The roles of the N-linked glycans and extension regions of soybean beta-conglycinin in folding, assembly and structural features

beta-Conglycinin, one of the dominant storage proteins of soybean, is a tri mer composed of three subunits, alpha, alpha' and beta. All subunits are N- glycosylated and alpha and alpha' contain extension regions in addition to the core regions common to all subunits. Non-glycosylatcd individual subuni ts and deletion mutants (alpha(c) and alpha'(c)) lacking the extension regi ons of alpha and alpha' were expressed in Escherichia coli. All recombinant proteins were purified to near homogeneity and appeared to have the correc t conformation, as judged by CD, density-gradient centrifugation and gel-fi ltration profiles, indicating that the N-linked glycans and extension regio ns are not essential for the folding and the assembly into trimers of beta- conglycinin. Density-gradient centrifugation, gel-filtration and differenti al scanning calorimetry profiles of the recombinant proteins and the native beta-conglycinin indicated that the N-linked glycans and extension regions contribute to the dimension of beta-conglycinin but not to the density and the thermal stability. Comparing the solubilities of the individual subuni ts with those of deletion mutants, only the alpha and alpha' subunits were soluble at lower ionic strength (mu < 0.25) at around the pH value of the e ndoplasmic reticulum. This suggests that the extension regions play an impo rtant role in the prevention of aggregation in the endoplasmic reticulum in analogy with the N-linked glycans.