Hexosamine biosynthesis and related pathways, protein N-glycosylation and O-GlcNAcylation: their interconnection and role in plants

Abstract

N-Acetylglucosamine (GlcNAc), a fundamental amino sugar moiety, is essential for protein glycosylation, glycolipid, GPI-anchor protein, and cell wall components. Uridine diphosphate-GlcNAc (UDP-GlcNAc), an active form of GlcNAc, is synthesized through the hexosamine biosynthesis pathway (HBP). Although HBP is highly conserved across organisms, the enzymes involved perform subtly distinct functions among microbes, mammals, and plants. A complete block of HBP normally causes lethality in any life form, reflecting the pivotal role of HBP in the normal growth and development of organisms. Although HBP is mainly composed of four biochemical reactions, HBP is exquisitely regulated to maintain the homeostasis of UDP-GlcNAc content. As HBP utilizes substrates including fructose-6-P, glutamine, acetyl-CoA, and UTP, endogenous nutrient/energy metabolites may be integrated to better suit internal growth and development, and external environmental stimuli. Although the genes encoding HBP enzymes are well characterized in microbes and mammals, they were less understood in higher plants in the past. As the HBP-related genes/enzymes have largely been characterized in higher plants in recent years, in this review we update the latest advances in the functions of the HBP-related genes in higher plants. In addition, HBP's salvage pathway and GlcNAc-mediated two major co- or post-translational modifications, N-glycosylation and O-GlcNAcylation, are also included in this review. Further knowledge on the function of HBP and its product conjugates, and the mechanisms underlying their response to deleterious environments might provide an alternative strategy for agricultural biofortification in the future.

Keywords: N-acetylglucosamine; N-glycosylation; O-GlcNAcylation; abiotic stress; hexosamine biosynthesis pathway; salvage pathway.

Figure 1

UDP-GlcNAc biosynthesis through hexosamine biosynthesis and salvage pathways. (A) Hexosamine biosynthesis and salvage pathways. The hexosamine biosynthesis pathway (HBP) is composed of four reactions catalyzed sequentially by glutamine:Fru-6-P amidotransferase (GFAT), glucosamine-6-P N-acetyltransferase (GNA), N-acetylglucosamine-phosphate mutase (AGM) and N-acetylglucosamine-1-P uridylyltransferase (GlcNAc1pUT or UAP) to synthesize uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). UDP-GlcNAc is presumably interconverted to UDP-N-acetylgalactosamine (UDP-GalNAc) by an uncharacterized UDP-Glc-4-epimerase (UGE) in plants. In the salvage pathway (purple lines), GlcN is used and converted to GlcN-6-P catalyzed by a hexokinase (HK), followed by entering HBP to form UDP-GlcNAc. In addition, GlcNAc can be converted to GlcNAc-6-P catalyzed by an N-acetylglucosamine kinase (GNK); GlcNAc-6-P further enters the HBP to form UDP-GlcNAc. The green dashed lines represent HBP in prokaryotes. This HBP diagram is modified from Furo et al. (2015) and Chen et al. (2022). (B) Biochemical structures of HBP. These chemical structures are derived from the BRENDA database (https://www.brenda-enzymes.org/fulltext.php?overall=1).

Figure 2

Schematic diagram of N-glycosylation and O-GlcNAcylation. GlcNAc is the fundamental amino sugar moiety essential for N-glycosylation and GlcNAcylation. (A) N-glycosylation. UDP-GlcNAc is generated by the hexosamine biosynthesis pathway (HBP) and provides GlcNAc for the initial biosynthesis of oligosaccharide precursors at the cytosolic side of the ER. The oligosaccharide precursor (Man5GlcNAc2-PP-Dol) enters the ER lumen for N-glycan modification and N-glycosylation of proteins. Complex and hybrid N-glycan processing occurs in the Golgi apparatus. Proteins with mature N-glycans will be secreted to their destinations. (B) O-GlcNAcylation. UDP-GlcNAc also provides the GlcNAc molecular unit directly to the Ser/Thr amino acids of proteins localized in the cytosol and nucleus. (1) Asparagine-linked glycosylation (ALG) enzyme ALG7, a UDP-N-acetylglucosamine:dolichol phosphate N-acetylglucosamine-1-P transferase; (2) ALG13 and ALG14, UDP-N-acetylglucosamine transferase subunits; (3) ALG1/2/11, mannosyltransferases; (4) Flippase-like protein; (5) ALG3/9/12, mannosyltransferases; (6) ALG6/8/10, glucosyltransferases; (7) OST, oligosaccharyltransferase complex; (8) GCSI/II, glucosidases; (9) MNS3, ER-α-mannosidase I; (10) MNS1/2, Golgi-α-mannosidase I; (11) GnTI, β-(1->2)-N-acetylglucosaminyltransferase I or COMPLEX GLYCAN LESS 1 (CGL1); (12) GMII, Golgi α-mannosidase II; (13) GnTII, β-(1->2)-N-acetylglucosaminyltransferase II; (14) XYLT, β-(1->2)-xylosyltransferase; FUT11/12, core α-(1->3)-fucosyltransferases; GALT1, β-(1->3)-galactosyltransferase 1; FUT13, α-(1->4)-fucosyltransferase; (15) SEC, SECRET AGENT (O-GlcNAc transferase, OGT); ROCK1, REPRESSOR OF CYTOKININ DEFICIENCY 1; UGNT1, UDP-GlcNAc transporter; NOPE1, NO PERCEPTION 1. The nomenclature of enzymes is generally based on the report by Strasser et al. (2021).

Figure 3

Hexosamine biosynthesis and related pathways in response to stresses. This diagram depicts that the HBP integrates several key metabolites to synthesize UDP-GlcNAc, an essential amino sugar moiety of glycosylation of proteins and lipids. Under stress conditions, HBP integrates endogenous metabolites and energy status to maintain UDP-GlcNAc homeostasis and reprogram metabolic pathways including glycosylation to benefit plant adaptation to deleterious environments. Fru-6-P, fructose-6-phosphate; L-Gln, L-glutamine; CoA, coenzyme A; UTP, uridine triphosphate; ABA, abscisic acid; JA, jasmonic acid; CK, cytokinin; GA, gibberellic acid.