Cloning of a novel alpha 2,3-sialyltransferase that sialylates glycoprotein and glycolipid carbohydrate groups.

Kitagawa, Hisato; Paulson, James C.

doi:10.1016/s0021-9258(17)42271-x

Cited by 207 publications

(30 citation statements)

References 54 publications

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“…5D). This was further corroborated by the finding that KO of ST3GAL4, but not ST3GAL6, which adds α2-3Sia to LacNAc on N-glycans and lactoseries glycolipids (60), resulted in loss of Siglec-3 binding and strongly reduced Siglec-8 binding (Fig. 5D).…”

Section: Siglec-7 Binds Dst and The Cancer-associated Stn O-glycan Withsupporting

confidence: 69%

Probing the binding specificities of human Siglecs by cell-based glycan arrays

Büll

Nason

Sun

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

125

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Siglecs are a family of sialic acid–binding receptors expressed by cells of the immune system and a few other cell types capable of modulating immune cell functions upon recognition of sialoglycan ligands. While human Siglecs primarily bind to sialic acid residues on diverse types of glycoproteins and glycolipids that constitute the sialome, their fine binding specificities for elaborated complex glycan structures and the contribution of the glycoconjugate and protein context for recognition of sialoglycans at the cell surface are not fully elucidated. Here, we generated a library of isogenic human HEK293 cells with combinatorial loss/gain of individual sialyltransferase genes and the introduction of sulfotransferases for display of the human sialome and to dissect Siglec interactions in the natural context of glycoconjugates at the cell surface. We found that Siglec-4/7/15 all have distinct binding preferences for sialylated GalNAc-type O-glycans but exhibit selectivity for patterns of O-glycans as presented on distinct protein sequences. We discovered that the sulfotransferase CHST1 drives sialoglycan binding of Siglec-3/8/7/15 and that sulfation can impact the preferences for binding to O-glycan patterns. In particular, the branched Neu5Acα2–3(6-O-sulfo)Galβ1–4GlcNAc (6′-Su-SLacNAc) epitope was discovered as the binding epitope for Siglec-3 (CD33) implicated in late-onset Alzheimer’s disease. The cell-based display of the human sialome provides a versatile discovery platform that enables dissection of the genetic and biosynthetic basis for the Siglec glycan interactome and other sialic acid–binding proteins.

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Section: Siglec-7 Binds Dst and The Cancer-associated Stn O-glycan Withsupporting

confidence: 69%

Probing the binding specificities of human Siglecs by cell-based glycan arrays

Büll

Nason

Sun

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

125

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“…a moderate strength hit (Dataset S1). ST3GAL2 has been reported to have similar biochemical activity toward O-linked glycans as ST3GAL1 in vitro, although this finding has never been confirmed in living cells (51,(53)(54)(55). Our results imply that ST3GAL2 could function redundantly with ST3GAL1 in cellular O-sialoglycan biosynthesis.…”

Section: Genome-wide Crispri Screens Reveal Genetic Determinants Of Siglecmentioning

confidence: 55%

“…4A), a motif commonly found on cell-surface O-glycoproteins, may be a component of the glycan epitope that binds Siglec-7 (50). The enzyme ST3GAL1 (which attaches sialic acid α-2,3-linked to galactose residues) has also been reported to be a key component of this biosynthetic pathway (51,52). Our screen, however, did not identify ST3GAL1 as a driver of Siglec-7 ligand expression.…”

Section: Genome-wide Crispri Screens Reveal Genetic Determinants Of Siglecmentioning

confidence: 57%

Genome-wide CRISPR screens reveal a specific ligand for the glycan-binding immune checkpoint receptor Siglec-7

Wisnovsky

Möckl

Malaker

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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Glyco-immune checkpoint receptors, molecules that inhibit immune cell activity following binding to glycosylated cell-surface antigens, are emerging as attractive targets for cancer immunotherapy. Defining biologically relevant ligands that bind and activate such receptors, however, has historically been a significant challenge. Here, we present a CRISPRi genomic screening strategy that allowed unbiased identification of the key genes required for cell-surface presentation of glycan ligands on leukemia cells that bind the glyco-immune checkpoint receptors Siglec-7 and Siglec-9. This approach revealed a selective interaction between Siglec-7 and the mucin-type glycoprotein CD43. Further work identified a specific N-terminal glycopeptide region of CD43 containing clusters of disialylated O-glycan tetrasaccharides that form specific Siglec-7 binding motifs. Knockout or blockade of CD43 in leukemia cells relieves Siglec-7-mediated inhibition of immune killing activity. This work identifies a potential target for immune checkpoint blockade therapy and represents a generalizable approach to dissection of glycan–receptor interactions in living cells.

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“…vST3Gal-I has high sequence homology with mouse ST3Gal-IV (Kono et al 1997) and human ST3Gal-IV (Sasaki et al 1993;Kitagawa and Paulson 1994). Similar to human placenta hST3Gal-IV expressed in COS-1 cells which showed marginal activity toward Le x with the relative rate of the reaction 33-fold less than that of type-II acceptor (Kitagawa and Paulson 1994), the recombinant MBP-Δ30vST3Gal-I-His 6 shows a 19-fold less efficiency in its sialyltransferase activity toward Le x acceptor (Le x βMU) compared to an acceptor (LacβMU) closely resembling type-II glycan.…”

Section: Discussionmentioning

confidence: 99%

“…The amino-acid sequence of vST3Gal-I shares high identity to mouse ST3Gal-IV (37%) (Sujino et al 2000), human ST3Gal-IV (38%) (Sasaki et al 1993;Kitagawa and Paulson 1994), human ST3Gal-III (36%) (Kitagawa and Paulson 1993) and porcine ST3Gal-I (26%) whose X-ray crystal structure was recently reported (Rao et al 2009). As shown in Figure 1, vST3Gal-I has all four conserved sialyl motifs including large (L), small (S), motif 3 and very small (VS) motifs identified previously (Datta and Paulson 1995;Geremia et al 1997;Datta et al 1998Datta et al , 2001Jeanneau et al 2004).…”

Section: Sequence Comparison Of Vst3gal-i and Mammalian St3gal-ivsmentioning

confidence: 99%

Cloning and characterization of a viral α2–3-sialyltransferase (vST3Gal-I) for the synthesis of sialyl Lewisx

Sugiarto

Lau

et al. 2010

Glycobiology

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Sialyl Lewis(x) (SLe(x), Siaα2-3Galβ1-4(Fucα1-3)GlcNAcβOR) is an important sialic acid-containing carbohydrate epitope involved in many biological processes such as inflammation and cancer metastasis. In the biosynthetic process of SLe(x), α2-3-sialyltransferase-catalyzed sialylation generally proceeds prior to α1-3-fucosyltransferase-catalyzed fucosylation. For the chemoenzymatic synthesis of SLe(x) containing different sialic acid forms, however, it would be more efficient if diverse sialic acid forms are transferred in the last step to the fucosylated substrate Lewis(x) (Le(x)). An α2-3-sialyltransferase obtained from myxoma virus-infected European rabbit kidney RK13 cells (viral α2-3-sialyltransferase (vST3Gal-I)) was reported to be able to tolerate fucosylated substrate Le(x). Nevertheless, the substrate specificity of the enzyme was only determined using partially purified protein from extracts of cells infected with myxoma virus. Herein we demonstrate that a previously reported multifunctional bacterial enzyme Pasteurella multocida sialyltransferase 1 (PmST1) can also use Le(x) as an acceptor substrate, although at a much lower efficiency compared to nonfucosylated acceptor. In addition, N-terminal 30-amino-acid truncated vST3Gal-I has been successfully cloned and expressed in Escherichia coli Origami™ B(DE3) cells as a fusion protein with an N-terminal maltose binding protein (MBP) and a C-terminal His(6)-tag (MBP-Δ30vST3Gal-I-His(6)). The viral protein has been purified to homogeneity and characterized biochemically. The enzyme is active in a broad pH range varying from 5.0 to 9.0. It does not require a divalent metal for its α2-3-sialyltransferase activity. It has been used in one-pot multienzyme sialylation of Le(x) for the synthesis of SLe(x) containing different sialic acid forms with good yields.

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Cloning of a novel alpha 2,3-sialyltransferase that sialylates glycoprotein and glycolipid carbohydrate groups.

Cited by 207 publications

References 54 publications

Probing the binding specificities of human Siglecs by cell-based glycan arrays

Probing the binding specificities of human Siglecs by cell-based glycan arrays

Genome-wide CRISPR screens reveal a specific ligand for the glycan-binding immune checkpoint receptor Siglec-7

Cloning and characterization of a viral α2–3-sialyltransferase (vST3Gal-I) for the synthesis of sialyl Lewisx

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