Human CLEC18 gene cluster contains C-type lectins with differential glycan-binding specificity

The human C-type lectin 18 ( clec18 ) gene cluster, which contains clec18a , clec18b and clec18c three loci, is located in human chromosome 16q22. Even though the amino acid sequences of CLEC18A, CLEC18B, and CLEC18C are almost identical, several amino acid residues located in the C-type lectin-like domain (CTLD) and the Sperm-Coating Protein/Tpx-1/Ag5/PR-1/Sc7 (SCP/TAPS) domain, also known as the cysteine-rich secretory proteins/antigen 5/pathogenesis-related 1 proteins (CAP) domain, are distinct from each other. Genotyping by real-time PCR and sequencing further shows the presence of multiple alleles in clec18a/b/c loci. Flow cytometry analysis demonstrates that CLEC18 (CLEC18A, B and C) are expressed abundantly in human peripheral blood cells. Moreover, CLEC18 expression is further upregulated when monocytes differentiate into macrophages and dendritic cells (DCs). Immunofluorescence staining reveals that CLEC18 are localized in the (ER),


INTRODUCTION
The superfamily of C-type lectin (CLEC) comprises a large group of glycoproteins with divergent functions, including host-pathogen interaction and cell-cell interaction (1,2). The However, whether this is a general rule to all the members of C-type lectins need to be further confirmed.
Recently, more and more evidence shows that not all proteins with CTLD interact with Ca 2+ in the long loop region (6). The classical CLECs (such as selectins, collectins, and mannose binding proteins) bind glycans in Ca 2+ -dependent manner, while members of Syk-coupled CLEC receptors (such as Dectin-1/CLEC7A and MDL1/CLEC5A) bind glycans in Ca 2+independent manner (7,8). Moreover, proteins with less closely related CTLDs (such as CD69, CD72, KLRF1 of NK receptor family) do not appear to have carbohydrate-binding activity.
In human genome, there are at least 57 CTLD-containing proteins divided into XVI groups (9). Among these proteins, we are especially interested in group XV, the CLEC18 family, for following reasons: 1) the clec18 gene   Polysaccharide binding assay--Samples of GLPS-F3 were weighed, dissolved, and diluted with 100 mM Tris buffer (pH 9.5) to give 20µg/ml as described previously (14). Briefly,   Figure 1C).

Alignment of human CLEC18 with CLEC18
of other species shows that CLEC18 is highly conserved in CTLD, but the predicted nine amino acid insertion is only found in human CLEC18B and chimpanzee CLEC18 ( Figure 1D). The typical motifs of CTLDs, such as "WIGL" (a.a.    Figure 4E), as well as in human sera (lower, Figure 4E) as determined by ELISA. This observation suggests that CLEC18A is a secretory protein.

Subcellular localization of CLEC18--Because
CLEC18 were detectable in cell lysates, we further investigated its subcellular localization.
To address this question, human CD14 + -derived M-M was incubated with anti-CLEC18A mAb, followed by incubation with Alex488-conjugated goat anti-mouse mAb. To determine its subcellular localization, M-M was also coincubated with both anti-GM 130 mAb (rabbit), anti-calreticulin mAb (rabbit), and anti-EEA1 mAb (rabbit), respectively, followed by incubation with TRITC-conjugated goat-antirabbit mAb. We found that CLEC18 were co-   Figure 7A). Similar observation was observed when incubating fusion proteins with zymosans ( Figure 7B) and house dust mite ( Figure 7C). Since Dectin-1.Fc binding to betaglucans is independent of Ca ++ (7), this observation suggests that CLEC18 binding to F3 polysaccharides and zymosans is also Ca ++independent, and amino acid residue S 339 is critical for binding to polysaccharides.

DISCUSSION
Several unusual features of CLEC18 are also noted in this study: 1) It is surprising to find that ( Figure 7A) and zymosans ( Figure 7B) in Ca ++independent manner, and display diverse binding specificity to various glycans ( Figure 7D and