Homologous and Heterologous Carboxyl Terminal Peptide (CTP) Linker Sequences Enhance the Secretion of Bioactive Single-Chain Bovine LH Analogs

Abstract

Single chain variants of the heterodimeric gonadotropins were engineered by tethering the genes of the individual subunits into one polypeptide. In tethered human (h) gonadotropins, the carboxyl terminal peptide (CTP) of the choriogonadotropin (CG) β subunit serves as an effective linker to enhance the secretion of the analogs compared to variants lacking the CTP. The gonadotropin subunits of non-primate, non-equid species lack a CTP domain that precludes the use of a homologous CTP in tethered analogs in many species. Here we used the bovine LH as a model to examine the impact of the CTP domain of the hCGβ subunit (denoted as huCTP) and of a previously untranslated CTP-like sequence decoded from the bovine LHβ gene on the secretion and bioactivity of tethered analogs. This cryptic CTP (designated boCTP) was incorporated into the bovine LHβ reading frame by deletion frame-shift mutations analogous to these that presumably occurred in primates and equids. We genetically engineered single chain variants in which the β and α subunit domains were linked directly or via the heterologous huCTP or the homologous boCTP sequences and expressed them in CHO cells. The data suggest that the tethered analogs were expressed and N-glycosylated, but unlike the huCTP, the boCTP appears as devoid of mucin O-glycans. The incorporation of the boCTP or huCTP linkers enhanced by about 3fold the rate and efficiency of secretion from the transfected cells. The tether variants were bioactive, as estimated by induction of steroid production in immortalized granulosa cells expressing the rat LH receptor. Furthermore, the variants were about equally potent, as judged by their EC50s (0.7 - 0.9 ng/ml). Thus, the hCGβ CTP maintains pro-secretory determinants without inhibiting receptor activation when applied as a linker in tethered bovine LH, implying that these CTP features are preserved when the domain is incorporated into non-primate single chain analogs. The study suggests that the boCTP and huCTP domains are advantageous for the secretion of tethered bovine gonadotropins, and also demonstrates strategies for the design of bioactive LH analogs in ruminant species.

Single chain gonadotropins - linker - cryptic CTP - recombinant - bovine LH

References

• 1 Ben-Menahem D. Single chain variants of the glycoprotein hormones and their receptors as tools to study receptor activation and for analogue design.  J Neuroendocrinol. 2004;  16 171-177
• 2 Ben-Menahem D, Boime I. Converting heterodimeric gonadotropins to genetically linked single chains: New approaches to structure activity relationships and analogue design.  Trends Endocrinol Metab. 1996;  7 100-105
• 3 Ben-Menahem D, Kudo M, Pixley M R, Sato A, Suganuma M, Perlas E, Hsueh A JW, Boime I. The biological action of single chain choriogonadotropin is not dependent on the individual disulfide bonds of the β subunit.  J Biol Chem. 1997;  272 6827-6830
• 4 Birken S, Maydelman Y, Gawinowicz M A, Pound A, Liu Y, Hartee A S. Isolation and characterization of human pituitary chorionic gonadotropin.  Endocrinology. 1996;  147 1402-1411
• 5 Birken S, Yershova O, Myers R V, Bernard M P, Moyle W. Analysis of human choriogonadotropin core 2 o-glycan isoforms.  Mol Cell Endocrinol. 2003;  204 21-30
• 6 Boime I, Ben-Menahem D, Olijve W. Studies of recombinant gonadotropins: interaction of basic sciences and therapeutics. Fauser BCJM, Rutherford AJ, Strauss JF, Van Streiteghem A Molecular Biology in Reproductive Medicine. New York, London; Parthenon Publishing 1999: 147-164
• 7 Bousfield G R, Liu W K, Sugino H, Ward D N. Structural studies on equine glycoprotein hormones.  Amino acid sequence of equine lutropin beta-subunit: J Biol Chem. 1987;  262 8610-8620
• 8 Boussif O, Lezouale'h F, Zanta M A, Mergny M D, Scherman D, Demeneix B, Behr J P. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine.  Proc Natl Acad Sci USA. 1995;  92 7297-7301
• 9 Chopineau M, Stewart F, Allen W R. Cloning and analysis of the cDNA encoding the horse and donkey luteinizing hormone beta-subunits.  Gene. 1995;  28 253-256
• 10 Dirnberger D, Steinkellner H, Abdennebi L, Remy J J, van de Wiel D. Secretion of biologically active glycoforms of bovine follicle stimulating hormone in plants.  Eur J Biochem. 2001;  268 4570-4579
• 11 Fares F A, Suganuma N, Nishimori K, LaPolt P S, Hsueh A J, Boime I. Design of a long-acting follitropin agonist by fusing the C-terminal sequence of the chorionic gonadotropin β subunit to the follitropin β subunit.  Proc Natl Acad Sci USA. 1992;  89 4304-4308
• 12 Fares F A, Yamabe S, Ben-Menahem D, Pixley M, Hsueh A J, Boime I. Conversion of thyrotropin heterodimer to a biologically active single-chain.  Endocrinology. 1998;  139 2459-2464
• 13 Fiddes J C, Goodman H M. The cDNA for the beta-subunit of human chorionic gonadotropin suggest evolution of a gene by readthrough into the 3′-untranslated region.  Nature. 1980;  286 684-687
• 14 Fidler A E, Lin J S, Lun S, Ng Chie W, Western A, Stent V, McNatty K P. Production of biologically active tethered ovine FSHβα by the methylotrophic yeast Pichia pastoris.  J Mol Endocrinol. 2003;  30 213-225
• 15 Fritz T A, Hurley J H, Trinh L B, Shiloach J, Tabak L A. The beginnings of mucin biosynthesis: the crystal structure of UDP-GalNAc:polypeptide alpha-N-acetylgalactosaminyltransferase-T1.  Proc Natl Acad Sci USA. 2004;  101 15307-15312
• 16 Furuhashi M, Shikone T, Fares F A, Sugahara T, Hsueh A J, Boime I. Fusing the carboxy-terminal peptide of the chorionic gonadotropin (CG) beta-subunit to the common alpha-subunit: retention of O-linked glycosylation and enhanced in vivo bioactivity of chimeric human CG.  Mol Endocrinol. 1995;  9 54-63
• 17 Galet C, Le Bourhis C M, Chopineau M, Le Griec G, Perrin A, Magallon T, Attal J, Viglietta C, Houdebine L M, Guillou F. Expression of a single βα chain protein of equine LH/CG in milk of transgenic rabbits and its biological activity.  Mol Cell Endocrinol. 2001;  174 31-40
• 18 Garcia-Campayo V, Sato A, Hirsch B, Sugahara T, Muyan M, Hsueh A J, Boime I. Design of stable biologically active recombinant lutropin analogs.  Nat Biotechnol. 1997;  15 663-667
• 19 Garcia-Campayo V, Boime I. Novel recombinant gonadotropins.  Trends Endocrinol Metab. 2001;  12 72-77
• 20 Garcia-Campayo V, Kumar T R, Boime I. Thyrotropin, follitropin and chorionic gonadotropin expressed as a single multifunctional unit reveal remarkable permissiveness in receptor-ligand interactions.  Endocrinology. 2002;  143 3773-3778
• 21 Grossmann M, Wong R, Szkudlinski M W, Weintraub B D. Human thyroidstimulating hormone (hTSH) subunit gene fusion produces hTSH with increased stability and serum half-life and compensates for mutagenesis-induced defects in subunit association.  J Biol Chem. 1997;  272 21312-21316
• 22 Heikoop J C, van Beuningen-de Vaan N MM, van den Boogart P, Grootenhuis P D. Evaluation of subunit truncation and the nature of the spacer for single chain human gonadotropins.  Eur J Biochem. 1997;  245 656-662
• 23 Hill Jr H D, Schwyzer M, Steinman H M, Hill R L. Ovine submaxillary mucin. Primary structure and peptide substrates of UDP-N-acetylgalactosamine:mucin transferase.  J Biol Chem. 1977;  252 3799-3804
• 24 Jackson A M, Berger P, Pixley M, Klein C, Hsueh A J, Boime I. The biological action of choriogonadotropin is not dependent on the complete native quaternary interactions between the subunits.  Mol Endocrinol. 1999;  13 2175-2188
• 25 Kaetzel D M, Virgin J B, Clay C M, Nilson J H. Disruption of N-linked glycosylation of bovine luteinizing hormone β-subunit by site-directed mutagenesis dramatically increases its intracellular stability but does not affect biological activity of the secreted heterodimer.  Mol Endocrinol. 1989;  3 1765-1774
• 26 Kanda M, Jablonka-Shariff A, Sato A, Pixley M R, Bos E, Hiro'oka T, Ben-Menahem D, Boime I. Genetic fusion of an alpha-subunit gene to the folliclestimulating hormone and chorionic gonatrophin-beta subunit genes: production of a bifunctional protein.  Mol Endocrinol. 1999;  13 1873-1881
• 27 Kasuto H, Levavi-Sivan B. Production of biologically active tethered tilapia LHβα by the methylotrophic yeast Pichia pastoris.  Gen Comp Endocrinol. 2005;  140 222-232
• 28 Keren-Tal I, Dantes A, Sprengel R, Amsterdam A. Establishment of steroidogenic granulosa cell lines expressing follicle stimulating hormone receptors.  Mol Cell Endocrinol. 1993;  95 R1-R10
• 29 Klein J, Lobel L, Pollak S, Lustbader B, Ogden R T, Sauer M V, Lustbader J W. Development and characterization of a long-acting recombinant hFSH agonist.  Hum Reprod. 2003;  18 50-56
• 30 Krichevsky A, Birken S, O'Connor J, Acevedo H F, Bikel K, Lustbader J, Hartree A, Canfield R E. Development, characterization and application of monoclonal antibodies to the native and synthetic beta COOH-terminal portion of human chorionic gonadotropin (hCG) that distinguish between the native and desialylated forms of hCG.  Endocrinology. 1994;  134 1139-1145
• 31 Mutayoba B M, Meyer H HD, Schams D, Schallenger E. Development of a sensitive enzyme immunoassay for LH determination in bovine plasma using the streptoavidin-biotin technique.  Acta Endocrinol. 1990;  122 227-232
• 32 Muyan M, Ruddon R W, Norton S E, Boime I, Bedows E. Dissociation of early folding events from assembly of the human lutropin beta-subunit.  Mol Endocrinol. 1998;  12 1640-1649
• 33 Nakav S, Jablonka-Shariff A, Kaner S, Chadna-Mohanty P, Grotjan H E, Ben-Menahem D. The LHbeta gene of several mammals embeds a carboxyl-terminal peptide-like sequence revealing a critical role for mucin oligosaccharides in the evolution of lutropin to chorionic gonadotropin in the animal phyla.  J Biol Chem. 2005;  280 16676-16684
• 34 Narayan P, Wu C, Puett D. Functional expression of yoked human chorionic gonadotropin in baculovirus-infected insect cells.  Mol Endocrinol. 1995;  9 1720-1726
• 35 Narayan P, Gray J, Puett D. A biologically active single chain human chorionic gonadotropin analog with altered receptor binding properties.  Endocrinology. 2000;  141 67-71
• 36 Peng K C, Puett D, Brewer J M. Homodimer formation by the individual subunits of bovine lutropin as determined by sedimentation equilibrium.  J Mol Endocrinol. 1997;  18 259-265
• 37 Pierce J G, Parsons T F. Glycoprotein hormones: structure and function.  Annu Rev Biochem. 1981;  50 465-495
• 38 Puett D, Wu C, Narayan P. The tie that binds: design of biologically active singlechain human chorionic gonadotropins and a gonadotropin-receptor complex using protein engineering.  Biol Reprod. 1998;  58 1337-1342
• 39 Risma K, Clay C M, Nett T M, Wagner T, Yan J, Nilson J H. Targeted overexpression of luteinizing hormone in transgenic mice leads to infertility, polycystic ovaries, and ovarian tumors.  Proc Natl Acad Sci USA. 1995;  92 1322-1326
• 40 Sairam M R. Role of carbohydrates in glycoprotein hormone signal transduction.  FASEB J. 1989;  3 1915-1926
• 41 Sato A, Perlas E, Ben-Menahem D, Kudo M, Pixley M R, Furuhashi M, Hsueh A J, Boime I. Cystine knot of the gonadotropin alpha subunit is critical for intracellular behavior but not for in vitro biological activity.  J Biol Chem. 1997;  272 18098-18103
• 42 Gupta Sen C, Dighe R R. Biological activity of single chain gonadotropin, hCGalphabeta, is decreased upon deletion of five carboxyl terminal amino acids of the alpha subunit without affecting its receptor binding.  J Mol Endocrinol. 2000;  24 157-164
• 43 Sherman G B, Wolfe M W, Farmeric T A, Clay C M, Threadgill D S, Sharp D C, Nilson J H. A single gene encodes the β-subunits of equine luteinizing hormone and chorionic gonadotropin.  Mol Endocrinol. 1992;  6 951-959
• 44 Sugahara T, Pixley M R, Minami S, Perlas E, Ben-Menahem D, Hsueh A J, Boime I. Biosynthesis of a biologically active single peptide chain containing the human common alpha and chorionic gonadotropin beta subunits in tandem.  Proc Natl Acad Sci USA. 1995;  92 2041-2045
• 45 Sugahara T, Sato A, Kudo M, Ben-Menahem D, Pixley M R, Hsueh A JW, Boime I. Expression of biologically active fusion genes encoding the common α subunit and the follicle-stimulating hormone β subunit: Role of a linker sequence.  J Biol Chem. 1996;  271 10445-10448
• 46 Suganuma N, Matzuk M M, Boime I. Elimination of disulfide bonds affects assembly and secretion of the human chorionic gonadotropin β subunit.  J Biol Chem. 1989;  264 19302-19307
• 47 Suh B S, Sprengel R, Keren-Tal I, Himmelhoch S, Amsterdam A. Introduction of a gonadotropin receptor expression plasmid into immortalized granulosa cells leads to reconstitution of hormone-dependent steroidogenesis.  J Cell Biol. 1992;  119 439-450
• 48 Talmadge K, Vamvakopoulos N C, Fiddes J C. Evolution of the genes for the beta subunits of human chorionic gonadotropin and luteinizing hormone.  Nature. 1984;  307 37-40
• 49 Ulloa-Aguirre A, Timossi C, Zdamian-Matsumura P, Dias J A. Role of glycosylation in function of follicle-stimulating hormone.  Endocrine. 1999;  11 205-215
• 50 Xing Y, Lin W, Jiang M, Myers R V, Cao D, Bernard M P, Moyle W R. Alternatively folded choriogonadotropin analogs. Implications for hormone folding and biological activity.  J Biol Chem. 2001;  276 46953-46960