Conversion of thyrotropin heterodimer to a biologically active single-chain.
TSH and the gonadotropins, FSH, LH, and CG are a family of heterodimeric glycoprotein hormones composed of a common alpha-subunit noncovalently linked to a hormone specific beta-subunit. Assembly of alpha- and beta-subunits is essential for hormone-specific posttranslational modifications, receptor binding, and bioactivity. Structure-function studies of TSH and gonadotropins using site-directed mutagenesis can often affect folding, assembly, and secretion of the hormone. To circumvent these difficulties, recently, the gonadotropin heterodimers were converted to single chains. Here we converted the hTSH heterodimer to a biologically active single chain by genetically fusing the amino terminal end of the common alpha-subunit to the carboxyl terminal end of hTSHbeta in the presence or absence of hCGbeta carboxyl terminal peptide (CTP), which was used as a linker. Wild-type hTSH and the single chains were expressed in Chinese hamster ovary (CHO) cells, and they were efficiently secreted. Although the secretion rate of the single chain was 3-fold higher than that of hTSH wild-type. Moreover, the secretion of the single chain in the presence of the CTP linker was dramatically increased. On the other hand, receptor binding and in vitro bioactivity of the single chains were similar to that of hTSH wild-type. These data indicate the potential of the single chain approach to further investigate structure-function relationships of TSH.
Fares
F A
FA
Department of Biochemistry, Carmel Medical Center, and the Rappaport Family Institute for Research in the Medical Sciences, Faculty of Medicine, Technion-Israel Institute of Technology, Haifa. biochem@actcom.co.il
Yamabe
S
S
Ben-Menahem
D
D
Pixley
M
M
Hsueh
A J
AJ
Boime
I
I
D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D018997:Chorionic Gonadotropin, beta Subunit, Human / Q000737:chemistry / Q000235:genetics; D006224:Cricetinae; D019281:Dimerization; D015870:Gene Expression; D006801:Humans; D016297:Mutagenesis, Site-Directed; D011869:Radioligand Assay; D011993:Recombinant Fusion Proteins; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D013961:Thyroid Gland / Q000378:metabolism; D013972:Thyrotropin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D018997:Chorionic Gonadotropin, beta Subunit, Human; D011993:Recombinant Fusion Proteins; D013972:Thyrotropin