Journal of Experimental Botany, Vol. 52, No. 358, pp. 911-917,
May 1, 2001
© 2001 Oxford University Press
Original Papers |
Deglycosylation is necessary but not sufficient for activation of proconcanavalin A
Laboratoire des Transports Intracellulaires, CNRS-UMR 6037, European Institute for Peptide Research (IFRMP No. 23), Université de Rouen, 76821 Mont Saint Aignan, France
Received 31 August 2000; Accepted 27 November 2000
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Concanavalin A (ConA), one of the most studied plant lectins, is formed in jack bean (Canavalia ensiformis) seeds. ConA is synthesized as an inactive glycoprotein precursor proConA. Different processing events such as endoproteolytic cleavages, ligation of peptides and deglycosylation of the precursor are required to generate the different polypeptides constitutive of mature ConA. Among these events, deglycosylation of the prolectin appears as a key step in the lectin activation. The detection of deglycosylated proConA in immature jack bean seeds indicates that endoproteolytic cleavages are not prerequisite for its deglycosylation. Both the structure of the lectin precursor N-glycans Man8–9GlcNAc2 and the capacity of Endo H to cleave these oligosaccharide from native proConA in vitro favoured Endo H-type glycosidases as candidates for proConA deglycosylation in planta. Evidence for pH-dependent changes in the prolectin folding were obtained from analysis of the N-glycan accessibility and activation of the deglycosylated lectin precursor in acidic conditions. These data are consistent with the observation that both deglycosylation and acidification of the pH are the minimum requirements to convert the inactive precursor into an active lectin.
Key words: Concanavalin A, lectin activity, endoglycosidase.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Concanavalin A (ConA), one of the major seed proteins of jack bean (Canavalia ensiformis), is a lectin studied extensively since its discovery in 1919. ConA presents a high affinity for high-mannose-type N-glycans. Two different binding sites for these glycans were identified. A high affinity site specific for the
-1,6 mannose linked to the core -mannose residue of the trimannosyl moiety of N-glycans (primary site), and a lower affinity for the 1,3 mannose of the same sequence (secondary site) (Bhattacharyya et al., 1987
; Naismith and Field, 1996). ConA has been shown to crystallize as a tetramer of identical 238 amino acid (Mr 25.500) monomers (Edelman et al., 1972). In solution, there is a dimer–tetramer equilibrium that is pH dependent. This lectin is a tetramer above pH 7 and a dimer below pH 6 (McKenzie et al., 1972). Molecular distribution analysis of ConA obtained by sedimentation equilibrium did not indicate the presence of any oligomer species larger than the tetramer. Furthermore, only the dimer and tetramer of ConA are observed by the electrospray ionization mass spectometry (Light-Wahl et al., 1993).
The lectin monomer is synthesized as a 34 kDa glycoprotein precursor, proconcanavalin A (proConA), which is transported from the lumen of the rough endoplasmic reticulum through the Golgi apparatus to the vacuolar compartment (Herman et al., 1985). The comparison of the amino acid sequence of proConA deduced from its cDNA sequence, with the amino acid sequence of mature ConA, indicates that the lectin precursor is processed to a protein with lectin activity through (i) the endoproteolytic cleavages of an internal peptide and a C-terminus peptide, and (ii) the ligation within the original polypeptide in circular permutation (Carrington et al., 1985; Chrispeels et al., 1986; Bowles et al., 1986). This proteolytic processing and the removal of the N-glycan convert the non-active glycoprotein lectin precursor into the carbohydrate binding ConA. Despite its complexity, this processing apparently occurs without a large change in the three-dimensional structure of proConA (Carrington et al., 1985).
Among the different post-translational modifications of ConA, deglycosylation appears to be a key step. In this respect, deglycosylation by a microbial peptide N-glycanase was found to be sufficient for the activation of the lectin precursor (Sheldon and Bowles, 1992). However, peptide N-glycanases isolated from plants are poorly active on native glycoproteins as compared to their high efficiency in glycopeptide deglycosylation (Taga et al., 1984). Recently, the purification and characterization of a jack bean N-glycanase was described, and the enzyme was shown to deglycosylate proConA in vitro (Sheldon et al., 1998). In this paper, the identification of N-glycans associated with the glycoprotein precursor proConA and the in planta characterization of a deglycosylaled form of proConA is reported. Experimental evidence shows that proConA N-glycans are fully accessible and that deglycosylation is necessary but is not sufficient for the activation of the inactive prolectin into a mannose-binding lectin.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Chemicals and materials
Jack bean (Canavalia ensiformis L. DC) seeds were obtained from the Central University of Venezuela. Endoglycosidase H was purchased from Boehringer, Bio-Gel P4 from Bio-Rad, ConA and
-mannosidase (jack bean) were from Sigma and ovalbumin was from Serva. CNBr-Sepharose and ConA-Sepharose were purchased at Pharmacia.
Preparation of seed extracts
Mature and immature (1–2 g fresh weight) jack bean seeds were lyophilized and stored at room temperature until use. Mature seeds were collected 120 d after flowering (daf). Immature seeds were collected between 40 and 50 daf. To study proConA maturation during seed development, flowers of jack bean plants were marked at flowering day in the field. Twenty-eight daf, one fruit was harvested every day until maturation of fruit. Seed cotyledons were homogenized in a cold mortar in phosphate-buffered saline (100 mM phosphate buffer pH 7.4, 145 mM NaCl, 1 mM MgCl2, 1 mM CaCl2). After removal of cell debris and walls by centrifugation (4000 rpmx20 min), the supernatant was either recovered, or frozen until use (crude extract) or used immediately to isolate microsomal (microsomal fraction), cytosolic and protein body proteins (soluble fraction) fractions as described previously (Chrispeels et al., 1986).
Antibody production
Commercial ConA (Sigma) was subjected to preparative SDS-PAGE and the major polypeptide (Mr 30 000) was electro-eluted for 18 h using a Bio Trap (Schleicher & Schüell) electro elution chamber. 200 µg of purified ConA were injected into New Zealand white rabbits subcutaneously with complete Freund's adjuvant. Three other injections with incomplete Freund's adjuvant were done 14, 28 and 42 d after the first injection. The antiserum was collected 8 and 15 d after the last injection. Specific immunoglobulins were then purified by affinity chromatography on a ConA-Sepharose column.
SDS-PAGE, immunoblotting and affinodetection
Polypeptides were separated by SDS-PAGE in a 15% polyacrylamide gel under reducing conditions (according to Laemmli, 1970). After electrophoresis, the proteins were transferred electrophoretically onto a nitrocellulose membrane (Schleicher & Schüell) as described previously (Faye and Chrispeels, 1985). Immunodetection on the blot was carried out using purified antibodies specific for ConA as previously reported (Fitchette-Lainé et al., 1994). Affinodetection of glycoproteins with high-mannose-type N-glycans was carried out using the concanavalin A/peroxidase method as described previously (Faye and Chrispeels, 1987).
Purification of proConA under denaturating conditions
A protein extract from immature seeds was applied to a ConA-Sepharose column (10 ml) equilibrated with 50 mM sodium phosphate buffer pH 7.4 containing 1 mM CaCl2 and 1 mM MgCl2. Bound material was then eluted with 0.2 M -methyl mannoside in the same buffer. Bound proteins were separated by SDS-PAGE on preparative gels. The polyacrylamide gel band containing proConA (Mr 34 kDa) was cut and the prolectin was electro-eluted for 4 h.
Deglycosylation of proConA
Deglycosylation of proConA (0.5 mg) with Endo H (10 mU) was carried out in a 0.1 M sodium acetate buffer pH 5.5, overnight at 37 °C. Free N-glycans were then separated from the polypeptide by gel filtration on a Bio-Gel P4 column (1x40 cm) equilibrated in a 10 mM sodium phosphate buffer pH 7.
Analysis of N-glycans
High pH Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD) was performed on a Dionex DX500 system equipped with a GP50 gradient pump, a ED40 detector and a CarboPac PA1 column (4.6x250 mm). Elution of N-glycans was carried out using a linear gradient from 0–100 mM NaOAc in 100 mM NaOH at 1 ml min-1 over 30 min.
Purification and deglycosylation of native proConA
Immature jack bean seeds (1–2 g fresh weight) were harvested and the microsomal fraction was immediately isolated as described previously (Chrispeels et al., 1986). Native proConA was purified by affinity chromatography on ConA-Sepharose as described above. Native proConA was then desalted by gel permeation on a P4 Bio Gel column. Deglycosylation of native proConA with Endo H (10 mU) was carried out at pH 5.5 and 7.4 in phosphate buffer for 24 or 40 h at 37 °C. For digestion with -mannosidase (Sigma) 0.25 mg native proConA was diluted in 0.5 ml of 50 mM sodium acetate buffer pH 5.8, containing 5 mM ZnSO4 and incubated for 48 h at 37 °C with 10 U jack bean -mannosidase. Before and after digestion fractions were analysed by SDS-PAGE, immunodetection and affinodetection.
Lectin assay
Ovalbumin-Sepharose affinity gel was obtained by coupling purified ovalbumin on CNBr-Sepharose according to the specification of the manufacturer. Commercial ConA, proConA and deglycosylated proConA were preincubated overnight in 10 mM phosphate buffers pH 5.5 or 7.4 and then applied to a ovalbumin–Sepharose column (0.3 ml) in the same buffer (new gel was used for each application). Bound material was then eluted with 0.3 M -methyl mannose. Retained and non-retained protein fractions were then analysed by SDS-PAGE.
In vitro labelling and purification of native proConA
Jack bean cotyledons at mid-maturation (approximately 0.6 g fresh weight) were incubated with radioactive [3H]GlcNH2 as described earlier (Spencer et al., 1980). After 6 h of labelling at 20 °C, 1 mm slice from the radiolabelled surface of each cotyledon were cut with a razor blade and radioactive slices from cotyledons were combined and homogenized in a cold mortar in 100 mM phosphate buffer pH 7.4. Purification of native proConA was carried out as described above.
Analysis of lectin activity of [3H]proConA
Deglycosylation of [3H]proConA with Endo H (10 mU) was carried out in a 0.1 M, pH 5.5, sodium acetate buffer, overnight at 37 °C. Free N-glycans were then separated from the polypeptide by gel filtration on a Bio-Gel P4 column (1x40 cm) equilibrated in a 100 mM sodium phosphate buffer pH 7. The fractions were counted for radioactivity and the deglycosylated [3H]proConA, eluted in the void volume, was incubated at pH 5.5 or at pH 7.4 overnight at 37 °C, and then applied to a ovalbumin–Sepharose column as described above. Retained and non-retained fractions were counted for radioactivity.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
In planta characterization of ConA and of its precursor
The characterization of ConA and its glycoprotein precursor was achieved by affino- and immunodetection on blots on protein extracts prepared either from mature seeds or from immature seeds collected between 32 and 43 d after flowering (daf). Rabbit antibodies directed against ConA were prepared by immunization with commercial ConA purified by preparative SDS-PAGE and electro-elution. After a further purification on a ConA-Sepharose column, the antibodies specifically recognize proConA, ConA and ConA fragments in the protein extracts from both the immature and mature seeds (Fig. 1B
). The ConA fragments detected on blot, are intermediate polypeptides arising from the proteolytic cleavages of proConA during the biosynthesis of the mature ConA (Bowles et al., 1986; Chrispeels et al., 1986). ProConA was detected only in immature seeds confirming that this precursor is completely processed to ConA in protein bodies. Proteins from immature and mature seeds were analysed by affinodetection using the ConA/peroxidase method (Faye and Chrispeels, 1987) for detection of high-mannose-type N-glycans (Fig. 1C). ProConA was found to be revealed in the affinoblot which confirms the presence of high-mannose-type glycans N-linked to the prolectin. In contrast, proConA neither reacts with anti-xylose nor with anti-fucose antibodies (Faye et al., 1993) which indicates that the lectin precursor does not contain complex N-glycans (not shown). Furthermore, an additional polypeptide of 32 kDa is detected by ConA-antibodies in immature seeds (Fig. 1B). This polypeptide, having a slightly higher electrophoretic mobility than proConA, was not affinodetected in Fig. 1C. As a consequence, this additional polypeptide was assigned to the deglycosylated form of proConA.
|
Immunodetection with anti-ConA antibodies and affinodetection of high-mannose-type N-glycans was carried out on microsomal and soluble fractions of immature seeds (not shown). Both the deglycosylated proConA and proConA as well as ConA were recovered in the soluble fractions. In contrast, only proConA was detected in the microsomal fraction, indicating that deglycosylation and then proteolytic maturation of the prolectin both occur in the protein storage vacuole.
N-glycosylation analysis of proConA
Denatured proConA was purified from proteins isolated from immature seeds (40–50 daf) by affinity chromatography on a Con A-Sepharose column followed by an electro-elution of the 34 kDa band from a preparative gel (Fig. 2). Affinity chromatography on immobilized ConA has been widely used for the purification of glycoproteins bearing high-mannose type N-glycans. ProConA was found to be retained on a Con A-Sepharose column and eluted with 0.2 M -methyl mannoside. In addition to proConA, ConA and fragments of ConA were also partially retained by affinity. As purified ConA did not bind to the affinity gel in the same conditions (data not shown), it was concluded that in immature seeds the lectin exists in multimeric forms containing both proConA and ConA. The retention of proConA by recognition of its glycan on the affinity column, results in the copurification of associated ConA polypeptides. Final purification of proConA was achieved by electro-elution from preparative SDS-PAGE resulting in a single polypeptide band immunodetected by anti-ConA antibodies and affinodetected using the ConA/peroxidase method (Fig. 2, lane 3).
|
The presence of high-mannose-type glycans N-linked to proConA was demonstrated by affinodetection (Figs 1
, 2). Consistent with this ‘on blot’ N-glycan characterization, deglycosylation of proConA was carried out with endoglycosidase H (Endo H), an endoglycosidase which cleaves the linkage between the two glucosamine residues of the chitobiose unit, specifically in high-mannose-type N-glycans. The complete deglycosylation of proConA by Endo H was confirmed by both a shift of the polypeptide on SDS-PAGE and the disappearance of any glycan detection by the ConA/peroxidase method after Endo H treatment (not shown), indicating that only Endo H-sensitive oligosaccharides are N-linked to proConA. Free N-glycans released from proConA were isolated by gel filtration and analysed by High pH Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD) (Fig. 3). Two N-glycans were detected and identified as the high-mannose-type Man8GlcNAc and Man9GlcNAc by comparison of their retention time with the ones of homologous structures isolated from a mammalian glycoprotein (Rayon et al., 1996).
|
N-glycan accessibility in native proConA
In order to analyse the accessibility of glycans N-linked to proConA, native proConA was purified in a one step procedure by affinity chromatography of a microsomal protein extract isolated from immature seeds on a ConA-Sepharose column. As illustrated in Fig. 4, only native proConA was found to be retained on the gel. This indicates that the Man8–9GlcNAc2 N-linked to proConA are fully accessible to the immobilized lectin. N-glycan accessibility on native proConA was also investigated by measuring their sensitivity to deglycosylating enzyme, such as -mannosidase and Endo H. After incubation of proConA with -mannosidase, proConA N-glycans lost their reactivity with ConA. The accessibility of N-glycans on native proConA was confirmed after incubation with Endo H (Fig. 5). The rate of deglycosylation was estimated by SDS-PAGE after 24 h and 40 h of incubation with the enzyme. As illustrated in Fig. 5, proConA is fully deglycosylated after denaturation and a 24 h incubation with Endo H either at pH 5.5 or at pH 7.4. When native proConA was incubated with Endo H, the efficiency of the deglycosylation was found to be pH dependent. Although the pH-optimun of the enzyme is at pH 5–6 (Boehringer Mannheim), Endo H appears to be less active on native proConA at pH 5.5 than at pH 7.4. This result clearly point out at pH-dependent changes in the accessibility of the core of proConA N-glycans. These changes are indirect evidence for pH-dependent conformational modifications of the prolectin whose N-glycans are more buried inside the protein at pH 5.5 than at pH 7.4.
|
|
Activation of proConA by deglycosylation
The major event which converts inactive proConA into active ConA has been investigated. Involvement of both deglycosylation and pH were evaluated by affinity chromatography on ovalbumin–Sepharose. As previously reported (Bowles et al., 1986; Chrispeels et al., 1986) it was also observed here that native proConA has no affinity for N-glycans at pH 5.5 or at pH 7.4 (Fig. 6). The activation of proConA was investigated when deglycosylation by Endo H was performed at pH 5.5 or at pH 7. (Fig. 4, panels A and B, respectively). On panel A (Fig. 6), the prolectin was first deglycosylated at pH 5.5 and one half of deglycosylated proConA was loaded on the ovalbumin–Sepharose column in the same pH condition, while the other half was ajusted to pH 7.4 and loaded on a second ovalbumin column previously equilibrated at pH 7.4. Results reported in Fig. 6A indicates that, as observed for glycosylated proConA, deglycosylated proConA does not show any affinity for ovalbumin at pH 7.4. In contrast, at pH 5.5, 30–40% of the deglycosylated proConA was found to be activated into a lectin. To confirm these results, deglycosylation was carried out at pH 7.4 (Fig. 6B). Data obtained in these conditions confirm that deglycosylation by itself is not sufficient to activate proConA into an active lectin. Indeed, it is clearly illustrated that when proConA is deglycosylated at pH 7.4 and maintained at this pH during affinity chromatography, the deglycosylated proConA does not show any affinity for ovalbumin. In contrast, acidification to pH 5.5 after deglycosylation at pH 7.4 revealed the lectin activity of deglycosylated proConA.
|
As proConA present N-glycans having high affinity for ConA, self-association between the N-glycan and the lectin site of the prolectin was suggested as an explanation for absence of lectin activity in proConA (Bowles, 1993
). To investigate this possibility, radiolabelled proConA was prepared by incubation of immature seeds with radioactive [3H]GlcNH2. Purified native [3H]proConA was treated with Endo H and then the digest was submitted to gel permeation to separate free N-glycan from the deglycosylated prolectin. Consistent with Endo H cleavage site between the two GlcNAc residues of the chitobiose unit of N-glycans, the radioactivity was found to be divided into two equal fractions, one half of the radioactivity being retained on the deglycosylated proConA while the other half was eluted with the free N-glycans. Deglycosylated proConA was further applied to affinity columns of ovalbumin–Sepharose either at pH 5.5 or 7.4. As observed above in Fig. 6, about 40% of [3H]-labelled deglycosylated proConA was retained on the ovalbumin column at pH 5.5 while deglycosylated proConA remained inactive at pH 7.4. These results clearly illustrate that an interaction between the lectin binding site and the glycan N-linked to the prolectin is not the reason why glycosylated proConA is inactive and deglycosylated proConA has lectin activity.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
As previously reported (Herman et al., 1985
; Sheldon et al., 1998), proConA was found to be N-glycosylated by high-mannose-type N-glycans. Analysis by HPAEC-PAD of the N-glycosylation of proConA has shown that the prolectin only bears Man8GlcNAc2 and Man9GlcNAc2. These N-glycans result from the processing of the N-glycan precursor in the ER by action of -glucosidases I and II and an ER-mannosidase (Lerouge et al., 1998). This limited processing could result either from the absence of N-glycan maturation machinery in jack bean seeds or from a low accessibility of proConA N-glycans to Golgi processing enzymes as previously illustrated for phytohemagglutinin (Faye et al., 1986). Recently, the structure of the glycan N-linked to jack bean -mannosidase was characterized as a fucosylated and xylosylated oligosaccharide (Kimura et al., 1999). This structure results from Golgi modifications which indicates that jack bean seeds contain all the glycan machinery required for conversion of high-mannose-type N-glycans into highly processed oligosaccharides. As a consequence, the accessibility of the oligosaccharides N-linked to proConA by affinity chromatography on ConA-Sepharose and by action of exo- or endoglycosidases was then investigated. Both approaches clearly show that Man8–9GlcNAc2 oligosaccharides are fully accessible in native proConA at pH 7.4. However, the decreased accessibility of the core of the N-glycans to Endo H after acidification to pH 5.5 indicates that changes in the prolectin folding in these pH conditions probably explains why proConA N-glycans are not maturated into complex structures when the prolectin is transported downstream by the secretory pathway. Indeed, it is generally accepted that pH decreases progressively from the ER to the Golgi apparatus, from the early to the late Golgi compartment and from the Golgi apparatus to the vacuole.
Removal of N-glycan from proConA is a key step in its conversion into an active lectin. It was previously suggested that proConA N-glycans could interact with the lectin binding site of the same or adjacent proConA monomers which could result in a self-inactivation process (Bowles, 1993). In this study using proConA labelled in the chitobiose unit of N-glycans it has been shown that glycans are not associated to the prolectin through linkages other than Endo H-sensitive ones between high-mannose oligosaccharides and Asn residues.
Several modifications such as post-translational proteolytic cleavages and ligation of peptides occurs in addition to deglycosylation when the prolectin is maturated into the active ConA. The in vitro activation of proConA after deglycosylation (Sheldon and Bowles, 1992) has focused the interest on the deglycosylation event in the proConA activation process. This study illustrates from a detailed glycan structural analysis and from glycosidase treatments that not only PNGases but also Endo H-type enzymes are sufficient to deglycosylate native proConA. Indeed, PNGases will generally cleave all N-glycan types from glycoproteins while Endo H greater specificity for high-mannose oligosaccharides is also perfectly consistent with the cleavage of Man8–9GlcNAc2 N-glycans identified on proConA. Both Endo H and PNGase activities have been characterized in jack bean (Yet and Wold, 1988). In planta deglycosylation of proConA by an Endo H-type enzyme looks more consistent than a deglycosylation with a PNGase-type enzyme. Indeed, native glycoproteins harbouring high-mannose-type N-glycans, and particularly proConA as illustrated in this study, are efficiently deglycosylated by Endo H. This is not the case with PNGase-type enzymes which are mostly active on glycopeptides and cleave N-glycans from native glycoproteins with a low efficiency (Taga et al., 1984). While it requires a high number of processing events involving deglycosylation, proteolysis and post-translational ligation of peptides, proConA processing to mature ConA is a very fast procedure which is not consistent with an in vivo deglycosylation depending on a low efficiency enzyme such as PNGase when this enzyme has native glycoproteins as substrates.
Interestingly, it has been shown that removal of the N-glycan from proConA is not sufficient to activate the lectin precursor. Indeed, while glycosylated proConA has no lectin activity either at pH 5.5 or at pH 7.4, deglycosylated proConA is converted into an active lectin only at pH 5.5 while it remains inactive when it is deglycosylated and kept at pH 7.4. This result and the differences in the N-glycan accessibility observed in the same pH range indicate that removal of the proConA N-glycan is necessary to refold the precursor into an active lectin. However, proConA deglycosylation is not sufficient to reveal the lectin activity when the pH conditions are not in the range of vacuolar content. Such an activation could be related to a dimer–tetramer equilibrium of the deglycosylated proConA in pH 5–7 range as observed for mature ConA (McKenzie et al., 1972).
The recent expression of proConA in transgenic BY2 tobacco cells will help to characterize whether the prolectin maturation machinery is specific to jack bean or is highly conserved in plants as shown for deglycosylating enzymes such as PNGases and endoglycosidases.
|
Acknowledgments |
|---|
This work was supported by the University of Rouen and the Centre National de Recherche Scientifique. This study was also supported by grants from Centro de Desarrollo Científico y Humanístico, Universidad Central de Venezuela, Consejo National de Investigacion Cientifica y Tecnologica (CONICIT-Venezuela) and Embassade of France in Venezuela.
|
Notes |
|---|
1 Present address: Instituto de Genética, Facultad de Agronomía, Universidad Central de Venezuela, Maracay, Aragua, Venezuela.
2 To whom correspondence should be addressed. Fax: +33 2 35 14 67 87. E-mail: lfaye{at}crihan.fr
|
Abbreviations |
|---|
ConA, concanavalin A; Endo H, endoglycosidase H; HPAEC-PAD, High pH Anion Exchange Chromatography with Pulsed Amperometric Detection; proConA, proconcanavalin A; PNGase, peptide-N-glycanase.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Bhattacharyya L, Ceccarini C, Lorenzoni P, Brewer F. 1987. Concanavalin A interactions with asparagine-linked glycopeptides. Journal of Biological Chemistry 262, 1288–1293.
Bowles DJ. 1993. Post-translational processing of concanavalin A. In: Battey NH, Dickinson HG, Hetherington AM, eds. Post-translational modifications in plants. Cambridge University Press, 257–266.
Bowles DJ, Marcus S, Pappin DJC, Findlay JB, Eliopoulos E, Maycox PR, Burgess J. 1986. Posttranslational processing of concanavalin A precursors in jackbean cotyledons. Journal of Cell Biology 102, 1284–1297.
Carrington DM, Auffret A, Hanke DE. 1985. Polypeptide ligation occurs during post-translational modification of concanavalin A. Nature 313, 64–67.[Medline]
Chrispeels MJ, Hartl PM, Sturm A, Faye L. 1986. Characterization of the endoplasmic reticulum-associated precursor of concanavalin A. Journal of Biological Chemistry 261, 10021–10024.
Edelman GM, Cunnigham BA, Reeke GN, Becker JW, Waxdal MJ, Wang JL. 1972. The covalent and three dimensional structure of concanavalin A. Proceedings of the National Academy of Sciences. USA 69, 2580–2584.
Faye L, Chrispeels MJ. 1985. Transport and processing of the glycosylated precursor of concanavalin A in jack bean. Planta 170, 217–224.
Faye L, Chrispeels MJ. 1987. Characterization of N-linked oligosaccharides by affinoblotting with concanavalin A-peroxidase and treatment of the blots with glycosidases. Analytical Biochemistry 149, 218–224.
Faye L, Gomord V, Fitchette-Lainé A-C, Chrispeels MJ. 1993. Affinity purification of antibodies specific for Asn-linked glycans containing 1–3 fucose or ß 1–2 xylose. Analytical Biochemistry 209, 104–108.[Web of Science][Medline]
Faye L, Sturm A, Bollini R, Vitale A, Chrispeels MJ. 1986. The position of the oligosaccharides side-chains of phytohemagglutinin and their accessibility to glycosidases determines their subsequent processing in the Golgi. European Journal of Biochemistry 158, 655–661.[Web of Science][Medline]
Fitchette-Lainé A-C, Gomord V, Chekkafi A, Faye L. 1994. Distribution of xylosylation and fucosylation in the plant Golgi apparatus. The Plant Journal 5, 673–682.
Herman EM, Shannon LM, Chrispeels MJ. 1985. Concanavalin A is synthesized as a glycoprotein precursor. Planta 165, 23–29.
Kimura Y, Hess D, Sturm A. 1999. The N-glycans of jack bean -mannosidase. European Journal of Biochemistry 264, 168–175.[Web of Science][Medline]
Laemmli U. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–687.[Medline]
Lerouge P, Cabanes-Macheteau M, Rayon C, Fitchette-Lainé A-C, Gomord V, Faye L. 1998. N-glycoprotein biosynthesis: recent development and future trends. Plant Molecular Biology 38, 31–48.[Web of Science][Medline]
Light-Wahl KL, Winger BE, Smith RD. 1993. Observation of the multimeric forms of concanavalin A by Electrospray Ionization Mass Spectrometry. Journal of American Chemical Society 115, 5869–5870.
McKenzie GH, Sawyer WH, Nichol LW. 1972. The molecular weight and stability of concanavalin A. Biochemica et Biophysica Acta 263, 283–293.
Naismith JH, Field RA. 1996. Structural basis of trimannoside recognition by concanavalin A. Journal of Biological Chemistry 271, 2972–2976.
Rayon C, Gomord V, Faye L, Lerouge P. 1996. N-glycosylation of phytohemagglutinin expressed in bean cotyledons or in transgenic tobacco cells. Plant Physiology and Biochemistry 64, 273–281.
Sheldon PS, Bowles DJ. 1992. The glycoprotein precursor of concanavalin A is converted to an active lectin by deglycosylation. EMBO Journal 11, 1297–1301.[Web of Science][Medline]
Sheldon PS, Keen JN, Bowles DJ. 1998. Purification and characterization of N-glycanase, a concanavalin A binding protein from jackbean (Canavalia ensiformis). Biochemistry Journal 330, 13–20.
Spencer D, Higgins TJV, Button SC, Davey RA. 1980. Pulse-labeling studies on protein synthesis in developing pea seeds and evidence for a precursor form of legumin small subunit. Plant Physiology 66, 510–515.
Taga EM, Waheed A, van Etten RL. 1984. Structural and chemical characterization of a homogeneous peptide: N-glycosidase from almond. Biochemistry 23, 815–822.[Medline]
Yet MG, Wold F. 1988. Purification and characterization of two glycopeptide hydrolases from jack beans. Journal of Biological Chemistry 263, 118–122.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  S.-J.-D. Claude, G. Marie-Agnes, R. Catalina, P. Nadine, K.-M. Marie-Christine, N. Jean-Marc, F. Loic, and G. Veronique Targeting of proConA to the Plant Vacuole depends on its Nine Amino-acid C-terminal Propeptide Plant Cell Physiol., October 1, 2005; 46(10): 1603 - 1612. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||