cytoplasmic domain. FcγRIIIB, found exclusively on neutrophils, is an activating glycosylphosphatidylinositol (GPI)-linked receptor.

Figure 6. IgG Glycoforms and their inflammatory properties

Ig heavy chain residue CH2-84.4 is post-translationally modified with the addition of an N-glycan, depicted here as a blue, green, and yellow Y-shaped structure between the two IgG heavy chains. This glycosylation site is conserved in all IgG subclasses (IgG1-4). To accommodate its CH2-84.4-linked glycan, IgG has a hydrophobic patch (not depicted). The CH2-84.4-linked glycan can be classified broadly as being either G0, G1, or G2. G0 glycans have a higher affinity for FcγRIII and are associated with a variety of autoimmune diseases. G0 glycans terminate with GlcNac residues and thus have zero galactose residues, hence their name. In contrast, G2 glycans terminate with two galactose residues. CH2-84.4 glycans can also be sialylated or fucosylated, which can bestow the antibody with anti-inflammatory properties because these modifications decrease Ig affinity for FcγRIII and also allow the antibody to interact with endogenous lectins on antigen presenting cells, e.g. sialylated antibodies likely bind to DC-SIGN.

Figure 7. Multiple Reaction Monitoring (MRM) to identify glycopeptide biomarkers of autoimmunity

A) Without the need for additional purification, serum or plasma from peripheral blood is digested with trypsin to yield peptide fragments, including glycopeptides. A C18 Nano-LC Chip is then employed to separate the peptides and glycopeptides form one another utilizing Ultra High-Pressure Liquid Chromatography. The separated sample is then ionized using electron spray ionization and analyzed using a triple quadrupole time of flight mass spectrometry (QqQ-MS) using multiple reaction monitoring (MRM). B) MRM requires prior knowledge of the collision-induced dissociation (CID) behavior of the peptides and glycopeptides of interest. This knowledge allows for the appropriate MRM transitions to be developed for QqQ-MS detection. The process also requires a great deal of instrument optimization and knowledge of the peptide and glycopeptide retention times, but once established MRM can rapidly identify peptide and glycopeptides from serum samples with great sensitivity. Depicted here, a tryptic peptide common to the Fc region of all four IgG subclasses (red arrow) is used for absolute quantitation of total IgG. IgG subclass-specific peptides (light green, purple, black, and dark green arrows) are then used for comparison to the common Fc region peptide to determine the relative abundance of the individual IgG subclasses. C) Using a data set of theoretical IgA values, no significant difference between total IgA or IgA subclass-specific titers is seen between healthy controls and patients with two different autoimmune diseases (AutoD1, AutoD2). However, when the data is graphed as the relative abundance of the different IgA subclasses, it becomes clear that patients with AutoD1 have an increase in IgA1 that is highly significant.