Tumor-associated Neu5Ac-Tn and Neu5Gc-Tn antigens bind to C-type lectin CLEC10A (CD301, MGL)

Abstract

In human tumors, glycoproteins often exhibit abnormal glycosylation patterns, e.g. certain Lewis structures, TF antigen, Tn antigen and/or their sialylated forms, creating additional binding sites for glycoreceptors. In the present study, we have analyzed the carbohydrate specificity of the C-type lectin CLEC10A using glycan profiling by enzyme-linked immunosorbent assay (ELISA). In addition to the known ligands, we show binding to two tumor-associated antigens, namely Neu5Acα2,6-Tn and Neu5Gcα2,6-Tn, with an affinity of CLEC10A in the micromolar range. Detailed analyses of the glycan-lectin interactions were carried out by surface plasmon resonance (SPR) and saturation transfer difference (STD) NMR. CLEC10A binds Neu5Acα2,6-Tn and Neu5Gcα2,6-Tn with dissociation constants of 297 and 80 µM, respectively, as determined by SPR. Comparison of the STD nuclear magnetic resonance (NMR) binding epitopes of Tn and Neu5Acα2,6-Tn revealed a constant binding mode of the N-acetylgalactosamine moiety. This finding is in good agreement with binding studies of CLEC10A transfectomas, which show a well-defined interaction of transmembrane CLEC10A with 6-sialylated-Tn structures. Since both Neu5Acα2,6-Tn and Neu5Gcα2,6-Tn together with the previously known Tn antigen are expressed in human tumors such as mammary carcinoma, the interaction with CLEC10A expressed by macrophages and dendritic cells could be of major functional significance in tumor progression.

Interactions between the breast cancer-associated MUC1 mucins and C-type lectin characterized by optical tweezers

“…The similarity of the rupture force and lifetimes of the MUC1(Tn) and MUC1(STn) interaction with MGL (Tables 2 and 3) is consistent with the main conclusions drawn based on previous NMR data, which indicated a similar binding mode for the Tn and the sialylated Tn antigen when interacting with MGL [30]. The NMR data indicated that the N-acetyl group and the H-2, H-3 and H-4 protons of the GalNAc residue made the major contribution to the interaction [30]. However, the NMR data also indicated a slightly lower affinity of the STn antigen for MGL compared to the Tn antigen [30].…”

“…Provided the molecular interaction possesses an inner energy barrier in the energy landscape, the characterization by AFM may yield lower x β values [49]. The similarity of the rupture force and lifetimes of the MUC1(Tn) and MUC1(STn) interaction with MGL (Tables 2 and 3) is consistent with the main conclusions drawn based on previous NMR data, which indicated a similar binding mode for the Tn and the sialylated Tn antigen when interacting with MGL [30]. The NMR data indicated that the N-acetyl group and the H-2, H-3 and H-4 protons of the GalNAc residue made the major contribution to the interaction [30].…”

“…Human MGL is a 40 kDa transmembrane glycoprotein consisting of a 39 amino acid (aa) cytoplasmic region, a 21 aa transmembrane segment and a 256 aa extracellular domain (ECD) with a carbohydrate recognition domain (CRD) and a neck region [28]. It is reported that MGL binds to the Tn antigen present on MUC1 [29], and NMR data indicate that MGL also binds the STn antigen [30]. Furthermore, based on NMR data it has been suggested that the affinity of the STn antigen to MGL is mainly mediated by the GalNAc moiety [30].…”

“…It is reported that MGL binds to the Tn antigen present on MUC1 [29], and NMR data indicate that MGL also binds the STn antigen [30]. Furthermore, based on NMR data it has been suggested that the affinity of the STn antigen to MGL is mainly mediated by the GalNAc moiety [30]. …”

“…Interestingly, monocytes stimulated in this way express MGL [33]. The interaction of MGL, expressed by M2 macrophages, with Tn and STn exposed by tumor cells, has been suggested to modulate the TAM phenotype and/or activity, and thus affect the progression of human tumors [30]. The importance of gaining further knowledge of the possible role of the STn structure is supported by the fact that the Tn glycan is mostly intracellular and not frequently on the carcinoma surface [34].…”