Analysis of carbohydrate residues on recombinant human thyrotropin receptor.
An investigation of the sugar groups on recombinant human TSH receptors (TSHR) expressed in CHO-K1 cells and solubilized with detergents is described. Western blotting studies with TSHR monoclonal antibodies showed that the receptor was present principally as two bands with approximate molecular masses of 120 and 100 kDa. Further blotting studies using lectins and/or involving treatment with different glycosidases indicated that the 100-kDa band contained about 16 kDa of high mannose-type sugars, and the 120-kDa band contained about 33 kDa of complex-type sugars. It was possible to separate the 120- and 100-kDa components of the TSHRs by lectin affinity chromatography. In particular, Galanthus nivalis lectin, which binds high mannose-type sugars, bound the 100-kDa band, but not the 120-kDa band, whereas Datura stramonium lectin, which binds complex-type sugars, bound the 120-kDa band, but not the 100-kDa band. 125I-Labeled TSH binding studies with the various lectin column fractions showed that TSH-binding activity was principally associated with the complex-type sugar containing the 120-kDa form of the receptor rather than the high mannose-containing 100-kDa form. During peptide chain glycosylation, high mannose-type sugar residues are attached first and then modified by the formation of complex type structures to form the mature glycoprotein. Our data suggest that in the case of the TSH receptor, this type of posttranslational processing has an important role in forming the TSH-binding site.
Oda
Y
Y
FIRS Laboratories, RSR Ltd., Llanishen, Cardiff, United Kingdom.
Sanders
J
J
Roberts
S
S
Maruyama
M
M
Kiddie
A
A
Furmaniak
J
J
Smith
B R
BR
D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002241:Carbohydrates / Q000032:analysis*; D002846:Chromatography, Affinity; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D027801:Galanthus; D006031:Glycosylation; D006801:Humans; D037102:Lectins; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D037121:Plant Lectins; D011989:Receptors, Thyrotropin / Q000032:analysis*; D011994:Recombinant Proteins / Q000032:analysis
D002241:Carbohydrates; D037102:Lectins; D037121:Plant Lectins; D011989:Receptors, Thyrotropin; D011994:Recombinant Proteins