Abstract
BackgroundCystine-knot (cys-knot) structure is found in a rather large number of secreted proteins and glycoproteins belonging to the TGFbeta and glycoprotein hormone (GPH) superfamilies, many of which are involved in endocrine control of reproduction. In these molecules, the cys-knot is formed by a disulfide (SS) bridge penetrating a ring formed by 8, 9 or 10 amino-acid residues among which four are cysteine residues forming two SS bridges. The glycoprotein hormones Follicle-Stimulating Hormone (FSH), Luteinizing Hormone (LH), Thyroid-Stimulating Hormone (TSH) and Chorionic Gonadotropin (CG) are heterodimers consisting of non-covalently associated alpha and beta subunits that possess cys-knots with 8-amino-acyl (8aa) rings. In order to get better insight in the structural evolution of glycoprotein hormones, we examined the number and organization of SS bridges in the sequences of human 8-aa-ring cys-knot proteins having 7 (gremlins), 9 (cerberus, DAN), 10 (GPA2, GPB5, GPHα) and 12 (GPHβ) cysteine residues in their sequence.DiscussionThe comparison indicated that the common GPH-alpha subunit exhibits a SS bridge organization ressembling that of DAN and GPA2 but possesses a unique bridge linking an additional cysteine inside the ring to the most N-terminal cysteine residue. The specific GPHbeta subunits also exhibit a SS bridge organization close to that of DAN but it has two additional C-terminal cysteine residues which are involved in the formation of the "seat belt" fastened by a SS "buckle" that ensures the stability of the heterodimeric structure of GPHs. GPA2 and GPB5 exhibit no cys residue potentially involved in interchain SS bridge and GPB5 does not possess a sequence homologous to that of the seatbelt in GPH β-subunits. GPA2 and GPB5 are thus not expected to form a stable heterodimer at low concentration in circulation.SummaryThe 8-aa cys-knot proteins GPA2 and GPB5 are expected to form a heterodimer only at concentrations above 0.1 microM: this would be consistent with a short-term paracrine role but not with an endocrine role after dilution in circulation. Consequently, GPA2 and GPB5 could exert separate endocrine roles either during development and/or during adult life of both vertebrates and invertebrates.
Highlights
Cystine-knot structure is found in a rather large number of secreted proteins and glycoproteins belonging to the TGFbeta and glycoprotein hormone (GPH) superfamilies, many of which are involved in endocrine control of reproduction
Because of our interest in the structure-function relationships of glycoprotein hormones that are cys-knot proteins with 8-aa-ring, we more closely examined and compared the distribution of cysteine residues along the amino acid sequences of all 8aa-ring cys-knot proteins. This comparison of the arrangements of cysteine residues and SS bridges in the amino acid sequences of 8-aa-ring cys-knot proteins was initiated in order to distinguish the specific structural features of the GPHα-related molecule (GPA2) and GPB5 molecules compared to glycoprotein hormones (GPH) subunits and to other 8aa-ring cys-knot proteins
The comparison of the arrangements of cysteine residues and SS bridges in the amino acid sequences of cys-knot proteins with 8-aa rings was initiated in order to distinguish the specific structural features of the GPA2 and GPB5 molecules compared to glycoprotein hormones (GPH) and to other 8-aa-ring cysknot proteins
Summary
Phylogenetic analyses of cys-knot proteins show that the 8-aa-ring proteins are more closely related to each other than to the 9-aa- and 10-aa ring subfamilies [6]. All 8-aa-ring cys-knot proteins with even numbers of cysteines (10 in DAN, GPA2, GPB5 and GPHα 12 in LHβ, FSHβ, TSHβ and CGβ) exhibit no unpaired cys residues and all of them are engaged in intra-chain SS bridges. Even after chemical cross-linking, only a faint spot was observed at the level expected for a dimer in SDS-PAGE whereas the majority of material was found at the monomer level [7] Expression patterns It has been known for a long time that GPH α and β subunits readily form a heterodimer only when they are expressed in the same cell and associate at high concentration and under moderate reducing redox potential in endoplasmic reticulum to permit opening of the seatbelt SS buckle [12,14,16,17]. Potential roles for GPA2 and GPB5 monomers should be searched [36]
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