Evaluation of Analogues of GalNAc as Substrates for Enzymes of the Mammalian GalNAc Salvage Pathway

Abstract

Changes in glycosylation are correlated to disease and associated with differentiation processes. Experimental tools are needed to investigate the physiological implications of these changes either by labeling of the modified glycans or by blocking their biosynthesis. N-Acetylgalactosamine (GalNAc) is a monosaccharide widely encountered in glycolipids, proteoglycans, and glycoproteins; once taken up by cells it can be converted through a salvage pathway to UDP-GalNAc, which is further used by glycosyltransferases to build glycans. In order to find new reporter molecules able to integrate into cellular glycans, synthetic analogues of GalNAc were prepared and tested as substrates of both enzymes acting sequentially in the GalNAc salvage pathway, galactokinase 2 (GK2) and uridylpyrophosphorylase AGX1. Detailed in vitro assays identified the GalNAc analogues that can be transformed into sugar nucleotides and revealed several bottlenecks in the pathway: a modification on C6 is not tolerated by GK2; AGX1 can use all products of GK2 although with various efficiencies; and all analogues transformed into UDP-GalNAc analogues except those with alterations on C4 are substrates for the polypeptide GalNAc transferase T1. Besides, all analogues that could be incorporated in vitro into O-glycans were also integrated into cellular O-glycans as attested by their detection on the cell surface of CHO-ldlD cells. Altogether our results show that GalNAc analogues can help to better define structural requirements of the donor substrates for the enzymes involved in GalNAc metabolism, and those that are incorporated into cells will prove valuable for the development of novel diagnostic and therapeutic tools.

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.…”

Changes in Metabolic Chemical Reporter Structure Yield a Selective Probe of O-GlcNAc Modification

“…To confirm this result, we subjected 6AzGlcNAc (Scheme S3A and Figures S13–S15 (SI) for details of synthesis and characterization) to in vitro phosphorylation by recombinant GalK2. 36 Specifically, GalK2 was incubated with 40 mM concentrations of GalNAc, GlcNAc, or 6AzGlcNAc and [ 32 P]γATP (5 mM). At these elevated substrate-concentrations, GalK2 readily phosphorylated both GalNAc and GlcNAc but gave no detectable modification of 6AzGlcNAc (Figure 3A).…”

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

“…Metabolic labeling to enable selective photocrosslinking of O-GlcNAc-modified proteins to identify their binding partners (Yu et al, 2012d) Glycopeptides, glycoproteins R-, L-TOF Evaluation of GalNAc analogues as substrates for enzymes of the mammalian GalNAc salvage pathway (Pouilly et al, 2012b) Glycoproteins TOF/TOF (Hart et al, 2011) Human -defensin TOF (sinapinic), peptides…”