The sugar binding activity of MR60, a mannose-specific shuttling lectin, requires a dimeric state.
MR60 is an intracellular membrane protein which has been shown to act as a mannoside specific lectin and to be identical to ERGIC-53, a protein characteristic of the endoplasmic reticulum-Golgi apparatus-intermediate compartment, acting as a shuttle. According to its primary sequence, this MR60/ERGIC-53 protein contains a luminal domain including the carbohydrate recognition domain, a stem, a transmembrane segment and a cytosolic domain. The endogenous MR60/ERGIC-53 protein is spontaneously oligomeric, (dimers and hexamers). In this paper, we study the relationship between the oligomerization state and the sugar binding capacity by using recombinant proteins. The expression of the recombinant proteins was evidenced by immunocytochemistry and by immunoprecipitation followed by SDS-PAGE analysis. The full size recombinant protein binds mannosides and is oligomeric, up to the hexameric form. Two truncated proteins lacking the transmembrane and the cytosolic domains were prepared and characterized. A long one, containing the cysteine 466 close to the C-terminal end of the recombinant protein but lacking the cysteine 475, close to the C-terminal end of the native protein, does bind mannosides and forms dimers but no higher oligomeric forms. A shorter one, lacking both the cysteines 466 and 475, does not bind mannosides and does not form dimers or higher polymers. The two cysteines in the carbohydrate recognition domain (C190 and C230) are not involved in the stabilization of oligomers. In conclusion, this study shows that the luminal moiety of MR60/ERGIC-53 contains a device allowing both its oligomeric pattern and its sugar binding capability.
Carrière
V
V
Centre de Biophysique Moléculaire, CNRS, Orléans, France.
Piller
V
V
Legrand
A
A
Monsigny
M
M
Roche
A C
AC
D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D050260:Carbohydrate Metabolism; D019281:Dimerization; D006367:HeLa Cells; D006801:Humans; D037102:Lectins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008358:Mannose / Q000378:metabolism*; D037241:Mannose-Binding Lectins; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020319:Oligodeoxyribonucleotides, Antisense / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
C084004:LMAN1 protein, human; D037102:Lectins; D037241:Mannose-Binding Lectins; D008565:Membrane Proteins; D020319:Oligodeoxyribonucleotides, Antisense; D011994:Recombinant Proteins; D008358:Mannose