ER-resident chaperone interactions with recombinant antibodies in transgenic plants.
In this study, we demonstrate that the folding and assembly of IgG in transgenic tobacco plants is orchestrated by BiP (binding protein), an endoplasmic reticulum resident chaperone. Expression of BiP and calreticulin was examined in transgenic tobacco plants that express immunoglobulin chains, either singly or in combination to form IgG antibody. BiP mRNA expression was lowest in wild-type nontransformed plants and those that expressed immunoglobulin light chain alone. Higher mRNA levels were detected in plants expressing fully assembled immunoglobulin (light and heavy chains), and the most abundant levels of RNA transcript were found in those plants that expressed immunoglobulin heavy chain alone. Estimation of total BiP demonstrated a similar pattern, with the highest levels detected in plants expressing immunoglobulin heavy chain alone. Immunoprecipitation studies demonstrated that BiP was associated with immunoglobulin chains extracted from protoplast lysates, but not from secreted fluids. Again, most BiP was coprecipitated from plants expressing heavy chain only and those that produced full length IgG. The binding of BiP to Ig heavy chains was ATP-sensitive. Co-expression of heavy and light chain resulted in IgG assembly and displacement of BiP from the heavy chain as the amount of light chain increased. Although calreticulin mRNA and total protein levels varied in a similar manner to those of BiP in the transgenic plants, there was no evidence for association between calreticulin and Ig chains, by coimmunoprecipitation. The results indicate that BiP, but not calreticulin, takes part in immunoglobulin folding and assembly in transgenic plants.
Nuttall
James
J
Department of Biological Sciences, University of Warwick, Coventry, UK.
Vine
Nicholas
N
Hadlington
Jane L
JL
Drake
Pascal
P
Frigerio
Lorenzo
L
Ma
Julian K-C
JK
D015152:Blotting, Northern; D015153:Blotting, Western; D037282:Calreticulin / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D007074:Immunoglobulin G / Q000378:metabolism; D018832:Molecular Chaperones / Q000378:metabolism; D030821:Plants, Genetically Modified; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics*; D014162:Transfection
D037282:Calreticulin; D002352:Carrier Proteins; D007074:Immunoglobulin G; D018832:Molecular Chaperones; D012333:RNA, Messenger; D012313:RNA