Heat‐induced synthesis and tunicamycin‐sensitive secretion of the putative storage glycoprotein conglutin γ from mature lupin seeds

Abstract

) -E.IB 94 0179/1 SDSRAGE, inmune blotting with specific antibodies and amino acid sequence analyses revealed that 90% of the protein released from Lupinus albus seeds incubated in water at 60°C for about 3 h was conglutin y , a putative storage glycoprotein already present in the protein bodies of mature seeds. Incorporation of ['4C]leucine into the protein demonstrated that conglutin y was newly synthesized during the treatment and the use of protein synthesis inhibitors ruled out the secretion of constitutive conglutin y. Synthesis and secretion took place only over a narrow temperature range, 57.5 -62.5 "C, and in a short time interval, 135 -180 min, of incubation of the seed. The amount of secreted conglutin y, i.e. 1 mg/seed, was about three times that present inside the treated or untreated seed.Secreted conglutin y contained covalently linked carbohydrate as well as the constitutive protein. Inhibition of the glycosylation by tunicamycin did not affect conglutin y synthesis, but prevented its secretion from the seed, as indicated by quantifying conglutin y remaining in the seed. An accumulation of the protein outside the protein bodies and at the cotyledonary cell periphery was shown in these samples by immunocytochemistry.Peptide mapping of the fragments obtained by incubation of constitutive and secreted conglutin y with trypsin and pepsin revealed no difference between the two proteins.Lupin seeds were still viable after the treatment. However no similarities between conglutin y and heat-shock proteins were observed either in the amino acid sequence or other molecular features.The essential role of seed storage proteins is to serve as nitrogen and carbon skeleton sources for the plantlet developing during germination [l].In lupin seeds, storage proteins are mainly represented by legumin-and vicilin-like proteins, also named 11s and 7s globulins respectively, which together account for about 80% of total globulins [2]. Other minor fractions have been found and some of them have also been isolated and partially char- Like most seed storage proteins, conglutin y is synthesized during early seed development as a precursor polypeptide of about 50 kDa, which is soon proteolytically cleaved to mature subunits [6, 71 and deposited in the protein bodies of lupin cotyledonary cells [7-91. However the amino acid Correspondence to M. Duranti,

Monoclonal Antibodies against the Candidate Lupin Allergens α-Conglutin and β-Conglutin

“…In the present study, the α- and β-conglutins were successfully separated from the total globulin fraction by anion exchange chromatography. Since the amount of γ-conglutin obtained by this procedure was small, a heat treatment of the lupin seeds was performed [32,] resulting in sufficient amounts of the protein for subsequent studies. Furthermore, the isolated globulin fractions corresponded in their molecular size to those reported by others [24, 31, 32, 36].…”

“…The release of γ-conglutin was induced by the heat treatment of mature L. angustifolius seeds as described in the literature [32]. In brief, dry seeds were soaked in deionized H 2 O (1:10 w/v) and heated for 4 h at 60°C under gentle shaking.…”

Soybean Basic 7S Globulin: Subunit Heterogeneity and Molecular Evolution

“…Like Bg, conglutin and carrot XEGIP bind insulin and leginsulin 2,17,22) and thus may be involved in signal transduction. Both conglutin and carrot XEGIP are synthesized in the responses of plants to abiotic stresses (heat treatment 23) and mechanical wounding 24) ). It would be interesting to test whether Bg and conglutin retain any residual xylanase inhibitory activity.…”

Molecular identification and chromosomal localization of genes encoding Triticum aestivum xylanase inhibitor I-like proteins in cereals

“…Moreover, a general protective role of XEGIP against biotic or abiotic stresses can be assumed, based on its wide expression in tomato vegetative tissues and high expression in cultured cells (Qin et al 2003). Leguminous seed proteins Bs7s and Cg have been considered as storage proteins, but more recent research indicates an additional physiological role as enhanced expression is observed in the response to heatshock (Duranti et al 1994;Kagawa and Hirano 1987) and both proteins have the capacity to bind specific proteins or peptides (Komatsu and Hirano 1991;Duranti et al 1995). Chromosomal location of TAXI-like genes PCR on nulli-tetrasomic lines located the identified TAXI-I sequence on wheat chromosome 3B of cv.…”