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 trimer 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-glycosylated individual subunits and deletion mutants (alpha(c) and alpha'(c)) lacking the extension regions of alpha and alpha' were expressed in Escherichia coli. All recombinant proteins were purified to near homogeneity and appeared to have the correct conformation, as judged by CD, density-gradient centrifugation and gel-filtration profiles, indicating that the N-linked glycans and extension regions are not essential for the folding and the assembly into trimers of beta-conglycinin. Density-gradient centrifugation, gel-filtration and differential 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 subunits 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 endoplasmic reticulum. This suggests that the extension regions play an important role in the prevention of aggregation in the endoplasmic reticulum in analogy with the N-linked glycans.
Maruyama
N
N
Research Institute for Food Science, Kyoto University, Uji, Japan.
Katsube
T
T
Wada
Y
Y
Oh
M H
MH
Barba De La Rosa
A P
AP
Okuda
E
E
Nakagawa
S
S
Utsumi
S
S
D000595:Amino Acid Sequence; D052179:Antigens, Plant; D002152:Calorimetry, Differential Scanning; D002499:Centrifugation, Density Gradient; D002942:Circular Dichroism; D003001:Cloning, Molecular; D005916:Globulins / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009994:Osmolar Concentration; D010940:Plant Proteins / Q000737:chemistry; D011134:Polysaccharides / Q000737:chemistry*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D055314:Seed Storage Proteins; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D012995:Solubility / Q000187:drug effects; D030262:Soybean Proteins; D013025:Glycine max / Q000378:metabolism*
D052179:Antigens, Plant; D005916:Globulins; D010940:Plant Proteins; D011134:Polysaccharides; D011994:Recombinant Proteins; D055314:Seed Storage Proteins; D030262:Soybean Proteins; C013894:beta-conglycinin protein, Glycine max