Concanavalin A is synthesized as a glycoprotein precursor

Abstract

Concanavalin A (Con A) is a tetrameric lectin which is synthesized in the cotyledons of developing jack-bean (Canavalia ensiformis (L.) D.C.) seeds and accumulates in the protein bodies of storage-parenchyma cells. The polypeptides of Con A have a molecular weight of 27000 and a relative molecular mass (Mr) of 30000 when analyzed by gel electrophoresis on denaturing polyacrylamide gels. In-vitro translation of RNA isolated from immature jack-bean cotyledons shows that Con A is synthesized as a polypeptide with Mr 34000. In-vivo pulse labeling of cotyledons with radioactive amino acids or glucosamine also resulted in the formation of a 34000-Mr polypeptide. In-vivo labeling with radioactive amino acids in the presence of tunicamycin yielded an additional polypeptide of 32000 Mr. Together these results indicate that Con A is cotranslationally processed by the removal of a signal sequence and the addition of an oligosaccharide side chain of corresponding size. Analysis of the structure of the oligogosaccharide side chain was accomplished through glycosidase digestion of glycopeptides isolated from [(3)H]glucosamine-labeled Con A. Incubation of the labeled glycopeptides with endoglycosidase H, α-mannosidase or β-N-acetylglucosaminidase, followed by gel filtration, allowed us to deduce that the oligosaccharide side chain of pro-Con A is a high-mannose oligosaccharide. Pulse-chase experiments with labeled amino acids are consistent with the interpretation that the glycosylated precursor of Con A is processed to mature Con A (Mr=30000). The 4000 decrease in Mr is interpreted to result from the removal of a small glycopeptide. The implications of the conversion of a glycoprotein pro-Con A to mature Con A are discussed in the context of the unique circular permutation of the primary structure of Con A.

Purification and characterization of N-glycanase, a concanavalin A binding protein from jackbean (Canavalia ensiformis)

“…The presence of a GlcNAc # Man ( glycan on the precursor would give a theoretical molecular mass change of 1541 Da on deglycosylation, which lies within the predicted accuracy of the measured values. This value of 1550 Da, corresponding to an oligosaccharide composition of GlcNAc # Man ( , contrasts with a previous report that the Nglycan on the Con A precursor had a composition of GlcNAc # Man * [7]. This discrepancy suggests that trimming by α-mannosidase of outer mannose residues has taken place, either during seed development, in precursor purification or in storage.…”

“…The jackbean lectin, Con A, is synthesized as an inactive Nlinked glycoprotein, which undergoes a number of processing events involving deglycosylation, proteolysis and post-translational ligation of peptides to yield the mature protein [3][4][5][6][7][8][9][10][11][12]. Previously it has been shown that the essential step in converting the inactive precursor into an active lectin is deglycosylation [13].…”

“…Con A biosynthesis is complex, involving virtually unprecedented processes [3][4][5][6][7][8][9][10][11][12]. It is thought that the nascent polypeptide is co-translationally segregated into the endoplasmic reticulum, glycosylated and the signal sequence cleaved to form a glycoprotein precursor which does not possess lectin activity [5,9].…”

The glycoprotein precursor of concanavalin A is converted to an active lectin by deglycosylation.

“…At seed maturity the lectin is located in protein bodies of the storage parenchyma cells of the cotyledons, where it constitutes 20% of the total protein (Agrawal and Goldstein, 1976;Bowles, 1990b). Accumulation of Con A in the seed starts during the midstages of development when the newly synthesized protein is transported from the lumen of the rough endoplasmic reticulum (RER) through the Golgi apparatus to the vacuolar compartment (Herman and Shannon, 1984;Herman et al, 1985;Maycox et al, 1988). Protein loading into the vacuole is accompanied by progressive fragmentation of the compartment to give rise to the protein bodies (Bowles 1990a).…”

Hippophae rhamnoides N-glycoproteome analysis: a small step towards sea buckthorn proteome mining

“…Concanavalin A precursor (spot 46), a precursor of nonenzymatic carbohydrate binding protein (lectin) present in the endoplasmic reticulum (Herman et al 1985) is a highmannose oligosaccharide which is processed to form mature con A which is non-glycosylated (Sheldon and Bowles 1992). Con A precursor in elute may be from the column itself as mature non-glycosylated con A is derived from its glycosylated precursor which upon modification forms the lectin (Chrispeels et al 1986).…”