Identification of Physiologically Relevant Substrates for Cloned Gal: 3-O-Sulfotransferases (Gal3STs)

Abstract

Sulfated glycoconjugates regulate biological processes such as cell adhesion and cancer metastasis. We examined the acceptor specificities and kinetic properties of three cloned Gal:3-O-sulfotransferases (Gal3STs) ST-2, ST-3, and ST-4 along with a purified Gal3ST from colon carcinoma LS180 cells. Gal3ST-2 was the dominant Gal3ST in LS180. While the mucin core-2 structure Gal␤1,4GlcNAc␤1,6(3-O-MeGal␤1,3)GalNAc␣-O-Bn (where Bn is benzyl) and the disaccharide Gal␤1,4GlcNAc served as high affinity acceptors for Gal3ST-2 and Gal3ST-3, 3-O-MeGal␤1,4GlcNAc␤1,-6(Gal␤1,3)GalNAc␣-O-Bn and Gal␤1,3GalNAc␣-O-Al (where Al is allyl) were efficient acceptors for Gal3ST-4. The activities of Gal3ST-2 and Gal3ST-3 could be distinguished with the Globo H precursor (Gal␤1,3GalNAc␤1,3Gal␣-O-Me) and fetuin triantennary asialoglycopeptide. Gal3ST-2 acted efficiently on the former, while Gal3ST-3 showed preference for the latter. Gal3ST-4 also acted on the Globo H precursor but not the glycopeptide. In support of the specificity, Gal3ST-2 activity toward the Gal␤1,4GlcNAc␤ unit on mucin core-2 as well as the Globo H precursor could be inhibited competitively by Gal␤1,4GlcNAc␤1,6(3-OsulfoGal␤1,3)GalNAc␣-O-Bn but not 3-O-sulfoGal␤1,-4GlcNAc␤1,6(Gal␤1,3)GalNAc␣-O-Bn. Remarkably these sulfotransferases were uniquely specific for sulfated substrates: Gal3ST-3 utilized Gal␤1,4(6-O-sulfo)-GlcNAc␤-O-Al as acceptor, Gal3ST-2 acted efficiently on Gal␤1,3(6-O-sulfo)GlcNAc␤-O-Al, and Gal3ST-4 acted efficiently on Gal␤1,3(6-O-sulfo)GalNAc␣-O-Al. Mg 2؉ , Mn 2؉ , and Ca 2؉ stimulated the activities of Gal3ST-2, whereas only Mg 2؉ augmented Gal3ST-3 activity. Divalent cations did not stimulate Gal3ST-4, although inhibition was noted at high Mn 2؉ concentrations. The fine substrate specificities of Gal3STs indicate a distinct physiological role for each enzyme.

Distinct substrate specificities of human GlcNAc-6-sulfotransferases revealed by mass spectrometry–based sulfoglycomic analysis

“…Only in one case here, when both termini of a core 2 O-glycan (m/z 1747) were sialylated, could the diagnostic ions at m/z 195/234 for an internal 6-sulfo GlcNAc (GlcNAc6S) be detected for the precursor derived from the non-GlcNAc6ST-transfected parent SW480 cells. The predominance of Gal3S is consistent with previous reports indicating the dominant presence of Gal3ST2 in normal and cancer colonic cells (30,44). Due to this lack of other sulfation possibilities, the monosulfated O-glycan structures that could be deduced for SW480 have the lowest isomeric variation, with the major ones shown by the cartoon drawings on their respective MS 2 spectra (Fig.…”

Synthesis of Fluorinated Mucin Core 2 Branched Oligosaccharides with the Potential of Novel Substrates and Enzyme Inhibitors for Glycosyltransferases and Sulfotransferases

“…A sulfated Lewis x determinant was identified as a major structural motif in mucins produced by human colon carcinoma cells 1b. Previous studies from our group 20a,b indicated that two distinct Gal 3- O -sulfotransferases (Gal3STs) in tumor tissues and cancer cells exhibited distinct acceptor preferences. Enzymes from colon cancer cell lines and colon tumor tissues prefer to sulfate the C-3 position of Gal in the Galβ1,4GlcNAcβ moiety of the mucin core 2 structure.…”

“…In contrast, enzymes from breast cancer cells prefer to act on the Galβ1,3GalNAcα moiety. Therefore, to determine the structural features of the mucin core 2 that influence Gal3ST activity, assay was undertaken with compounds 4 and 5 for various sulfotransferases 20c. As compared to their activities toward the acceptor Galβ1,4GlcNAcβ1,6(3-OMeGalβ1,3)GalNAcα-O-Bn, LS180 Gal3ST, Gal3ST-2, and Gal3ST-3 exhibited poor activity (12−16%) toward compound 4 and as anticipated, negligible activity (1−2%) toward compound 5 ; Gal3ST4 showed only 23% activity with compound 5 as compared to its activity toward 3-OMeGalβ1,4GlcNAcβ1,6(Galβ1,3)GalNAcα-OBn.…”

DNA methylation status of a distinctively different subset of genes is associated with each histologic Lauren classification subtype in early gastric carcinogenesis

“…Little is known about the functional role of GAL3ST2, a member of the galactose-3-O-sulfotransferase protein family, in human types of cancers. However, a recent series of evidence indicates that downregulation of GAL3ST2 expression is observed in colon cancers, and GAL3ST2 has also been implicated in the metastatic potential of breast and laryngeal cancers (23)(24)(25). Considering the mounting evidence on the central role of selectins such as galectin-3 in thyroid, pancreatic, colon and even GCs in relation to several relevant genes, the associative function of Gal:3-O-sulfotransferase in GCs should be emphasized and evaluated in more detail (26)(27)(28)(29).…”