Abstract
Sulfated glycans are known to be involved in several glycan-mediated cell adhesion and recognition pathways. Our mRNA transcript analyses on the genes involved in synthesizing GlcNAc-6-O-sulfated glycans in human colon cancer tissues indicated that GlcNAc6ST-2 (CHST4) is preferentially expressed in cancer cells compared with nonmalignant epithelial cells among the three known major GlcNAc-6-O-sulfotransferases. On the contrary, GlcNAc6ST-3 (CHST5) was only expressed in nonmalignant epithelial cells, whereas GlcNAc6ST-1 (CHST2) was expressed equally in both cancerous and nonmalignant epithelial cells. These results suggest that 6-O-sulfated glycans that are synthesized only by GlcNAc6ST-2 may be highly colon cancer-specific, as supported by immunohistochemical staining of cancer cells using the MECA-79 antibody known to be relatively specific to the enzymatic reaction products of GlcNAc6ST-2. By more precise MS-based sulfoglycomic analyses, we sought to further infer the substrate specificities of GlcNAc6STs via a definitive mapping of various sulfo-glycotopes and O-glycan structures expressed in response to overexpression of transfected GlcNAc6STs in the SW480 colon cancer cell line. By detailed MS/MS sequencing, GlcNAc6ST-3 was shown to preferentially add sulfate onto core 2-based O-glycan structures, but it does not act on extended core 1 structures, whereas GlcNAc6ST-1 prefers core 2-based O-glycans to extended core 1 structures. In contrast, GlcNAc6ST-2 could efficiently add sulfate onto both extended core 1- and core 2-based O-glycans, leading to the production of unique sulfated extended core 1 structures such as R-GlcNAc(6-SO3-)β1-3Galβ1-4GlcNAc(6-SO3-)β1-3Galβ1-3GalNAcα, which are good candidates to be targeted as cancer-specific glycans.
Highlights
Sulfated glycans are known to be involved in several glycanmediated cell adhesion and recognition pathways
Our mRNA transcript analyses on the genes involved in synthesizing GlcNAc-6-O–sulfated glycans in human colon cancer tissues indicated that GlcNAc6ST-2 (CHST4) is preferentially expressed in cancer cells compared with nonmalignant epithelial cells among the three known major GlcNAc-6-O-sulfotransferases
RT-PCR analyses and results summarized from 15 patients (Fig. 1A (a)) showed that a marked decrease of GlcNAc6ST-3 mRNA was registered in cancer cells compared with nonmalignant epithelial cells, whereas GlcNAc6ST-2 mRNA showed a drastic increase in colon cancer tissues
Summary
GlcNAc6(Gal3)GalNAc-pNP, not core 3–based structure, GlcNAc2Man␣6Man1-octyl, GlcNAc2(GlcNAc6)-Man␣6Man1-octyl. The prime advantage of MS analysis resides in its ability to offer a global view of the relative abundance of all major sulfated glycans, including any unanticipated novel structures This is in contrast to the use of mAbs, limited by availability against few known sulfated glycotopes, or an HPLC-based mapping method, limited by the need for reference glycan standards [42]. By MS/MS analysis, we demonstrated that GlcNAc6ST-2 supports the synthesis of 6-sulfo LacNAc on the extended core 1 O-glycan structures and MECA-79 epitope, corroborated by a strong staining of colon cancer tissues by the MECA-79 mAb. GlcNAc6ST-3, on the other hand, was shown to be unable to efficiently put a sulfate on the extended core 1. We further identified a series of new cancer-associated carbohydrate antigens represented by GlcNAc-6 –sulfated extended core 1 structures, namely R-GlcNAc(6-SO3Ϫ)1–3Gal1– 4GlcNAc(6-SO3Ϫ)1–3Gal13GalNAc␣, which can only be synthesized by GlcNAc6ST-2 due to its unique substrate specificity
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