Critical relationship between glycosylation of recombinant lutropin receptor ectodomain and its secretion from baculovirus-infected insect cells

The lutropin receptor ectodomain overexpressed under the control of the pow erful polyhedrin promoter in baculovirus-infected Sf9 insect cells, is main ly found in an inactive, intracellularly-aggregated form. It is secreted in an active form under the control of the P10 promoter, a somewhat weaker an d earlier promoter, at the pride of a lower production. The apparent molecu lar masses of the two species encoded by the same cDNA are 48 kDa and 60-68 kDa, respectively. The relationship between the extent and type of glycosy lation and the extracellular targeting for the recombinant lutropin recepto r ectodomains was investigated precisely with endoglycosidases, lectins of various specificities, and a glycosylation inhibitor, and tested with monoc lonal and polyclonal antibodies. The results indicate that the strong polyh edrin promoter probably overwhelms the processing capacity of the ER in Sf9 cells, so that only a high- mannose precursor is expressed in large amount s. Only a minute amount of protein is secreted, which has been processed by Sf9 exoglycosidases/glycosyltransferases and bears complex/hybrid oligosac charides. The weaker P10 promoter allows secretion of a mature and active r eceptor ectodomain, bearing complex glycosylation. An important O-linked gl ycosylation is also added post-translationally on this species. In particul ar, beta-galactose and sialic acid residues were specifically detected in t he secreted species, evidence of the induction of the corresponding glycosy ltransferases or of their genes. These results suggest that Sf9 cells should eventually be engineered with c haperones and glycosyltransferases in order to improve the production of de manding glycoproteins such as the porcine lutropin ectodomain, so as to ope n the way to resolution of the three-dimensional structures of these recept ors.