Table 1.
Fish species and Genbank accession numbers for the sequences used in the amino acid percentage identity and phylogenetic analyses of the FSH and LH glycoprotein beta subunits.
Table 2.
Primers used in cloning A. gigas FSHβ and LHβ subunits cDNAs.
Fig 1.
Nucleotide and deduced amino acid sequence of the cDNA encoding for the FSHβ-subunit of A. gigas.
M, inside a square, start coding region; G, inside a rhombus, first amino acid of the mature peptide; N I T, inside a rectangle, glycosylation site; C, inside a circle, conserved cysteine residues; P, inside a triangle, conserved proline residues; attaaa, designated with a black square, polyadenylation signal; aaaaaaaaaaaa, designated with a gray square, poly(A+) tail. GenBank Accession number: KJ729119.
Fig 2.
Alignment of the FSHβ mature peptides of three Chondrostei and 38 teleosts, including A. gigas.
The Genbank accession numbers for these sequences are given in Table 1. Below the alignment the conserved cysteines(*), prolines (P) and the putative N-linked glycosylation sites (___) are highlighted.
Fig 3.
Nucleotide and deduced amino acid sequence of the cDNA encoding for the LHβ-subunit of A. gigas.
M, inside a square, start coding region; V, inside a rhombus, first amino acid of the mature peptide; N Q T, inside a rectangle, glycosylation site; C, inside a circle, cysteine residues; P, inside a triangle, proline residues; aataaa, designated with a black square, polyadenylation signal; aaaaaaaaaaaa, designated with a gray square, poly(A+) tail. GenBank Accession number: KJ741848.
Fig 4.
Alignment of the LHβ mature peptides of three Chondrostei and 38 teleosts, including A. gigas.
The Genbank accession numbers for these sequences are given in Table 1. Below the alignment the conserved cysteines (*), prolines (P) and the putative N-linked glycosylation site (___) are highlighted.
Table 3.
Percentage identity of FSHβ and LHβ peptides among fish orders (FSHβ above the diagonal).
Fig 5.
Tridimensional models obtained after molecular dynamics simulations.
On the top, the ag-FSH and ag-LH structures highlighting the α-subunit (green cartoons) and the β-subunit (blue for FSH and magenta for LH) and the predicted dissulfide bonds of the β-subunits are shown. On the bottom, the 3D models are presented in cartoon style and colored according to the secondary structure (helices in red, sheets in yellow and loops in green), highlighting the predicted loops of the β-subunits and the residues predicted in the outlier regions of the Ramachandran plot.
Fig 6.
On the top, electrostatic potential surface of ag-FSH (left) and ag-LH (right) and surface of α- and β-subunits.
The surface is colored in blue (positively charged groups) and red (negatively charged groups), while neutral groups are in white. The black arrow indicates the “seat-belt” structure of the β-subunits. On the bottom, localization of 5 (ag-FSH) and 7 (ag-LH) conserved proline residues in the hormone structures. Each proline is identified according to its position in relation to the previous cysteine residue.
Fig 7.
First purification step of HEK293F conditioned medium via RP-HPLC.
The chromatogram is derived from the application of 5 ml of ag-FSH transfection conditioned medium, in comparison with an analogous chromatogram derived from the application of 5 ml of the negative control of transfection.
Fig 8.
HPSEC obtained upon application of: (A) 0.25 ml of a pool of ag-FSH-derived material from RP-HPLC (see Fig 7) and already purified once on the same HPSEC; (B) 5μg/5μl of human pituitary FSH reference preparation from NHPP, run under identical conditions.
Fig 9.
RP-HPLC, run under dissociating conditions as described [64], after application of a sample of ag-FSH obtained from HPSEC (see Fig 8) and incubated overnight with 5M acetic acid at 37°C.