Recent insights into structures and functions of C-type lectins in the immune system

The majority of the C-type lectin-like domains in the human genome likely to bind sugars have been investigated structurally, although novel mechanisms of sugar binding are still being discovered. In the immune system, adhesion and endocytic receptors that bind endogenous mammalian glycans are often conserved, while pathogen-binding CRDs on cells of the innate immune system are more divergent. Lack of orthology between some human and mouse receptors, as well as overlapping specificities of many receptors and formation of receptor hetero-oligomers, can make it difficult to define the roles of individual receptors. There is good evidence that C-type lectins initiate signalling pathways in several different ways, but this function remains the least well understood from a mechanistic perspective. *Manuscript Click here to view linked References

At another extreme, a proposed binding interaction of sialylated glycans on IgG with SIGNR1, one of the mouse homologs of human DC-SIGN, is quite minimal: in the crystals, just the carboxyl group of the sialic acid interacts with the protein by making coordination bonds to the bound Ca 2+ (Figure 2(b)) [16••]. However, a role for glycan recognition in binding of IgG to DC-SIGN has been difficult to document [17••]. While it may be that this controversy reflects difference between human DC-SIGN and mouse SIGNR1, it is also interesting to note that the position and ligation of Ca 2+ in the crystals of mouse SIGNR1, obtained in the presence of high sulphate and low Ca 2+ , differs from that observed in most other CRD-sugar complexes and no adjacent second Ca 2+ near the sugar-binding site is observed. This latter observation may be significant, because the four amino acid side chains that form this second Ca 2+ site in human DC-SIGN and many other C-type CRDs are all present in SIGNR1 (Fig 2(c)). If the CRD in the structure of mouse SIGNR1 is not fully ligated with Ca 2+ , the conformation in the crystal may not reflect the organization of the binding site under physiological conditions. At low Ca 2+ concentration, crystals of the CRD from human and mouse mincle, a macrophage receptor that binds glycoconjugates on the surface of mycobacteria and fungi, similarly lack the second Ca 2+ , resulting in a rearrangement of the primary Ca 2+ site [18-20•]. However, bound sugar ligand is only observed under conditions of higher Ca 2+ , when the second Ca 2+ is occupied and the primary site takes on its canonical geometry.
In proteins such as mouse DCIR2 and BDCA-2, which lack the accessory Ca 2+ , residues that would have ligated this Ca 2+ are changed so that a basic amino acid side chain takes up the position of the missing Ca 2+

Relationships of CRDs across species
Comparison of C-type lectins in the immune systems of humans and mice shows that there are several distinct patterns of evolution ( Figure 3). Conservation is observed for many receptors that bind endogenous glycans and function in adhesion and glycoprotein clearance by endocytosis. Examples of proteins for which it is possible to identify welldefined one-to-one orthologs between species include the selectin cell adhesion molecules [24] as well as the mannose receptor and the scavenger receptor C-type lectin, which function in clearance of serum glycoproteins released at sites of infection or inflammation [25,26].
In contrast, many receptors that bind to pathogen glycans have undergone recent dramatic evolutionary changes, resulting in the absence of simple orthology between mice and humans. The differences include absence of specific proteins in one of the species and very recent duplications and divergences. For example, the human genome encodes two closely related proteins DC-SIGN and DC-SIGNR (L-SIGN) while there are eight genes for mouse SIGN proteins, none of which is organized in the same way as DC-SIGN and DC-SIGNR with extended neck domains between the CRD and the membrane [27,28]. Similarly, in the collectin family, mice express two different forms of mannose-binding protein, while there is only one functional gene in humans [29]. It appears that there has been significant evolutionary pressure on some of these receptors during the recent evolution of mammals, possibly reflecting the fact that these receptors are targets for viruses that may have selected for changes in or loss of these genes. However, it is not an absolute rule that only receptors that bind endogenous ligands are conserved, since langerin and mincle both bind pathogen glycans but their properties are very similar across mammalian species [30,31].
The absence of orthology between mice and humans for some proteins puts restrictions on the use of mouse models in understanding the functions of C-type lectins. In some cases, such as for the selectins and the mannose receptor, mice in which a particular gene is knocked out will provide information about the function of a well-defined human orthologue, while in other cases, including DC-SIGN and the mouse SIGNS, care must be taken to select an appropriate mouse model [28].

Functions of C-type lectins in signalling
Many C-type lectins found on cells in the immune system have been reported to initiate intracellular signalling, but this remains the least well understood function of these receptors.
The different arrangements of these receptors in the plasma membrane reflects the fact that there are several distinct mechanisms by which sugar binding at the cell surface leads to events on the cytoplasmic side of the membrane (Figure 4).

Conclusions
The broad outlines of the mechanism by which simple sugars are bound to C-type CRDs, established roughly twenty years ago, remain valid. However, the ways that accessory binding sites lead to selectivity for specific classes of oligosaccharide ligands continue to be elucidated. While further structures of CRDs with bound ligand are needed, the current state of structural information makes it possible to envision that a relatively complete description of the sugar-recognition aspect of C-type lectin function can be achieved. The mechanisms by which these lectins participate in cell adhesion and in glycoprotein clearance are also now relatively well understood. In contrast, roles of C-type lectins in signalling are continuing to emerge and description of the mechanisms by which glycan binding leads to initiation of signalling pathways remains an area of active investigation.   Summary of evolution of vertebrate C-type lectin-like domains. Common domain organizations were established early. However, recent evolution makes it difficult to define specific orthologues for some proteins, even between mammals such as humans and mice.

Figure 4
Organization of proteins containing extracellular C-type CRDs and intracellular domains involved in signalling. Sequence motifs in the cytoplasmic domains include immunotyrosine activation motifs (ITAMs), in which the tyrosine residues become phosphorylated, making them targets for binding to Src homology type 2 domains (SH2).