Total synthesis of the α-subunit of human glycoprotein hormones: toward fully synthetic homogeneous human follicle-stimulating hormone.

Described herein is the first total chemical synthesis of the unique α-subunit of the human glycoprotein hormone (α-hGPH). Unlike the biologically derived glycoprotein hormones, which are isolated as highly complex mixtures of glycoforms, α-hGPH obtained by chemical synthesis contains discrete homogeneous glycoforms. Two such systems have been prepared. One contains the disaccharide chitobiose at the natural N-glycosylation sites. The other contains dodecamer oligosaccharides at these same sites. The dodecamer sugar is a consensus sequence incorporating the key features associated with human glycoproteins.

D-amino acid substitution of residues 32 to 46 of the glycoprotein hormone common alpha-subunit: development of a synthetic glycoprotein hormone antagonist.

We have used single- or double-point D-amino acid substitutions to study the structure-function relationships involving residues 32 to 46 of the glycoprotein hormone common alpha-subunit (GPHa) and the testicular follicle-stimulating hormone (FSH) and luteinizing hormone (LH/hCG) receptors. D-Amino acid substitution analogs of GPHa(32-46) were synthesized and tested in both FSH and hCG radioligand receptor assays using bovine calf testis membranes as receptor source. Correct orientation of the amino acid side chains was generally of paramount importance for peptide interaction with receptor and bioactivity. Most substitutions with corresponding D-amino acids did not enhance the potency of native GPHa(32-46). A significant increment in peptide potency, however, was observed by inversion of configuration at positions Ser-34 and Thr-39 with D-amino acid isoforms. Based on the relative potency of each peptide analog. [D-Ser-34, D-Thr-39]GPHa(32-46) was approximately twofold more potent than native peptide GPHa(32-46) in both FSH and hCG radioligand receptor assays. [D-Ser-34, D-Thr-39]GPHa(32-46) also markedly inhibited FSH-stimulated estradiol biosynthesis in cultured rat Sertoli cells. The present study is unique in that it represents the first report of utilizing D-amino acid substitution to develop more potent peptide analogs related to the glycoprotein hormone common alpha-subunit region 32-46. Our results offer hope for the development of more potent and stabile peptide antagonists of possible usefulness in fertility regulation and control.

Identification of subunit contact sites on the alpha-subunit of lutropin.

Peptides corresponding to the entire sequence of the alpha-subunit of the human glycoprotein hormones were synthesized by using standard solid-phase procedures. Purified peptides were incubated in the presence of alpha- and beta-subunits of bovine lutropin, and subunit recombination was monitored by difference spectroscopy, reverse-phase high-pressure liquid chromatography, and gel filtration chromatography. Although the binding of alpha-peptides to either subunit could not be detected by these techniques, it was possible to demonstrate that some peptides could inhibit the recombination of alpha- and beta-subunits. Specifically, alpha-peptide 33-58 allowed only 0-11% of subunit recombination in 24 h (38-56% after 48 h), while alpha-peptide 51-65 allowed 10-60% of subunits to recombine in 24 h (65-94% in 48 h). Peptides 1-15, 11-27, 22-39, 61-78, and 73-92 of the alpha-subunit could not inhibit subunit recombination at any time or at any concentration tested. The data suggest that at least a portion of the alpha-subunit contact site has been identified, and results are discussed in terms of protein structure assessment tools.

Topographic analysis of the alpha-subunit of human follicle-stimulating hormone using site-specific antipeptide antisera.

Structure-function investigations were undertaken to increase understanding of the surface topology of the alpha-subunit of human FSH (hFSH). The objectives were to determine which sequences of the alpha-subunit of hFSH are surface-oriented (exposed to antibody) and to identify which of these surface-oriented sequences are in contact with the beta-subunit of hFSH in the alpha/beta heterodimer. Seven overlapping synthetic peptides spanning the primary structure of hFSH alpha were used for immunizing rabbits to generate site-specific antipeptide antisera. The antisera were characterized with respect to their reactivity to the seven synthetic peptides, as well as hFSH, hFSH alpha, hFSH beta, and hFSH alpha r/a (reduced and alkylated), using an enzyme-linked immunosorbent assay. All of the peptides successfully generated antipeptide antisera with titers that range from 1:1,600 to 1:80,000. Anti-1-15 bound exclusively to hFSH alpha. Anti-11-27 and anti-33-58 bound to hFSH alpha to a much greater extent than to hFSH. In contrast, anti-73-92 had only slightly higher binding to hFSH alpha than to hFSH. Anti-22-39, anti-51-65, and anti-61-78 all failed to bind to either hFSH or hFSH alpha. With the exception of anti-22-39, all of the remaining antisera bound to hFSH alpha r/a. None of the antisera bound to hFSH beta. These data strongly suggest the following. Sequences 1-15, 11-27, and 33-58 contain residues that are masked by hFSH beta and are thus in or near the alpha/beta-subunit interface. In addition, sequences 11-27 and 33-58 contain other distinct residues that are surface-oriented in the hFSH heterodimer. In contrast, sequence 73-92 appears to be surface-oriented in the hFSH heterodimer. Lastly, sequences 51-65 and 61-78 appear to be buried within the native alpha-subunit and, thus, are unable to interact with antibodies. These results agree with and extend previous findings and will prove useful to those currently investigating the surface topology and structure-function relationships of the glycoprotein hormones.