The Saccharide Chain of Lupin Seed Conglutin γ 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

Abstract

Native glycosylated and enzymically deglycosylated conglutin γ (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 γ 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 γ forms. Therefore, it was concluded that the saccharide chain of conglutin γ 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.

Antimicrobial and Functional Properties of Duckweed (Wolffia globosa) Protein and Peptide Extracts Prepared by Ultrasound-Assisted Extraction

“…Burgos-Diaz et al [ 11 ] reported that the positive charge of proteins interacted with the negative charge of polysaccharides to form a stable electrostatic complex when the pH decreased below the pI of proteins, resulting in improvement of protein solubility. The oligosaccharide chain of polysaccharides also contributed to the solubility of proteins [ 27 ].…”

Direccionamiento de proteínas recombinantes a vacuolas de reserva

“…Para probar este impacto, se han utilizado diferentes enfoques, como el uso del antibiótico tunicamicina que inhibe la síntesis del precursor de glicanos (Faye and Chrispeels, 1989) o, el estudio de glicoproteínas donde el sitio de N-glicosilación ha sido removido por mutagénesis sitio dirigida. Varias proteínas candidato, han sido secretadas bajo estas condiciones (Ravi et al, 1986;Faye and Chrispeels, 1987;Driouich et al, 1989;Cordewener et al, 1991;Duranti et al, 1995), en lugar de ser retenidas en el ER (Faye and Chrispeels, 1987) o han formado agregados (Ceriotti et al, 1998). Sin embargo en algunos casos las proteínas no-glicosiladas fueron correctamente dirigidas y permanecieron activas (Sonnewald et al, 1990;Wilkins et al, 1990;Bustos et al, 1991;Santino et al, 1992).…”