It is reasonable to review the correlation of infection/inflammation with the glycome, but in the absence of functional consequences associated with those changes, the point is moot. Unfortunately, none of the referenced findings above reported even an attempt to explain what changes in biology might accompany the observed host glycosylation differences, so we must move to another set of findings in the literature on the role glycans play in the function of the immune system to make our argument.
Simply based on the fact that the ablation of glycosylation-related genes leads to immune pathology, as mentioned earlier, it logically follows that the function of at least some glycoproteins and glycolipids is altered when the glycans change, otherwise defects would not occur. The fucosylation and Mgat5 stories represent two clear examples of where glycan changes alter the function of the underlying protein, in those cases Notch and the TCR, respectively, but there are many more to support the notion that the nature and composition of protein glycosylation directly impacts function in ways that are grossly underappreciated by the general research community. Furthermore, it is important to differentiate between studies where sites of glycosylation are removed by mutagenesis and studies where the “nature of the attached glycan” changes. Although the removal of a glycosylation site from a protein may arise due to genetic mutation associated with cancer or other insults, this event is not biologically equivalent and rare in vivo compared with the actively regulated changes in glycan composition that are in focus here. Our emphasis is how changes in glycan composition affect the function of the glycoprotein as a whole.
The best studied and highly regulated glycan change known to alter function is sialylation. The fundamental biology of sialic acids have been reviewed previously (Troy 1992
; Vimr et al. 2004
; Severi et al. 2007
; Lewis et al. 2009
; Schauer 2009
; Schauer et al. 2011
), but the central theme is that sialylation tends to alter a molecule's binding partners and/or affinity for a given ligand. This can take more forms than can be adequately covered here, so we will limit the discussion to a few canonical and recent immune system-specific findings that will serve as the basis for our unifying hypothesis. It might also strike the reader that sialylation is the glycosylation step most obviously manipulated by microbes (e.g. by microbial neuraminidases, sialyltransferases and trans-sialidases), as discussed in the previous section.
One of the more established examples within the immunological setting is the impact of sialylation upon the synthesis of selectin ligands (Schauer 2009
). As already mentioned, selectins require the negative charge provided by terminal 2,3-linked sialic acids on cell surface molecules like P-selectin glycoprotein ligand-1 (PSGL-1) and mucin-like glycoproteins (Cummings and Smith 1992
; Ellies et al. 2002
; Lowe 2003
). Removal of these residues through mutation or neuraminidase treatment alters the selectin-dependent cellular homing properties such that pathogens can evade the brunt of the immune response. In this sense, the presence or the absence of sialic acids alters the function of PSGL-1 (for example) in that it can no longer bind well to the necessary selectin to promote appropriate leukocyte homing to sites of infection and inflammation. Indeed, this provides a nice example whereby a microbe-encoded neuraminidase could dramatically impact the host's immune response through the direct enzymatic modulation of selectin ligands.
A somewhat more esoteric example is CD22/Siglec-2. This B cell-specific surface glycoprotein is a member of the B cell receptor complex and is now recognized to be a sialic acid-binding lectin (O'Reilly et al. 2011
). What is particularly remarkable about CD22 is that it binds to as of yet unidentified ligands (possibly in cis
orientations) carrying α2,6-linked sialic acids, rather than the more common α2,3-linked sialic acids (Grewal et al. 2006
). To date, it is unknown whether specific molecules carrying such glycan structures are critical, or whether any specific glycoprotein or glycolipid with 2,6-linked sialic acids are particularly important; however, the ablation of the lone enzyme that creates this linkage (ST6Gal1) creates significant B cell defects that include suppressed B cell receptor signaling, reduced serum IgM and reduced antibody responses to both T cell-dependent and T cell-independent antigens (Hennet et al. 1998
). Remarkably, removing CD22 from the ST6Gal1 knockout background normalized B cell function (Collins et al. 2006
). This story provides evidence that unknown ligands carrying α2,6-linked sialic acids are necessary for proper B cell function via recognition by CD22, yet it remains a mystery as to the identity or nature of these ligands outside of the presence of the sialic acid-containing glycans. Likewise, the other members of the Siglec family are still described as “cellular adhesion” or “cellular interaction” molecules, and specific targets have not yet been fully explored, though it is clear that ligand activity is exquisitely sensitive to sialic acid content.
Although the CD22/Siglec and selectin data show that ligands and their interactions with key immune proteins are modulated by sialylation, there is evidence to show that the function of immune-associated glycoproteins themselves are altered by sialylation and glycan composition. Two examples, where the functional differences are largely attributed to the galectin family of molecules, can be found in studies of CD45 and the TCR complex. For both of these systems, it is important to note that galectins bind to LacNAc disaccharide units in N
- and O
-linked glycans, but this binding can be strongly inhibited for some galectins by the presence of terminal sialic acids on those glycans (Liu and Rabinovich 2010
). As such, the TCR complex could be an example for how the addition or the removal of terminal sialic acids on the glycans decorating the TCR might be used to modulate T cell responsiveness. More specifically, the increased expression of the appropriate sialyltransferase in a T cell leading to increased TCR sialylation could conceivably reduce the formation of the galectin-TCR lattice at the cell surface through the inhibition of galectin binding, thereby mimicking the loss of Mgat5 and the associated reduction in TCR threshold discussed earlier (Demetriou et al. 2001
). This could be equally true in the reverse, with decreased sialylation leading to increased threshold for signaling. As a result, the level of sialylation and the presence of the LacNAc motif on the TCR glycans could potentially act as a rheostat for T cell activation via galectin-mediated effects, which makes this pathway an attractive target for pathogens seeking to evade the immune response.
CD45 serves as another prominent example of the galectin-mediated glycosylation-dependent functional effect. While this has been reviewed in detail recently (Earl and Baum 2008
), several salient points are of specific interest in terms of the present line of reasoning. CD45 is a cell surface receptor expressed in hematopoietic cells in a number of isoforms (RA, RB, RBC, RABC and RO) and is a critical factor in cellular development, activation and cell death through its tyrosine phosphatase activity (Hermiston et al. 2003
). All isoforms of CD45 include both O- (primarily core 1 and core 2) and complex N
-linked glycans. Importantly, during T cell development and within the various T cell lineages (memory, activated, naïve etc.), the glycosylation of CD45 and other cell surface glycoproteins changes in a way that is characteristic for each lineage (Earl and Baum 2008
). For example, Th2 cells express ST6Gal1, whereas Th1 cells do not, resulting in Th2 cells (but not Th1 cells) carrying CD45 with α2,6-linked sialic acids (Toscano et al. 2007
). This is a critical observation because of the impact of sialylation on gal-1 binding to CD45. Without the terminal sialic acids, gal-1 binds and cross-links CD45, whereas the presence of terminal sialic acids inhibits this interaction (Earl et al. 2010
). Gal-1-mediated cross-linking of CD45 can have a number of effects, depending on the context, but often leads to the apoptosis of Th1 and Th17 cells which lack terminal 2,6-linked sialic acids on CD45, whereas Th2 cells are protected from this effect due to the sialic acids on surface glycans (Toscano et al. 2007
). In a biological setting, gal-1 can skew the cytokine response of T cells toward Th2, possibly through the cell death pathway of Th1 cells. Furthermore, changes in CD45 and CD43 glycosylation, specifically sialylation, are associated with aging and the age-related reduction in CD4+
T cell responsiveness (Abdul-Salam et al. 2000
). In total, it is now clear that the active modulation of CD45 glycosylation in T cells is a galectin-dependent regulatory pathway during T cell development and apoptosis.
Outside of the galectin effects that almost certainly include many other cell surface receptor molecules not yet identified or studied, it has been recently discovered that antibodies themselves depend on glycosylation for the determination of their function. Textbooks teach that the function of an antibody is determined by the constant (Fc) domain of antibodies (Abbas et al. 2000
). The Fc domain carries a single but highly conserved site (asparagine 297) of N-linked glycosylation that has long been recognized as critical for antibody structural stability (Arnold et al. 2007
). Although a few early studies hinted at this, a number of recent findings now reveal that the composition of the glycans at this site in the Fc domain has a dramatic impact on the overall function of antibodies. As early as 1987 (Roitt and Cooke 1987
), it was found that IgG molecules isolated from patients with rheumatoid arthritis have galactose-deficient Fc domain glycans (Roitt et al. 1988
; Bond et al. 1990
). Since galactose is the target for ST6Gal1, these antibodies also must have lacked terminal 2,6-linked sialic acids on these structures. Indeed, this interpretation was only recently confirmed 2 years ago (van de Geijn et al. 2009
). More recently, changes in Fc glycosylation were aligned with another autoimmune disease, Wegener's granulomatosis, where the level of 2,6-linked sialic acid-containing IgG molecules was significantly reduced (Espy et al. 2011
These observations are highly significant in light of the findings that have focused upon the biological activity of IVIg, where it was shown that terminal 2,6-linked sialic acids decorating the IgG Fc glycan (2,6-sialyl-IgG) alter the Fc receptor affinity and therefore antibody function (Kaneko et al. 2006
; Anthony, Nimmerjahn, et al. 2008
; Anthony, Wermeling, et al. 2008
; Anthony et al. 2011
). This discovery was observed within the context of IVIg, which is a treatment approach used for over two decades as an effective means to suppress autoimmunity. This pioneering work has now shown that the anti-inflammatory activity of IVIg in autoimmune patients can be highly enriched by lectin affinity using Sambucus nigra
lectin (SNA), which binds to 2,6-linked sialic acids. The enriched sialic acid-containing IgG pool showed a 100-fold increase in activity over IVIg by weight (Kaneko et al. 2006
). In addition, the presence of 2,6-linked sialic acid shifted the affinities of the IgG molecules for various Fc receptors such that asialo-IgG bound tighter to the activating pro-inflammatory FcγRIII receptor whereas 2,6-sialyl-IgG preferentially associated with FcγRIIB (Kaneko et al. 2006
; Anthony, Nimmerjahn, et al. 2008
; Anthony et al. 2011
) as well as DC-SIGN (or SIGN-R1, the murine homolog; Wieland et al. 2007
; Anthony, Wermeling, et al. 2008
), both of which send inhibitory signals into responding cells. Thus, IgG molecules can be pro-inflammatory (asialo-IgG) or anti-inflammatory (2,6-sialyl-IgG), and this is exquisitely regulated by the glycan composition of the Fc domain.
The alignment of autoimmunity and aberrant antibody glycosylation was also recently seen in IgA molecules from autoimmune glomerulonephritis patients. IgA nephropathy is an autoimmune disease characterized by mesangial immunodeposits containing high concentrations of IgA1. Investigators have now discovered that the IgA1 from these deposits are deficient in galactose in the hinge-region O
-glycans (Novak et al. 2011
). This altered that IgA1 is bound by glycan-specific antibodies and seems to arise from plasma cells with aberrant glycosyltransferase expression. Although this appears to affect IgA through a different mechanism than described for IgG molecules earlier, these data further support the notion that glycosylation composition alters antibody function and fitness.
Finally, our own work on the MHCII-dependent presentation of bacterial polysaccharide “glycoantigens” has recently revealed that the nature of the N
-linked glycans on MHCII modulates antigen-binding properties. We previously discovered that zwitterionic polysaccharides isolated from the capsules of commensal bacteria are processed and presented by MHCII to T cells for recognition and activation (Cobb et al. 2004
; Cobb and Kasper 2008
; Kreisman and Cobb 2011
; Ryan et al. 2011
). Through our attempts to better understand how these unusual glycoantigens associate with MHCII, we found that preventing the formation of complex-type N
-glycans on MHCII using both pharmacologic and genetic approaches resulted in defects in both the amount of presented antigen at the cell surface as well as the overall T cell response to these antigens (Ryan et al. 2011
). In vitro binding experiments with recombinant MHCII confirm that this presentation defect was due to a loss of interactions between glycoantigens and MHCII when the MHCII N
-glycans were comprised of only high-mannose or hybrid structures. Moreover, mimicking the CDG-IIa in vitro through ablation of the Mgat2 locus, which encodes the GlcNAc transferase II enzyme responsible for initiating branched complex N
-glycan synthesis (Figure ), resulted in a lack of T cell response to commensal glycoantigens (Ryan et al. 2011
). These data show that the function of MHCII and the nature of the presented antigens at the cell surface can be modulated or regulated via changes in the N-glycosylation pathway of antigen presenting cells.
Collectively, the data are strong in support the general conclusion that glycosylation impacts the function and binding interactions of immune proteins. In most cases, changes in glycosylation alter the interactions between various molecules, be they lectins that associate directly with the glycans or other ligands whose affinity changes for reasons that remain unclear (e.g. the glycoantigen binding to MHCII). The biophysics of how glycosylation alters this second group of examples is a major challenge to dissect since most structural work relies on crystallography, which often fails with fully glycosylated molecules and is further confounded by the natural heterogeneity of the system. Still, it is clear that glycosylation is linked to glycoprotein function.