Secretion of biologically active glycoforms of bovine follicle stimulating hormone in plants.
We chose the follicle stimulating hormone (FSH), a pituitary heterodimeric glycoprotein hormone, as a model to assess the ability of the plant cell to express a recombinant protein that requires extensive N-glycosylation for subunit folding and assembly, intracellular trafficking, signal transduction and circulatory stability. A tobacco mosaic virus (TMV) based transient expression system was used to express a single-chain (sc) version of bovine FSH in the tobacco related species Nicotiana benthamiana. Preparations of periplasmic proteins from plants infected with recombinant viral RNA contained high levels of sc-bFSH, up to 3% of total soluble proteins. Consistently, in situ indirect immunofluorescence revealed that the plant cell secreted the mammalian secretory protein to the extracellular compartment (EC). By mass spectrometric analysis of immunoaffinity purified sc-bFSH derived from EC fractions, we found two species of the plant paucimannosidic glycan type, truncated forms of complex-type N-glycans. Stimulation of cAMP production in a CHO cell line expressing the porcine FSH receptor acknowledged the native-like structure of sc-bFSH and a sufficient extent of N-glycosylation required for signal transduction. Furthermore, in superovulatory treatments of mice, sc-bFSH displayed significant in vivo bioactivity, although much lower than that of pregnant mare serum gonadotropin. We conclude that plants may have a broad utility as hosts for the recombinant expression of proteins even where glycosylation is essential for function.
Dirnberger
D
D
Zentrum für Angewandte Genetik, Universität für Bodenkultur-Wien, Wien, Austria.
Steinkellner
H
H
Abdennebi
L
L
Remy
J J
JJ
van de Wiel
D
D
D000818:Animals; D002417:Cattle; D005260:Female; D005640:Follicle Stimulating Hormone / Q000096:biosynthesis* / Q000494:pharmacology; D005822:Genetic Vectors; D006031:Glycosylation; D051379:Mice; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D010060:Ovulation / Q000187:drug effects; D011962:Receptors, FSH / Q000187:drug effects; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000494:pharmacology
D011962:Receptors, FSH; D011994:Recombinant Proteins; D005640:Follicle Stimulating Hormone