Metabolic glycoengineering through the mammalian GalNAc salvage pathway

Abstract

GalNAc is the initial sugar of mucin‐type O‐glycans, and is a component of several tumor antigens. The aim of this work was to determine whether synthetic GalNAc analogs could be taken up from the medium and incorporated into complex cellular O‐glycans. The cell line employed was CHO ldlD, which can only use GalNAc and Gal present in the medium for the synthesis of its glycans. All GalNAc analogs with modified N‐acyl groups (N‐formyl, N‐propionyl, N‐glycolyl, N‐azidoacetyl, N‐bromoacetyl, and N‐chloroacetyl) were incorporated into cellular O‐glycans, although to different extents. The GalNAc analogs linked to Ser or Thr could be extended by the β3‐galactosyltransferase glycoprotein‐N‐acetylgalactosamine 3β‐galactosyl transferase 1 in vitro and in vivo and by α6‐sialyltransferase α‐N‐acetylgalactosaminide‐α‐2,6‐sialyltransferase 1. At the surface of CHO ldlD cells, all analogs were incorporated into sialylated O‐glycan structures like those present on wild‐type CHO cells, indicating that the GalNAc analogs do not change the overall structure of core‐1 O‐glycans. In addition, this study shows that the unnatural synthetic GalNAc analogs can be incorporated into human tumor cells, and that a tumor antigen modified by an analog can be readily detected by a specific antiserum. GalNAc analogs are therefore potential targets for tumor immunotherapy.

Metabolism of Vertebrate Amino Sugars with N-Glycolyl Groups

“…In addition, our accompanying paper (5) demonstrates that UDP-GlcNGc can also successfully enter the O-GlcNAcylation pathway resulting in O-GlcNGc modifications. We found GalNGc to be incorporated into O-glycans as also reported very recently by another group (36,37). Going beyond these published reports, we also isolated cellular O-glycans from cells grown in the presence of either GalNAc or GalNGc and performed structural analyses using mass spectrometry.…”

“…Future studies will reveal the impact of media supplemented GalNAc derivatives onto the overall complexity of O-glycan structural diversity in cells. After this study was finalized, another research group showed predominantly by lectin binding analyses that various artificial GalNAc derivatives, including GalNGc, seem to incorporate into O-glycans via the GalNAc salvage pathway (36,37).…”

“…This finding already suggests a certain level of tolerance of basic metabolic enzymes toward the N-glycolyl substituent and raises the question of whether the N-glycolylated breakdown products of Neu5Gc are able to be salvaged. The GalNAc salvage pathway has already been described to be very efficient in introducing artificial GalNAc derivatives, resulting in modified O-glycans on glycoproteins and nucleocytoplasmic proteins (35)(36)(37)(38)(39)(40). In this study, we use exogenous GalNGc for metabolic labeling and demonstrate its incorporation in nearly all of the major glycans present in vertebrates.…”

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2011–2012

“…(1 2)-Linked nonamannoside TOF Use of ionic liquid support for the synthesis of large and complex oligosaccharides (Ma et al, 2011a) N-O-linked glycans MALDI Use of glycosyl ortho-hexynylbenzoates as donors under the catalysis of PPh3AuOTf O-Glycans with unnatural GalNR groups TOF Use of CHO IdID cell line fed with GalNR molecules so that they could be incorporated into O-glycans (Pouilly et al, 2012a) Oligosaccharides TOF (DHB) New method for regioselective glycosylation employing saccharide oxyanions (Matwiejuk & Thiem, 2011) Oligosaccharides TOF C5-acetamide sialic acid donor for -selective (Uchinashi et al, sialylation 2011) Oligosaccharides TOF/TOF (DHB)…”

The Expanding World of Tissue Engineering: The Building Blocks and New Applications of Tissue Engineered Constructs

“…As a result of the growing knowledge on signaling and metabolic pathways in mammalian cells, pathway engineering approaches have recently been extended to mammalian cells [160-162]. It has also been considered for tissue engineering and regenerative medicine applications [163].…”