Mutagenesis and chimeric genes define determinants in the beta subunits of human chorionic gonadotropin and lutropin for secretion and assembly.

Abstract

Abstract. Chorionic gonadotropin (CG) and lutropin (LH) are members of a family of glycoprotein hormones that share a common a subunit but differ in their hormone-specific fl subunits. The glycoprotein hormone ~ subunits share a high degree of amino acid homology that is most evident for the LHfl and CGfl subunits having >80% sequence similarity. However, transfection studies have shown that human CGfl and ot can be secreted as monomers and can combine efficiently to form dimer, whereas secretion and assembly of human LHfl is less efficient. To determine which specific regions of the LHfl and CGfl subunits are responsible for these differences, mutant and chimeric LH/%CGfl genes were constructed and transfected into CHO cells. Expression of these subunits showed that both the hydrophobic carboxy-terminal seven amino acids and amino acids Trp s, Ile ~5, Met 42, and Asp 77 together inhibit the secretion of LHfl. The carboxy-terminal amino acids, along with Trp s, Ile ~, Met 42, and Thr 5s are implicated in the delayed assembly of LHfl. These unique features of LHfl may also play an important role in pituitary intracellular events and may be responsible for the differential glycosylation and sorting of LH and FSH in gonadotrophs. HUMAN chorionic gonadotropin (CG), t lutropin (LH), follitropin (FSH), and thyrotropin (TSH) are a family of heterodimeric glycoprotein hormones that share a common t~ subunit but differ in their hormonespecific fl subunits (8,45,52). Combination of the ot and subunits begins in the endoplasmic reticulum (18, 43) and for CG, dimerization is completed before the addition of the O-linked oligosaccharides in the Golgi (43). Although the fl subunits determine biological specificity of the hormones, there is a high degree of amino acid homology between these subunits (50), which is most apparent for LHfl and CGfl. They are 85 % homologous in the first 114 amino acids (51), and this relationship is responsible for the binding of CG and LH to a common gonadal receptor (8, 45, 52). However, CGfl and LHfl contain two prominent structural differences: (a) LHfl contains one N-linked oligosaccharide at position 30, whereas CGfl contains two N-linked units at sites 13 and 30; and (b) CGfl contains a 31-amino acid hydrophilic COOH-terminal extension with four O-linked oligosaccharides (3, 7, 21, 23) compared with a shorter, 7-amino acid, hydrophobic stretch on LH/~

The LHβ 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

“…Fusing the hCG␤-CTP to the bovine LH␤ subunit resulted in a hormone with a longer halflife and various abnormalities related to the LH excess when this LH␤-CTP chimera was expressed in a transgenic mouse model (15). Although the hydrophobic carboxyl-terminal end of the hLH␤ subunit is involved in the intracellular retention of the protein, the hydrophilic CTP stretch of hCG␤ contains determinants that are favorable for secretion and, when fused to the ␤-subunits of human LH and human follitropin, enhance the release from transfected cells (14,16,17). In vivo, the storage and secretion profiles of the hormones differ; hLH is stored in secretagogue-sensitive granules, and there are data showing it is secreted from the basolateral surface of the pituitary gonadotropes (18 -20).…”

Processing of O-linked Glycosylation in the Chimera Consisting of .ALPHA.-Subunit and Carboxyl-terminal Peptide of the Human Chorionic Gonadotropin .BETA.-Subunit is Affected by Dimer Formation with Follicle-stimulating Hormone .BETA.-Subunit

“…The plasmids for chimeric a were transfected alone or with pM 2 FSHb into the cells by the calcium phosphate method [5]. The stable clones were selected with 0.25 mg/ml of the neomycin analog G418 (Life Technologies, Gaithersburg, MD, USA) as described previously [15].…”

“…1). It has been shown that the CTP does not play roles in the assembly, secretion and in vitro bioactivity of hCG [5][6][7], but is important for maintaining the longer halflife of hCG [6]. The structures of O-linked carbohydrates in hCGb-subunit have been elucidated using the urine of patients with trophoblastic diseases and healthy pregnant women [8], and the choriocarcinoma cell line, BeWo [9].…”

Human Chorionic Gonadotropin .BETA.-Subunit Affects the Folding and Glycosylation of .ALPHA.-Cys Mutants.

“…The BamHI-BglII fragment for each mutant was inserted into the compatible BamHI site of the eukaryotic expression vector, pM2 [8], downstream from the Harvey murine sarcoma virus long terminal repeat [9] . Expression vector for hCGpWT (pM2CGjI) was also described previously [10].…”

“…The plasmids described above were transfected alone or with pM2CGj3 into the cells by the calcium phosphate method [10]. The stable clones were selected with 0.25 mg/ml of the neomycin analog G418 (Life Technologies, Gaithersburg, MD).…”