The saccharide chain of lupin seed conglutin gamma is not responsible for the protection of the native protein from degradation by trypsin, but facilitates the refolding of the acid-treated protein to the resistant conformation.

Native glycosylated and enzymically deglycosylated conglutin gamma (a lupin seed oligomeric protein) both showed an unusual resistance to tryptic degradation. The result of this treatment was that a single 40-residue peptide was cleaved from the N-terminus of conglutin gamma light subunit. Acid treatment of the two protein forms led to their substantial unfolding, as indicated by CD spectra. After this treatment, both polypeptides were completely degraded by trypsin after a few minutes of incubation. Conversely, trypsin pulse experiments run under renaturing conditions demonstrated a different refolding behaviour of the two proteins: the glycosylated form became resistant to trypsin after a 7-h renaturation, while the deglycosylated form required 42 h renaturation. These results were confirmed by CD spectra and reverse-phase HPLC analyses of the glycosylated and deglycosylated conglutin gamma forms. Therefore, it was concluded that the saccharide chain of conglutin gamma increased the rate of formation of a trypsin-resistant conformation upon refolding of the acid-treated protein, without playing any direct role in the protection of the native protein from proteolysis.

Heat-induced synthesis and tunicamycin-sensitive secretion of the putative storage glycoprotein conglutin gamma from mature lupin seeds.

SDS/PAGE, immune 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 degrees C for about 3 h was conglutin gamma, a putative storage glycoprotein already present in the protein bodies of mature seeds. Incorporation of [14C]leucine into the protein demonstrated that conglutin gamma was newly synthesized during the treatment and the use of protein synthesis inhibitors ruled out the secretion of constitutive conglutin gamma. Synthesis and secretion took place only over a narrow temperature range, 57.5-62.5 degrees C, and in a short time interval, 135-180 min, of incubation of the seed. The amount of secreted conglutin gamma, i.e. 1 mg/seed, was about three times that present inside the treated or untreated seed. Secreted conglutin gamma contained covalently linked carbohydrate as well as the constitutive protein. Inhibition of the glycosylation by tunicamycin did not affect conglutin gamma synthesis, but prevented its secretion from the seed, as indicated by quantifying conglutin gamma 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 gamma with trypsin and pepsin revealed no difference between the two proteins. Lupin seeds were still viable after the treatment. However no similarities between conglutin gamma and heat-shock proteins were observed either in the amino acid sequence or other molecular features.