Sialylation and fucosylation at the non-reducing end of glycans often form meaningful glycan determinants, such as blood-type antigens and tumour-associated glycan antigens. Alterations of these glycosylations have been reported in malignant transformation11,12,13. Increased sialylation is generally relevant to malignant tumours and promotes invasiveness and metastasis, leading to cancer progression and poor prognosis14,15. We previously found that sialylation on mucins increased in MEC. In this study, lectin blotting using MAL-II revealed that the mucins in MEC tissues were distinctively modified with sialic acids through an α2,3-linkage but not through an α2,6-linkage. Therefore, the glycans were found to be sialylated at the 3-position of the Gal residues. BC2LCN specifically recognises Fucα1-2Galβ1–3 structures that are synthesised in human salivary glands by the product of FUT2, a gene responsible for secretion. Approximately 20% of the Japanese population are non-secretors who cannot make the Fucα1-2Galβ1–3 structures due to inactivation of the FUT2 product16. In this experiment, 5 among 10 samples were BC2LCN negative and considered to be of non-secretor origin. AAL broadly recognises fucose residues, including Lewis fucose (Fucα1–3/4GlcNAc) and core fucose (Fucα1-6GlcNAc) of N-linked glycans. AAL stained all three patients (1–3) with MEC and any bands of NSGs (7,9,10) (Fig. 1D). Thus, these results suggested that glycans of mucins in MEC tissues had Lewis fucose and also had Fucα1-2Galβ1–3 in the secretors. In addition, all origin spots except for sample 8 were also stained with AAL. During the SMME with the 0.1 M pyridine-formic acid buffer (pH 4.0), many proteins with pI values ≥ 4.0 do not migrate from the origin. AAL might bind fucosylated N-linked glycans on these proteins. The α2,3-linked sialylation competes with α1,2 fucosylation at the same Gal that forms type-H antigen. BC2LCN that binds the type-H antigen, however, stained the MUC1 bands (samples 1 and 3) that were also stained with MAL-II (Fig. 1C). This finding implies that plural Gal residues were present in the individual glycans, and they are likely to occur in core-2 glycans that have a branching structure. Tandem mass spectrometry of the sialylated glycans in our previous study also suggested that the glycans were core-2 glycans. O-glycans in humans are classified into four types: cores 1–4 based on a di- or trisaccharide structure at the reducing end17. Among them, core-2 glycans are known to be elongated to form the glycan antigens, such as sialyl LewisX and sialyl Lewisa 18. These glycan antigens on mucins and glycolipids have been used as serum tumour markers for several decades. In addition, sialyl LewisX and sialyl Lewis a are also known to be the ligands of selectins that are a family of cell-adhesion molecules expressed on endothelial cells, leukocytes and platelets. These glycans on tumour cells have been implicated in tumour cell metastasis through adhesion to the vascular endothelium19. The mRNA of C2GnT-1, the gene responsible for core-2 trisaccharide synthesis, increases in pancreatic cancer and lung cancer, whereas it decreases in breast cancer20. In this study, the location of cells expressing C2GnT-1 mRNA overlapped with MUC1-positive areas in MEC. These results suggested that MUC1-positive cells in MEC also synthesise core-2 O-glycans. This possibility would support the idea that these cells express MUC1 modified with core-2 glycans. It has been reported that core-2 O-glycans suppress the immune function of natural killer (NK) cells, contributing to tumour cells to evade the host’s cancer-cell elimination mechanism21. The expression of MUC1 with core-2 O-glycans in MEC may be related to the poor prognosis of MEC.
In normal human tissues, membrane-bound MUC1 is known to be expressed at the apical surface of glandular or luminal epithelial cells, such as the lung, mammary gland, stomach and salivary glands5,12,22. We confirmed restricted expression of MUC1 at the apical membranes in the surrounding normal tissue of MEC in this study. In contrast, diffuse staining was observed in the MEC. These results are consistent with those of previous reports5,23. Therefore, MUC1 with sialylated core-2 glycans is a potential biomarker for MEC in serum or saliva. However, it is not known whether MUC1 modified with this glycan can be detected in serum or saliva, so additional research is needed. In addition, whether or not MUC1 is localised in mucous cells and non-mucous cells of MEC is unknown and needs to be investigated.
Expression of sialyl-Tn, a well-known tumour-associated glycan antigen, was also evaluated and compared with expression of MUC1. Sialyl-Tn was only detected in one-third of the patients with MEC (Fig. 2C). This finding was consistent with our previous data showing that sialyl-Tn was rarely detected by mass spectrometry of the glycans from MUC1 separated by SMME of the MEC homogenate [our unpublished data]. The mass spectral data showed that sialylated core-2 O-glycans were major species of glycans on MUC1. Furthermore, we tried to stain the sections with MAL-II to reveal the locations of α2,3-linked sialic acid residues. Unfortunately, the background was too high to visualise the location of the sialic acid residues.
One limitation of this study was that only three samples from patients with MEC were used. In addition, the MECs were from minor salivary glands, whereas NSGs include the major and minor salivary glands. However, all three patients with MEC showed similar results. Additionally, there was a clear difference in the results between MEC and NSGs. Given these differences, it would be worthwhile to increase the number of samples and collection of additional data in the future. Another limitation of this study was that the SMME used tissue homogenates. Therefore, an unknown effect of a tissue other than MEC and NSGs could not be completely excluded. However, results of IHC and ISH were considered to be acceptable to support the results of lectin blotting on the SMME membrane.
These results suggest that MUC1 modified with core-2 O-glycans containing α2,3-linked sialic acid is expressed in mucous cells and non-mucous cells of MEC.