Carbohydrate variant of the recombinant beta-subunit of human choriogonadotropin expressed in baculovirus expression system

Abstract

The beta-subunit of human choriogonadotropin (hCG) has two complex type N-linked and four O-linked carbohydrate chains. To further evaluate the specificity of the carbohydrate moiety on the hCG function, we have expressed hCG beta subunit in the baculovirus insect cell system to modify its carbohydrate structures. The recombinant hCG beta (rhCG beta) was efficiently secreted in the medium and was purified to homogeneity by immunoaffinity chromatography using a highly specific monoclonal antibody against hCG beta. The homogeneity of the recombinant subunit was established by sodium dodecyl sulfate-polyacrylamide gel electrophoresis performed under reducing and nonreducing conditions and reverse phase high performance liquid chromatography. rhCG beta had molecular weights of 22,500 and 33,000 under reducing and nonreducing conditions, respectively. Digestion with N-glycanase cleaved the Mr = 22,500 protein to 18,000, while digestion with Endo H or Endo F yielded an additional protein band of 20,500 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The carbohydrate analysis by pulse amperometry yielded the relative number of 2.5, 2.4, 3.7, and 11.3 residues of fucose, N-acetylgalactosamine, galactose, and mannose, respectively, based on a value of 4 residues for N-acetylglucosamine. Lectin binding studies showed rhCG beta to bind with concanavalin A with a high affinity and not with wheat germ agglutinin. In the studies with endoglycosidases together with the carbohydrate analysis and lectin binding properties, rhCG beta appears to have two high mannose-type N-linked and three to four O-linked carbohydrate simple disaccharide chains. The carbohydrate modification of the beta-subunit did not alter its immunopotency and its ability to combine with hCG alpha. The reconstituted hormone made up of rhCG beta and hCG alpha was found to be similar to hCG in biological properties such as receptor binding and in its ability to stimulate cAMP and steroidogenesis.