Molecular cloning of pituitary glycoprotein alpha-subunit and follicle stimulating hormone and chorionic gonadotropin beta-subunits from New World squirrel monkey and owl monkey

Abstract

The goal of this study was to characterize the gonadotropins expressed in pituitary glands of the New World squirrel monkey (Saimiri sp.) and owl monkey (Aotus sp.). The various subunits were amplified from total RNA from squirrel monkey and owl monkey pituitary glands by reverse transcription-polymerase chain reaction and the deduced amino acid sequences compared to those of other species. Mature squirrel monkey and owl monkey glycoprotein hormone alpha-polypeptides (96 amino acids in length) were determined to be 80% homologous to the human sequence. The sequences of mature beta subunits of follicle stimulating hormone (FSHbeta) from squirrel monkey and owl monkey (111 amino acids in length) are 92% homologous to human FSHbeta. New World primate glycoprotein hormone alpha-polypeptides and FSHbeta subunits showed conservation of all cysteine residues and consensus N-linked glycosylation sites. Attempts to amplify the beta-subunit of luteinizing hormone from squirrel monkey and owl monkey pituitary glands were unsuccessful. Rather, the beta-subunit of chorionic gonadotropin (CG) was amplified from pituitaries of both New World primates. Squirrel monkey and owl monkey CGbeta are 143 and 144 amino acids in length and 77% homologous with human CGbeta. The greatest divergence is in the C terminus, where all four sites for O-linked glycosylation in human CGbeta, responsible for delayed metabolic clearance, are predicted to be absent in New World primate CGbetas. It is likely that CG secreted from pituitary of New World primates exhibits a relatively short half-life compared to human CG.

Figure 1

(A) Alignment of the predicted amino acid sequences of squirrel monkey (SqM α-glyc) and owl monkey (OM α-glyc) glycoprotein hormone α-subunits with those of common marmoset (Mar, U04446), human (Hum, NM_000735), rhesus monkey (RhM, (Golos et al., 1991)), cynomolgus monkey (CM, AJ781394), and rat (P11962) glycoprotein hormone α-subunits. Four amino acids deleted in the human glycoprotein hormone α-subunit sequence are indicated by asterisks. (B) Alignment of the predicted amino acid sequences of squirrel monkey (SqM FSHβ) and owl monkey (OM FSHβ) FSH β-subunits with those of common marmoset (Mar, (Wolfgang et al., 2001)), pygmy marmoset (PygM, (Wolfgang et al., 2001)), human (Hum, NM_000510), cynomolgus monkey (CM, AJ781395), rhesus monkey (RhM, AY083550 and (Wolfgang et al., 2001)), and rat (NM_001007597) FSH β-subunits. Dashes indicate amino acid residues that are identical to the squirrel monkey sequences. Numbering indicates residues in the mature subunit sequences. Consensus sequence (in bold) was obtained from completely conserved residues among mammalian species identified by Dias et al. (Dias et al., 2002) and squirrel monkey and owl monkey subunits. Conserved N-linked glycosylation sites are underlined in the consensus sequence.

Figure 2

(A) Alignment of the predicted amino acid sequences of the squirrel monkey (SqM CGβ) and owl monkey (Aotus nancymaae) (OMna CGβ) CG β-subunits with those of owl monkey Aotus trivirgatus (OMtr CGβ, AF397604, partial sequence), common marmoset (Mar CGβ, U04447), dusky titi monkey (Titi CGβ , AF397603, partial sequence), human (Hum CGβ, NM_000737), baboon (Bab CGβ, M14966), and rhesus monkey (RhM CGβ, AF397601 and AY011015) CG β-subunits, and human LH (Hum LHβ, NM_000894) β-subunit. Dashes indicate amino acid residues that are identical to the squirrel monkey CG β-subunit sequence. Deleted amino acids are indicated with an asterisk. Numbering indicates residues in the mature CG β-subunits. Consensus sequence in CGβ among all primate species described here is indicated in bold. N-linked glycosylation sites are underlined. (B) Schematic showing predicted glycosylation sites in New World squirrel monkey, owl monkey, common marmoset, and dusky titi monkey CG (SqM, OM, Mar, and Titi CGβ, respectively). Human CGβ possesses N-linked oligosaccharide chains at positions 13 and 30 and O-linked oligosaccharide chains at positions 121, 127, 132, and 138. No sites for O-linked glycosylation are predicted to exist in New World monkey CGβ. Rather, New World primate CGβs have gained a unique recognition sequence for N-linked glycosylation in the C terminus.

Figure 3

Phylogenetic tree of the β sub-units of chorionic gonadotropin (CG-beta), luteinizing hormone (LH-beta), follicle stimulating hormone (FSH-beta), and of α-glycopeptide (alpha-glyc) sequences from selected primates and rat. The phylogenetic tree was constructed with Mega version 3.1 software (Kumar et al., 2004). Minimum evolution, neighbor-joining and maximum parsimony methods were used with Poisson correction for multiple amino acid substitution and with 1000 random bootstrap replications. The figure shows the neighbor-joining tree (the three methods produced very similar topologies). Brackets to the right indicate the monophyletic groups encompassing New World monkey (NWM) subunits. Bootstrap values ≥ 50% are shown. The human transforming growth factor β (TGF-β) is used as an outgroup to root the tree (Li and Ford, 1998). The scale at the bottom left is in units of amino acid substitutions per site.