Human chorionic gonadotropin: An update on its receptor binding regions

448-455Human chorionic gonadotropin (hCG), an important member of the glycoprotein hormone family, plays a crucial role in the establishment and maintenance of pregnancy. Glycoprotein hormones are all heterodimers composed of a common α subunit and a hormone-specific β-subunit noncovalently linked. These hormones exert their action by binding to specific receptors on the target cells. As both the subunits are involved in receptor binding, the binding sites on the hormones have to be topographical extending across both subunits. This article attempts to critically review the work on the receptor binding regions of hCG in the α- and β-subunits. A variety of approaches like chemical modification, site-directed mutagenesis, hormone chimeras and synthetic peptides have been used to map the receptor binding regions of hCG. There is agreement on the involvement of a number of regions in the α- and β- subunits in receptor binding but, there are also some contrasting observations. The regions 30-45 and 81-92 in the α-subunit appear to be involved in receptor binding whereas 8-22, 85-95, 93-100 and 100-110 are the consensus receptor binding regions in the β-subunit. Using disulphide peptides of the β-subunit as probes, recently the regions around Cys(9-57) and Cys(23-72) disulphide bonds have been identified to be important for receptor binding. After sifting through the available data two potential receptor binding sites in the β-subunit have been proposed

Disulphide bond reduction and S-carboxamidomethylation of PSP94 affects its conformation but not the ability to bind immunoglobulin

Prostate secretory protein of 94 amino acids (PSP94) is a small non-glycosylated, cysteine rich protein with a molecular mass of 10 kDa. It has also been referred to as β-microseminoprotein (β-MSP) and proteins homologous to it have been reported in a number of species. Comparison of the amino acid sequence of these proteins suggests that, it is a rapidly evolving protein. However, all the ten cysteine residues are well conserved in these homologues, indicating their possible role in maintaining the structure and function of these proteins. In the present study, PSP94 was purified from human seminal plasma and characterized further and it showed the presence of five disulfide bonds. Reduction of disulphide bonds of PSP94 led to significant changes in the secondary and tertiary structure of PSP94. CD of disulphide bond reduced PSP94 indicates an overall decrease in the beta sheet content from 79.8% to 46.4%. Tertiary structural changes as monitored by fluorescence quenching reveal that reduction of disulphide bonds of PSP94 followed by the modification of the free thiol groups leads to complete exposure of Trp32 and Trp92 and that one or more side chain carboxyl groups move closer to their indole side chains. Antibodies against native and modified PSP94 demonstrated that the changes following reduction of disulphide linkages are within the immunodominant region of the protein. Changes induced in the functional properties of PSP94, if any, by modification were investigated with respect to IgG binding as PSP94 has been reported to be similar to immunoglobulin binding factor purified from seminal plasma. A novel finding from this study is that both native PSP94 as well as modified protein have the ability to bind human IgG, suggesting the involvement of sequential epitopes of PSP94 in IgG binding

Expression of Bioactive Callithrix jacchus Follicle-Stimulating Hormone in Pichia pastoris

Callithrix jacchus (common marmoset) is a New World primate monkey, used as an animal model in biomedical research. Marmoset-specific follicle-stimulating hormone (FSH) preparation is required to improve superovulation protocols and to develop homologous FSH monitoring assays in these monkeys. In this study, we document the large-scale expression of recombinant marmoset FSH in methylotropic yeast, Pichia pastoris. The recombinant preparation was found to be immunologically active in Western blotting and radioimmunoassay. The preparation displayed receptor binding ability in radioreceptor assay. Based on the receptor binding ability, the yield of fermentation was estimated to be 7.2 mg/L. FSH-induced cAMP assay and estradiol assay revealed that the recombinant hormone is able to induce signal transduction. Both immunological and in vitro biological activity of marmoset FSH was found to be comparable to purified human pituitary FSH, which served as reference hormone for these assays. Thus, the study suggests that a Pichia expression system can be used for large-scale expression of bioactive recombinant marmoset FSH

Prostate Secretory Protein of 94 Amino Acids (PSP94) Binds to Prostatic Acid Phosphatase (PAP) in Human Seminal Plasma

<div><p>Prostate Secretory Protein of 94 amino acids (PSP94) is one of the major proteins present in the human seminal plasma. Though several functions have been predicted for this protein, its exact role either in sperm function or in prostate pathophysiology has not been clearly defined. Attempts to understand the mechanism of action of PSP94 has led to the search for its probable binding partners. This has resulted in the identification of PSP94 binding proteins in plasma and seminal plasma from human. During the chromatographic separation step of proteins from human seminal plasma by reversed phase HPLC, we had observed that in addition to the main fraction of PSP94, other fractions containing higher molecular weight proteins also showed the presence of detectable amounts of PSP94. This prompted us to hypothesize that PSP94 could be present in the seminal plasma complexed with other protein/s of higher molecular weight. One such fraction containing a major protein of ∼47 kDa, on characterization by mass spectrometric analysis, was identified to be Prostatic Acid Phosphatase (PAP). The ability of PAP present in this fraction to bind to PSP94 was demonstrated by affinity chromatography. Co-immunoprecipitation experiments confirmed the presence of PSP94-PAP complex both in the fraction studied and in the fresh seminal plasma. In silico molecular modeling of the PSP94-PAP complex suggests that β-strands 1 and 6 of PSP94 appear to interact with domain 2 of PAP, while β-strands 7 and 10 with domain 1 of PAP. This is the first report which suggests that PSP94 can bind to PAP and the PAP-bound PSP94 is present in human seminal plasma.</p> </div

Two-dimensional gel electrophoresis profile of fraction III.

<p>The 2D gel resolved by isoelectricfocusing in the first dimension (pH range 3–10) followed by SDS-PAGE in the second dimension was stained with silver nitrate. The protein spots (circled) were excised and subjected to MS and MS/MS analysis (Data shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058631#pone-0058631-t001" target="_blank">Table 1</a>). Molecular weight markers shown are in kDa.</p

Identification of proteins in fraction III.

<p><b>A.</b> MALDI-TOF mass spectra of fraction III from preparative RP-HPLC showing a major peak of molecular mass 46753 Da. The peak at 10772 Da is probably of PSP94. <b>B.</b> Immunoblot analysis of fraction III probed with anti-PAP antibody. Molecular weight markers shown are in kDa.</p

Interaction of pure PSP94 and PAP proteins in vitro.

<p>500 ng of PSP94 incubated with or without PAP (500 ng) (lane 1 and 2 respectively) and immunoprecipitated using anti-PAP antibody. Pure PSP94 (20 ng; lane 3) and PAP (500 ng; lane 4) proteins were loaded as input. Lanes 1, 2 and 3 were immunoblotted with anti-PSP94 antibody, while lane 4 was immunoblotted with anti-PAP antibody. The immunoreactive band of PSP94 (∼17 kDa) is detected only in lane 1 and not in lane 2. Molecular weight markers shown are in kDa.</p