Journal of Experimental Botany, Vol. 51, No. 346, pp. 839-845,
May 2000
© 2000 Oxford University Press
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
Laboratoire de Biochimie Moléculaire et Cellulaire, Université d'Artois, Faculté J Perrin, rue J Souvraz, SP18, 62307 Lens, France
Received 28 September 1999; Accepted 29 December 1999
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Abstract |
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The occurrence of two enzymes performing de-N-glycosylation of glycoproteins, namely, endo-N-acetyl-ß- D-glucosaminidase (ENGase, EC 3.2.1.96) and peptide-N4-(N-acetyl-ß-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.
Key words: Glucoamidase, barley, de-N-glycosylation, ENGase, germination, PNGase.
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Introduction |
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Many proteins entering the endomembrane system of the secretory pathway are N-glycosylated, presenting a glycan linked to an asparagine residue of the peptide chain. The carbohydrate part is constituted of a pentasaccharide inner core composed of two N-acetylglucosamine (GlcNAc 1 and 2) and three mannose (Man 3, 4 and 4') residues (Fig. 1
). Two types of N-glycans can be found in plants. The oligomannoside-type presents an inner core substituted (on Man 4 and 4') by 2–6 additional mannosyl residues, and the complex-type glycans wich also contain other sugars such as additional GlcNAc, galactose, xylose (ß1,2 linked to Man 3) or fucose (
1,3 linked to GlcNAc 1) (reviewed in Lerouge et al., 1998). Two enzymes perform the removal of N-glycans. Endo-N-acetyl-ß-D-glucosaminidase (ENGase, EC 3.2.1.96) hydrolyses the di-acetylchitobiosyl linkage giving an oligosaccharide deprived of one GlcNAc and the peptide chain with GlcNAc 1 still linked to asparagine residue (Fig. 1). The peptide-N4-(N-acetyl-ß-D-glucosaminyl)asparagine amidase (PNGase, EC 3.5.1.52) cleaves the glycosylamine linkage between GlcNAc 1 and the asparagine residue, thus releasing the entire N-glycan, the peptide with an aspartic acid residue and ammonia (Fig. 1). The occurrence of ENGases and/or PNGases has been demonstrated in several plants such as almond, bamboo, fig, jack bean, pea, radish, Silene alba (reviewed in Berger et al., 1995) and, more recently, in Ginkgo biloba, soybean, and tomato (Kimura and Ohno, 1998; Kimura et al., 1998a, b). Due to their in vitro specificity of substrate (Kimura and Ohno, 1998; Kimura et al., 1998b) and to the nature of the unconjugated N-glycans found in plants (Priem et al., 1993; Kimura et al., 1997) scientists consider that ENGases are probably involved in the in vivo de-N-glycosylation of oligomannoside-type glycans containing proteins whereas the complex-type glycans containing glycoproteins could only be hydrolysed appreciably by PNGases.
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If several functions of the N-glycosylation such as modification of the protein conformation, protection against proteolysis and protein targeting have been put forward even with conflicting results (recently reviewed in Lerouge et al., 1998
), the functions of the de-N-glycosylation remain to be elucidated. Rare are the models offering a clear relation between de-N-glycosylation and a specific metabolic effect. Among them figures the processing of concanavalin A, a lectin synthesized as a glycoprotein, but incapable of interacting with sugars until de-N-glycosylation occurs (reviewed by Bowles, 1993). Alternatively, the reason for de-N-glycosylation could be found in the physiological functions of the N-glycans released, now considered as signalling molecules (reviewed in Morvan and Lhernould, 1996). Usually, the de-N-glycosylation of proteins is associated with the mobilization of storage glycoproteins. Hence, carbon starvation in Silene alba cell-suspension cultures was concomitant with the production of de-N-glycosylation activities and the subsequent release of unconjugated N-glycans in the culture medium (Lhernould et al., 1994). Efforts were made in order to verify, in seeds containing ENGase or PNGase activities, the presence of the corresponding unconjugated N-glycans and the occurrence of storage glycoproteins containing the N-glycan-type in relation to the respective enzyme substrate specificity as in pea seedlings (Kimura et al., 1995, 1996, 1997). However, this relationship was not clearly demonstrated in Ginkgo biloba which shows high ENGase activity, although the predominant storage glycoproteins contained N-glycans with xylose, a type known to be resistant to this ENGase activity (Kimura et al., 1998a). Until a clear demonstration that specific storage glycoproteins are in vivo substrates of ENGases or PNGases has been put forward, the implication of these enzymes in the recruitment of storage proteins during embryo development remains hypothetical.
Therefore, physiological approaches could provide some additional evidence. A large increase in ENGase and PNGase activities was observed during germination of radish seeds, a period marked by use of storage protein (Berger et al., 1996). This investigation was pursued on barley grains, a model with a large physiological and molecular background dealing with the mobilization of hydrolytic enzymes during germination. In this study, it appeared that both ENGase and PNGase activities are present in dry barley grains. These activities were monitored during grain formation and germination. Accurate information on their localization within the grain was given.
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Materials and methods |
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Plant materials
The experiments concerning dry grains were carried out on cv. Plaisant, a winter six rowed barley, harvested in July 1998. The grains were first soaked in sodium hypochlorite 1% (w:v) for 10 min before being rinsed for three periods of 20 min with deionized water. Samples of seeds were then placed on wet filter paper in closed darkened boxes and maintained at 16 °C until extraction.
Plaisant barley spikes were cut off once a week from the anthesis stage until harvest, from a selection field during spring and summer 1998. Their dry matter content was measured by an infrared humidity analyser and samples were frozen until enzyme extraction was performed.
Enzyme extraction
Enzyme extraction was performed by a Ultra-turraxTM homogenizer with 6 vols g-1 of fresh matter weight (at least 2 g in 12 ml) of a 5 mM sodium acetate buffer (pH 5.2) containing 50 mM EDTA, 1 mM PMSF and 1 mM 2-mercaptoethanol. Cells debris were spun down by centrifugating at 4000 g for 15 min at 4 °C. The supernatant was collected and centrifugating once more in microtubes at 13 000 g for 15 min at 4 °C. The final supernatant was conserved on ice until enzyme measurements.
Assay of the de-N-glycosylation enzymes
The activity of de-N-glycosylation enzymes was quantified using fluorescent substrates in an HPLC assay (Bourgerie et al., 1992, 1994): Man5GlcNAc2Asn(Ala, Thr, Ser)-resorufin (substrate S1) for the PNGase activity and Man7GlcNAc2Asn-resorufin (substrate S2) for the ENGase activity. Twenty microlitres of enzyme preparation were added to 20 µl (200 ng) of the appropriate resorufin-labelled substrate in 100 mM sodium acetate (pH 4.0) for PNGase or in 50 mM sodium phosphate (pH 7.0) for ENGase activity. After incubation at 37 °C the reaction was stopped by adding 95 µl of 4% trifluoroacetic acid and 50 µl was injected into a reverse phase C18 column (25x0.46 cm, 5 µm, Discovery, Sigma-Aldrich Co., USA). The substrate and the products of the enzymatic reaction were then separated using 0.07% trifluoroacetic acid as solvent A and acetonitrile containing 0.07% trifluoroacetic acid as solvent B in an isocratic elution (flow 1 ml min-1) at 85 : 15% (A : B) for PNGase assay and 88 : 12% (A : B) for ENGase assay. Resorufin-labelled products and substrates were detected by a fluorescence detector (excitation: 497 nm, emission: 559 nm). One unit of PNGase and ENGase activity was defined as the amount of enzyme required to convert 1 µmol of the substrate to the final product in 1 min at 37 °C in the appropriate buffer.
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Results |
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Demonstration of PNGase and ENGase activities in barley grains
Using fluorescent substrates, the glycopeptide Man5GlcNAc2Asn(Ala, Thr, Ser)-resorufin (substrate S1) and the glycoasparagine Man7GlcNAc2Asn-resorufin (substrate S2), the occurrence of enzyme activities performing the hydrolysis of the glycan part of each of them was investigated in barley grain extracts. Specific hydrolysis of substrate S1 by a PNGase activity and of substrate S2 by an ENGase activity was demonstrated in HPLC assays by the detection of the respective products. In fact, substrate S2 was hydrolysed by ENGase, but not by PNGase (not shown), consistent with previous data for PNGase from other sources (Berger et al., 1995
; Lhernould et al., 1995). ENGase can act on both substrates. However, the good chromatographic resolution of the products obtained by the two enzymes and the differences in their optimum pH (pH 4.0 for PNGase, pH 7.0 for ENGase) guarantee accurate quantification. An example is presented in Fig. 2, showing chromatograms corresponding to the digestion of substrate S1 by purified ENGase (Fig. 2A, trace 1) and PNGase (Fig. 2A, trace 2). The naturally occurring pigments in barley extracts (Fig. 2A, trace 3) give no interference to the assays. In the same figure, typical chromatograms obtained with a crude extract are presented, showing that not only PNGase (Fig. 2B) and ENGase (Fig. 2C) are detectable in barley, but also that there is no cross-interference in the detection of the activities. Hence, both de-N-glycosylation enzymes exist in barley grains. The activities in extracts were not susceptible to storage at 4 °C for several days in the presence of PMSF, ß-mercaptoethanol and EDTA. Raw material could be frozen several weeks before extraction without affecting the enzyme activities.
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PNGase and ENGase activities during germination of barley grains
The de-N-glycosylation enzyme activities were monitored during the germination process. The results were expressed with reference to the dry matter content instead of fresh weight in order to reduce discrepancies due to the rate of water absorption. Indeed, the dry matter content per grain was reduced by a factor of three during this period (from 89% DM to 28%). Both activities were already present in dry grains with a larger amount of PNGase, 9.56 mU g-1 DM compared to only 0.61 mU g-1 DM for ENGase (Fig. 3). PNGase activity was enhanced after imbibition from day 1 to day 3 of culture, reaching 16 mU g-1 DM, and then decreased to about 14 mU g-1 DM after 4 d of culture. ENGase activity greatly increased, from 0.61 mU g-1 DM until 2.84 mU g-1 DM, so roughly a 4.6-fold increase over the four days. It is noteworthy that the ENGase activity was significantly higher after the third day whereas PNGase activity stabilized.
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Partition of PNGase and ENGase activities between endosperm and embryo
Four separate physiological stages during the germination process were selected: the dry grain, the imbibed grain (16 h in the presence of water), the germinated stage-I with protruding radicle observed after 1–2 d, and a post-germinated stage-II attained by the fourth day, marked by the growth of the plumule which protruded at least 20 mm outside the pericarp, but was still covered by the coleoptile. For each stage, the grains were dissected in two fractions, endosperm (pericarp included) and embryo, before extraction and PNGase and ENGase activities were measured in each extract. These values were compared to those obtained for the whole grain (Table 1).
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In the dry grain, even if the endosperm and the embryo showed similar PNGase activities, respectively, 9.70 mU g-1 DM and 7.06 mU g-1 DM, ENGase activity was more than 10-fold higher in the embryo compared to the endosperm (6.59 mU g-1 DM versus 0.51 mU g-1 DM). On taking the contribution of the endosperm to the dry matter of the whole grain (typically 95% of the DM) into consideration, it is clear that the endosperm made the main contribution to the ENGase and PNGase activity of the grain. Indeed, these activities measured on whole grain extracts were not significantly different from those measured on dissected endosperm only (9.56 mU g-1 DM and 0.68 mU g-1 DM versus 9.70 mU g-1 DM and 0.51 mU g-1 DM).
After imbibition, the two enzyme activities in the grain increased (30% for PNGase and 44% for ENGase). Such increases in PNGase and ENGase activities were found in the endosperm but not in the embryo. This could correspond to de novo synthesis or to an enzyme activation, but a better extractibility of enzymes after imbibition can not be excluded, especially from the starchy endosperm. After germination, PNGase activity was slightly higher in the whole grain due to the endosperm contribution whose activity was 55% higher than in the imbibed grain. ENGase activity doubled during the same period of time. However, in each individual part, only a relatively weak increase in ENGase activity was observed (about 16%). In fact, the large amount of ENGase enhancement in the whole grain was explained by the increasing contribution of the embryo to the total DM (from 5% in the imbibed grain to 11% of total DM in the germinated stage-I grain). Indeed, the ENGase activity remained 10-fold higher in the embryo than in endosperm.
In the post-germinated stage-II, ENGase still increased by 50% in the whole grain and PNGase stabilized according to the standard error. In the embryo, both activities increased. In this stage-II, the embryo or plantlet represented 50% of the fresh matter and 16% of the dry matter and so, according to the ENGase activities in each part, contributed up to 74% of the whole grain ENGase.
Distribution of ENGase and PNGase activities within the plantlet
The localization of de-N-glycosylation activities was investigated inside the plantlet at the stage-II previously defined. For this purpose, the plantlet was divided into the following organs: radicle (and adventitious rootlets), plumule without the coleoptile, coleoptile alone, and scutellum. Further dissection of the stem axis was not investigated (mesocotyl for instance). The enzyme activities of each part were measured and those of the endosperm were recorded as a comparison (Fig. 4a). Radicles were quite different from other parts of embryo as shown by a very high ENGase activity (up to 19.76 mU g-1 DM) and a low PNGase activity (only 2.83 mU g-1 DM). All other parts showed similar amounts of both activities, but that of ENGase was a little higher than that of PNGase. By contrast, the endosperm showed more PNGase than ENGase activity. As the dry and fresh matter contents were quite different for these organs, it was also decided to express the activities as a percentage of the activity for the whole grain (Fig. 4b). The PNGase activity was mainly localized in the endosperm (92%), but the scutellum contained a significant amount (2.5%). The ENGase activity was almost equally distributed in the endosperm, the radicle and the scutellum. The radicle contributed 31% of total ENGase activity because of its high ENGase activity expressed as dry matter content and its low DM (only 4%) balanced by its high fresh matter content per grain (26% of total fresh matter). While the contribution of both plumule and coleoptile was low (respectively, 9% and 2%), the scutellum appeared to be an important contributor of ENGase activity (up to 27%) due to its high DM (representing roughly 8% of total DM of the grain).
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Expression of PNGase and ENGase activities during the formation of grains
The de-N-glycosylation activities were monitored during grain formation (Fig. 5). As the ear emerged, the PNGase activity was 5.6-fold higher in spikes than in leaves, 8.52±0.42 mU g-1 DM compared to only 1.50±0.135 mU g-1 DM (means ±standard error, n=3). By contrast, the ENGase activity in the spikes was not significantly different from the activity measured in leaves, respectively 3.107±0.166 and 3.301± 0.431 mU g-1 DM. Until 24 d after the ear emergence, ENGase and PNGase activities were quite stable, but as soon as the dry matter content increased, ENGase activity rapidly decreased from nearly 3 mU g-1 DM to below 1 mU g-1 DM and then stabilized (Fig. 5). During the same time, the PNGase activity remained at about 8 mU g-1 DM which represented a net increase per grain, when taking the higher DM content into account.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Dry barley grains from the Plaisant cultivar showed both PNGase and ENGase activities, respectively, 9.56 mU g-1 DM and 0.61 mU g-1 DM. PNGase was already found in barley (Plummer et al., 1987
) but it is the first time that ENGase has been in evidence. Moreover, these two enzyme activities have also been found in 25 other varieties of barley including spring and winter, six and two rowed types, and therefore seem uniformly spread (Vuylsteker et al., 1999). During germination, PNGase activity doubled after 3 d then stabilized, but ENGase activity increased 5-fold. The fact that both activities were pre-existent to the imbibition of grains represented a major difference with the radish situation where only PNGase activity was detected in dry seeds (Berger et al., 1996). Studying the repartition of PNGase and ENGase activities during germination, a net difference between endosperm and embryo enzymatic capacities was observed. At the beginning, in the dry grain, PNGase activity was equally present in the endosperm and embryo (9.70 mU g-1 DM and 7.06 mU g-1 DM, respectively). Thereafter, the PNGase activity doubled in the endosperm but remained stable in the embryo. If the distribution of endosperm in the total DM within the grain is considered, over 90% of PNGase activity in the grain, during the whole germination process, was thus localized in the endosperm. As the endosperm contains large amounts of reserves, this could support the hypothesis of the involvement of PNGase in the mobilization of some storage glycoproteins to support embryo development.
If, among seed proteins of cereals, prolamins and glutelins appear unglycosylated (reviewed in Müntz, 1998), glycoproteins were evidenced in barley globulin/albumin fractions by lectin affino-blotting (Weiss et al., 1991).
By contrast, ENGase activity was primarily associated with the embryo which showed a 10-fold higher activity than the endosperm (6.59 mU g-1 DM versus 0.51 mU g-1 DM in the dry grain). This situation persisted after germination during the growth and development of embryo. In fact, the contribution of the embryo to the ENGase activity of the grain was largely responsible for the overall increase in ENGase activity observed during the germination and post-germination stages. The segregation between high ENGase activity in the embryo and PNGase activity in the endosperm was reminiscent to the radish seeds situation. Indeed Berger et al. mentioned that ENGase was particularly high in the hypocotyl fraction, often referred to as the embryo part, and PNGase in the cotyledon fraction which represents the main storage organ in dicotyledonous seeds (Berger et al., 1996). This clear distinction between the endosperm and the embryo in terms of ENGase/PNGase ratios could reflect specific functions for each enzyme depending on the organ considered. The major challenge will be to identify the substrates of these enzymes in order to discover their respective functions.
More precise localization has been made in the plantlet. In this study, radicle and rootlets with 7-fold higher ENGase compared to PNGase activity, greatly deviated from other parts of the plantlet which contained similar amounts of both activities. When taking into account the dry matter distribution, the scutellum made a very large contribution in the ENGase activity (27%) equivalent to those of the radicle (31%) or the endosperm (31%). As the scutellum participates in the release of enzymes such as proteases towards the endosperm (Harris, 1962), an interesting point to clarify would be to question its role as a source of de-N-glycosylation enzymes.
On following the grain formation from anthesis to maturation, it was observed that the PNGase activity accumulated at the same rate as dry matter content, in agreement with its endosperm distribution, although ENGase activity began to decrease as soon as the grain filled up. This decrease in ENGase activity in terms of mU g-1 DM came from its localization in the embryo whose contribution to total grain DM rapidly dropped on expansion of the endosperm.
An interesting point was that spikes showed higher PNGase activity than the leaves even before fertilization and grain formation. A specific function of PNGase activity during flowering may be investigated. As ENGase and PNGase activities were present in all parts of the barley examined (flowers, immature and mature grains, embryo, roots, plumule, mature leaves) it can be concluded that de-N-glycosylation activities are well spread throughout the whole barley plant and throughout its life cycle. On considering previous studies made on seeds (reviewed in Berger et al., 1995), the existence of de-N-glycosylation enzyme activities in all parts of the plant and their maintainance throughout its life cycle should lead scientists to diversify the panel of potential functions of de-N-glycosylation. This evolution was already perceptible in free N-glycans purification. In tomatoes, previous purification of free N-glycans was made on ripening fruits due to their high dry matter and glycan contents (Priem et al., 1993) and, recently, their leaves and stems were shown to contain similar N-glycans (Yunovitz et al., 1996; Faugeron et al., 1997).
Future efforts should be made on the purification of ENGase and PNGase present in barley in order to specify their substrate specificity and to perform immunolocalization. An alternative way of providing proof of in vivo functions of these enzymes in barley, under investigation in this laboratory, is to verify the release of free N-glycans in the plant.
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Acknowledgments |
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This work was supported by grants from the Fonds pour l'Industrialisation des Bassins Miniers (FIBM 97.13.01.1640) and from Fonds Européen pour le Développement Régional (FEDER OBJ 2–98.1–01a-No. 33). We are grateful to the Laboratoire de Chimie Biologique of USTL, Lille, for the use of their premises during this study. We thank Florimond Desprez Co for barley spike collection during grain formation and Alastair Balloch for linguistic advice.
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Notes |
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1 Present address: Equipe de Physiologie et de Génétique Moléculaire Végétale, Université des Sciences et Technologies de Lille, 59 655 Villeneuve d'Ascq, France.
2 To whom correspondence should be addressed. Fax: +33 3 21791736. E-mail: yannis.karamanos{at}univ-artois.fr
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Abbreviations |
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DM, dry matter content; ENGase, endo-N-acetyl-ß-D-glucosaminidase; PNGase, peptide-N4-(N-acetyl-ß-D-glucosaminyl) asparagine amidase; GlcNAc, N-acetylglucosamine..
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