Evidence of two enzymes performing the de‐N‐glycosylation of proteins in barley: expression during germination, localization within the grain and set‐up during grain formation

Abstract

The occurrence of two enzymes performing de-N-glycosylation of glycoproteins, namely, endo-N-acetyl-beta-D-glucosaminidase (ENGase, EC 3.2.1.96) and peptide-N(4)-(N-acetyl-beta-D-glucosaminyl) asparagine amidase (PNGase, EC 3.5.1.52) was investigated in barley, cv. Plaisant (a winter six rowed variety). The dry grain showed both activities according to the HPLC detection of the hydrolysis of fluorescent resorufin-labelled substrates. However, PNGase activity was 16-fold higher than ENGase activity. During germination, both activities increased, PNGase by only 1.5-fold compared to nearly 4.8-fold for ENGase over the 4 d following imbibition. The localization of these activities within the grain showed that the major contribution of PNGase was due to the endosperm, typically representing over 90% of the whole grain activity. In contrast, ENGase activity was especially high in the embryo and, later, in the developing plantlet (10-fold higher than in the endosperm), particularly in the rootlets and scutellum. In developing spikes, PNGase activity was 5.6-fold higher than in the leaves, but similar ENGase activity was measured in both organs. During grain formation, PNGase activity followed dry matter increase together with endosperm development. In contrast, ENGase activity dropped by 66% at the beginning of grain filling before stabilizing until harvest. The occurrence of de-N-glycosylation-performing enzymes throughout the development of barley raises the question of the nature of their natural substrates. Moreover, the prevalence of one of these enzymes over the other depending on the organ and the developmental stage, could represent the first evidence of specific functions for each enzyme.

The dynamic behavior of storage organelles in developing cereal seeds and its impact on the production of recombinant proteins

“…Single GlcNAc residues have been detected on glycoproteins produced in maize, and may even represent the predominant glycoform suggesting a high level of ENGase) activity in cereal seeds (Rademacher et al, 2008; Ramessar et al, 2008a; Arcalis et al, 2010). Indeed, ENGase activity has been observed in cereal seeds (Vuylsteker et al, 2000), and free oligomannosidic glycan structures, which are released by ENGase activity, have been identified in seeds of diverse plant species (Kimura and Kitahara, 2000; Kimura et al, 2002) and a physiological role for them in plant development, fruit and seed maturation has been proposed (Priem and Gross, 1992; Kimura and Kitahara, 2000). However, Arabidopsis plants lacking ENGase activity showed no obvious morphological phenotype (Fischl et al, 2011; Kimura et al, 2011).…”

Heritable Genomic Fragment Deletions and Small Indels in the Putative ENGase Gene Induced by CRISPR/Cas9 in Barley

“…ENGase hydrolyses the bond between two GlcNac residues, leaving the peptide chain with the proximal GlcNAc still linked to the asparagine residue, whereas PNGase releases the entire N-glycan and changes the asparagine to an aspartic acid residue. Both activities have been detected in mature barley seeds, and there is a positive correlation between the removal of N-glycans from proteins and the mobilization of storage glycoproteins, reflecting the fact that both ENGase and PNGase activities increase during germination (Vuylsteker et al, 2000). Recombinant glycoproteins produced in cereal grains often carry a single GlcNAc linked to the asparagine residue, possibly reflecting endogenous ENGase activity (Rademacher et al, 2008; Hensel et al, 2015; Vamvaka et al, 2016).…”

The Seed Proteome Web Portal

“…Yet, at the seed proteome level, post-translational modifications by cleavage are not the most abundant modification as we found a good correlation between the theoretical and the observed protein molecular weight (Figure 3). In contrast, we found a very poor correlation between the proteins theoretical and the observed isoelectric point (Figure 3; Table S1 in Supplementary Material) illustrating the major impact of post-translational modifications, e.g., phosphorylation (Arc et al, 2011), glycosylation(Vuylsteker et al, 2000), or oxidation (Job et al, 2005) on the seed proteome.…”