No abstract

Pairs of cystine residues were introduced in the alpha- and beta-subunits of human choriogonadotropin at positions with optimal geometries for the formation of disulfide bonds. Using the homology with luteinizing hormone and follicle stimulating hormone, similar mutations were carried out in these glycoprotein hormones. In nearly all mutants the corresponding disulfide bonds were formed leading to a non-natural, covalent linkage between the alpha- and beta-subunits. The mutants typically display wild-type receptor binding and bioactivity. The mutants with non-natural intersubunit disulfide bonds display enhanced thermostabilities relative to the corresponding heterodimeric glycoprotein hormones, rendering them candidates for long acting gonadotropins with enhanced shelf lives.

Several studies indicate that in human choriogonadotropin the N-linked oligosaccharide at position 52 of the A-subunit is important for bioactivity. We have generated choriogonadotropin mutants in which the A52 glycosylation site is removed and the A and β subunits are covalently linked by intersubunit disulfide bonds. These mutants display wild-type receptor binding and bioactivity. Furthermore, we show that removal of the A52 sugar leads to instability of heterodimeric choriogonadotropin. Therefore, we conclude that the A52 oligosaccharide of choriogonadotropin is not involved in signal transduction, but in the stability of the heterodimer.Keywords : human choriogonadotropin; bioactivity ; signal transduction ; protein engineering; glycoprotein.Human choriogonadotropin (CG), luteinizing hormone (LH) position plays a major role in signal transduction in CG [5] and FSH [6, 7]. and follicle-stimulating hormone (FSH) form the family of gonadotropins, which is involved in the regulation of the cellular Recently, we have shown that it is feasible to introduce covalent bonds between the A and β subunits of CG by introducing and endocrine function of the reproductive organs [1]. They consist of A and β subunits that are associated by hydrogen bonds intersubunit disulfide bonds [8]. This class of glycoprotein hormones displays wild-type receptor binding and biological activand hydrophobic contacts. In a given species the A subunit is identical in all glycoprotein hormones, while the β subunits are ity, whereas the thermostability of the hormones is enhanced considerably. Furthermore, these disulfide-bonded mutants were unique and confer receptor specificity on the hormones.A substantial part of the molecular mass of gonadotropins is designed to have overall conformations minimally disturbed relative to that of wild-type CG. Therefore, such molecules provide attributed to complex carbohydrates; in CG approximately 34% of the mass of the glycoprotein is due to complex carbohydrates. a very attractive system to study effects of mutations that in a heterodimeric context could interfere with the stability of the The β subunit of CG has four O-linked carbohydrates at Ser121, Ser127, Ser132 and Ser138. In addition, N-linked oligosaccha-heterodimer. In this paper we analyze the role of the AsnA52 glycosylation site in signal-transducing activities using this class rides are present at Asn52 and Asn78 on the A subunit and at Asn13 and Asn30 on the β subunit. Several studies have ad-of disulfide-bonded CG molecules. We will show that in stable CG, the A52 glycosylation site is not involved in signal transducdressed the role of the oligosaccharide chains on the gonadotropins [2]. It has been shown that the carbohydrate moieties are tion. involved in the folding and secretion of gonadotropins. Furthermore, glycosylation is essential for the plasma half-life of the glycoprotein hormones.MATERIALS AND METHODS The gonadotropins are amongst the most prominent examMaterials. Enzymes for preparing the expression vectors ples in glycobi...

Three single chain gonadotropins were designed based on the three-dimensional-structure of human choriogonadotropin and structure/activity relationships of the glycoprotein hormones. In each single chain, the C-terminal end of the human choriogonadotropin /? subunit is connected via Ser-Gly repeats to the N-terminal end of the a subunit. In addition, two of the single chains have truncated subunits. The three mutants were expressed in CHO cells. In vitro binding of two of the three mutants to the human lutropinl choriogonadotropin receptor was found to be comparable to wild-type lutropin. In contrast, both the receptor binding and the in vitro bioactivity of the mutant with truncated (I and /? subunits in which the p:26-110 disulphide bond cannot be formed, are lowered relative to wild-type lutropin. The fact that this mutant still displays biological activity shows that the seat-belt arrangement proposed by Isaacs and coworkers [Lapthorn, A. J., Harris, D. C., Littlejohn, A., Lustbader, J. W., Canfield, R. E., Machin, K. Keywords: gonadotropin ; single chain ; structure-based design; spacer; truncation.The glycoprotein hormones 11 1 form a family of structurally related proteins. Members include choriogonadotropin, follitropin, lutropin and thyrotropin. Follitropin, lutropin and thyrotropin are present in most vertebrate species and are synthesized and secreted by the pituitary. Choriogonadotropin has so far been found only in primates, including humans, and in horses, and is synthesized by placental tissue. The glycoprotein hormones are heterodimers composed of two dissimilar subunits, named 0: and /?, which are associated by non-covalent bonds.Within a species, the a subunit is the same for each member of the gonadotropin family. The , / 3 subunits are different for each member, i.e. choriogonadotropin, follitropin, thyrotropin and lutropin, and confer receptor-binding specificity on the hormones.The association of the (x and the p subunit is a very important step in the biosynthesis of the glycoprotein hormones, since only the intact dimers are biologically active. The correct assembly of the heterodimer is essential for efficient secretion, receptor binding, and signal transduction of the hormone. Free a and / 3 subunits are biologically inactive 121. Numerous mutation studies have shown that the dimerization is a highly specific process and can easily be prohibited by single point mutations. Structure/function analysis of the glycoprotein hormones is often hampered by unwanted secondary effects on the assembly of the subunits.Therefore, we and others [3 -71 have pursued approaches to determine whether the active conformation of the glycoprotein hormones can also be formed when the two subunits are synthesized in tandem on a single polypeptide chain. It has been dem- onstrated that covalently linked u and /j subunits are efficiently secreted and able to fold in a conformation that binds the lutropin/choriogonadotropin receptor with high affinity and stimulates adenylate cyclase. Thus, it can be concluded th...

cDNA encoding an immunogenic protein from partially sporulated oocysts of Eimeria acervulina was cloned and used to search for the homologous counterpart in Eimeria tenella. Monospecific antibodies were raised against the recombinant expression product. Using these antibodies, the parasite proteins were found to be localised in the refractile bodies. The derived amino-acid sequences were compared by computer using the SWISSPROT protein database. In addition to high homology between the Eimeria species, extensive similarity was found with pyridine nucleotide transhydrogenase from Escherichia coli. Comparison with the sugar signature database also resulted in a possible sugar binding domain present only in the Eimeria proteins. It is possible that the corresponding parasite proteins play a role in the recently discovered mannitol cycle of Eimeria.