Glycoprotein II The Isolation and Characterization of a Major Antigenic and Non-Haemagglutinating Glycoprotein from Phaseolus Vulgaris

The glycoprotein nature of legumin and vicilin, the reserve globulins in the cotyledons of Pisum sativum was studied. Legumin from mature seed was found to contain 1% neutral sugars (mannose and glucose) and 0.1% amino sugar (glucosamine), whereas vicilin contained 0.3% neutral sugar (mannose) and 0.2% amino sugar (glucosamine). On the basis of the incorporation of 'IC-labeled glucosamine, it appeared that not alH of the component subunits of the reserve proteins are glycosylated to the same extent. In addition, it has been established that glycosylation occurs after peptide synthesis. During seed development there was a change in neutral sugars and amino sugar ratio in vicilin. During germination, the neutral sugars and the amino sugar content of the glycoproteins declined. These findings are discussed in relation to the synthesis and degradation of the glycosyl component of the glycoproteins.Glucosamine has been reported to be a constituent of the glycoproteins of many seeds (10). Additionally, on the basis of the incorporation of radioactive glucosamine, this amino sugar is apparently a constituent of the glycoproteins occurring in root tissue (15). The glucosamine containing glycoproteins from the seeds of Phaseolus vulgaris (11)(12)(13)(14) and Phaseolus aureus (3) have been characterized and they correspond in part to the cotyledonary reserve proteins. As part of our studies on the metabolism of the reserve proteins of Pisum sativum, we have investigated the glycoprotein nature of legumin and vicilin. Similar to Phaseolus, the reserve proteins of Pisum sativum have been found to contain glucosamine and mannose. We have followed the changes in the glucosamine-containing glycoproteins during seed development and germination. These changes are described below. (20). The amino sugar content in the remaining seven fractions was determined by the modified Rondle and Morgan method (16). For this purpose, the 2-ml fractions of the eluate were neutralized to the phenolphthalein end point. One ml of acetyl acetone reagent (2% acetyl acetone in 0.5 N Na2CO3) was added, and the mixture was heated in a boiling water bath for 20 min and cooled to room temperature. One ml of Ehrlich's reagent (0.8 g of recrystalized p-dimethylaminobenzaldehyde in 30 ml of ethanol and 30 ml of concentrated HCI) was added; the mixture was heated for 10 min at 60 to 65 C, cooled to room temperature, and the absorbance was measured at 530 nm. Glucosamine hydrochloride was used as a standard. Identification of Carbohydrates. One hundred ul of the concentrated samples of neutral and amino sugar fractions were spotted on Whatman No. 1 chromatogram paper strips of 6.25 cm width, and the sugars were separated by single dimensional descending chromatography using the solvent system I-propanol-ethyl acetate-water (7:4:2) as recommended by Koshiyama (6). After 18 hr, the paper strips were dried and rechromatographed once again in the same direction with the same solvent. After another 18 hr, the paper strips were dried at 90 C for 5 min and...

Section: Discussionmentioning

confidence: 90%

mentioning

confidence: 99%

Glycoprotein Metabolism in the Cotyledons of Pisum sativum during Development and Germination

Basha¹,

Beevers²

1976

“…It is surprising, however, for the P. vulgaris system, because although endogenous phaseolin was an acceptor of GlcNAc in vitro (4), we could find no significant terminal GlcNAc in phaseolin from mature seeds. It is possible that, in vivo, terminal GlcNAc residues may be subsequently removed in a processing event, analogous to those described for terminal mannose and glucose in other systems (for examples, see 23).…”

Section: Discussionmentioning

confidence: 97%

Two Kinds of Protein Glycosylation in a Cell-Free Preparation from Developing Cotyledons of Phaseolus vulgaris

Davies¹,

Delmer²

1981

Membrane preparations from developing cotyledons of red kidney bean (Phaseolas vulgaris L) transferred radioactive mannose from GDP-mannose (U-I'4Clmannose) to endogenous acceptor proteins. The transfer was inhibited by the antibiotic tunicamycin, suggesting the involvement of lipidoLigosaccharide intermediates typical of the pathway for glycosylation of asparagine residues. This was supported by the similarity of the linkage types of radioactive mannose in lipid-oligosaccharide and glycoprotein products; both contained labeled 2-linked mannose, 3,6-inked and terminal mannose typical of glycoprotein "core" oligosaccharides. As expected for "core" glycosylation, the transfer of labeled N-acetylgucosamine (GlcNAc) from UDP-GlcNAc (613HIGLcNAc) to 4-linkage in endogenous glycoproteins could also be demonstrated. However, most of the radioactive GlcNAc was incorporated into terminal linkage, in a reaction insensitive to tunicamycin. The proteins receiving "core" oligosaccharide in vitro were heterogeneous in size, in contrast to those receiving most of the GkcNAc (which chiefly comprised the seed reserve-proteins phaseolin and phytohemagglutinin). It is suggested that following "core" glycosylation, single GkcNAc residues are attached terminally to the oligosaccharides of these seed proteins, without the involvement of lipid-linked intermediates. Phaseolin from mature seeds does not possess a significant amount of terminal GIcNAc and so it is possible that these residues are subsequentiy removed in a processing event.cotyledons, is the identity of the endogenous glycoprotein products. Previous studies from our laboratory showed that crude cellfree extracts from this tissue synthesized lipid-linked GlcNAc4, dolichol monophosphoryl mannose, and lipid-oligosaccharide when supplied with UDP-GlcNAc and GDP-mannose, and also transferred GlcNAc and mannose to endogenous acceptor proteins (5, 9, 10). The lipid-linked sugars showed turnover, consistent with their roles as intermediates in protein glycosylation (9, 10). Recently we obtained evidence that the endogenous acceptor proteins for the in vitro transfer of GlcNAc are chiefly the principal reserve proteins of this seed, i.e. phaseolin5 and phytohemagglutinin (4). Both of these proteins contain typical asparagine-linked, "high mannose" type, oligosaccharide groups containing GlcNAc and mannose as sole constituent sugars (10, 17). We therefore expected concomitant incorporation of GlcNAc and mannose, via transfer of "core" oligosaccharides from lipid carriers in the usual pathway (7,22). However, in contrast to the results with GlcNAc, only a trace of incorporation of mannose into phaseolin could be shown (4). We suggested, therefore, the bulk of the transfer of GlcNAc to phaseolin and PHA did not represent "core" glycosylation. In this paper we present confirmatory evidence ofthis, demonstrating that two apparently independent kinds of glycosylation occur in this in vitro system, i.e. the attachment of GlcNAc residues to the identifiable (glyco)proteins phaseol...

“…Only a few of these glycoproteins have been characterized (12,23). Recently, Pusztai and Watt (18) (19) observed a similar protein in the cotyledons of this species and suggested that it was a storage protein. However, they were unable to show that either the protein or the glucosamine disappeared from the cotyledons during germination and subsequent growth.…”

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confidence: 96%

Isolation and Characterization of Glucosamine-containing Storage Glycoproteins from the Cotyledons of Phaseolus aureus

Ericson¹,

Chrispeels²

1973

Cotyledons of Phaseolus aureus contain protein-bound glu. cosamine which is metabolized during germination. The glucosamine is present in storage glycoproteins, and these are concurrently metabolized along with the glucosamine. These glycoproteins are associated with protein bodies. Characterization of the glucosamine-containing storage proteins showed them to be identical with vicilin and legumin, the major storage proteins of the Leguminosae. Extraction of Proteins. The cotyledons were homogenized either in 0.4 M NaCl or in 20 mm sodium borate, pH 9.0, using 2 ml of medium per g of tissue. The homogenate was centrifuged at 30,000g for 20 min, and the supernatant was collected. The pellet was extracted three times with the same medium, and the extracts were combined with the initial supernatant.Quantitative Determination of Protein. Proteins were usually assayed according to the procedure of Lowry et al. (13) using bovine serum albumin as a standard. The protein content of peak III glycoprotein was also determined with ninhydrin (27) after acid hydrolysis of the protein.Quantitative determination of hexosamine. The samples to be assayed for protein-bound hexosamine were precipitated with 7.5% trichloroacetic acid, washed with 5% trichloroacetic acid, alcohol, and ether. The dried samples were resuspended in 1 N HCI. Samples to be assayed for total hexosamine were homogenized in 1 N HCl. Hydrolysis was carried out at 90 C for 24 hr. Under these conditions there was no detectable loss of hexosamine. Hexosamines were separated from interfering chromogens by passage through a small Dowex 50 cation exchange column according to the method of Boas (4). This step is absolutely necessary to obtain reproducible results. Eluates from the columns were dried by evaporation under vacuum at 85 C, and the residue was dissolved in water. The hexosamine content was determined by the method of Elson-Morgan as modified by Johnson (11