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

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Not AvailableThe inhibins are disulphide-linked heterodimeric glycoproteins that belong to the TGFb superfamily. Inhibins have been well studied in mammals but the information about their structure and function is very limited in lower vertebrates. The aim of the present study was to characterize inhibin-A and to understand its receptor binding interaction, and to evaluate its biological function in Clarias batrachus. Structure prediction of inhibin-A revealed two glycosylation sites on inhibin-a (Asp262 and Asn334). Docking of inhibin-A with its receptor; betaglycan and Act RIIA showed that residues Ser321, Gly324 and Leu325 of inhibin-a are involved in high affinity binding with betaglycan while inhibin-bA bound to Act RIIA by forming hydrogen bonds. The mRNA transcript analysis of various tissues indicated the presence of higher to moderate expression of inhibin-a and inhibin-bA in the gonads and the extra- gonadal tissues. Further, stage specific expression showed decreased levels of inhibin-a in the gonads during the annual reproductive cycles. Inhibin-bA, activin-bB and Act RIIA increased in the brain during spawning while FSHr increased in the gonads during the preparatory phase. Our study provides molec- ular, structural and functional insights of inhibin-A for the first time in C. batrachus.Not Availabl

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

Co-immunoprecipitation of PSP94 and PAP proteins from fraction III.

<p><b>A.</b> PSP94-PAP complex from fraction III (50 µg) co-immunoprecipitated with anti-PAP antibody showing the presence of PSP94 (lane 3). Protein G beads incubated with either fraction III in buffer alone (lane 2) or fraction III incubated with mouse isotype control antibody (lane 1) served as controls. 10 µg of fraction III was loaded in lane 4 as input and the immunoblot was probed with anti-PSP94 antibody. <b>B.</b> PSP94-PAP complex from fraction III (50 µg) was co-immunoprecipitated with anti-PSP94 antibody showing the presence of PAP (lane 3). Protein G beads incubated with either fraction III in buffer alone (lane 2) or fraction III incubated with normal rabbit serum (lane 1) served as controls. 10 µg of fraction III was loaded in lane 3 as input and the immunoblot was probed with anti-PAP antibody. Molecular weight markers shown are in kDa.</p

Detection of PSP94-PAP complex in human seminal plasma.

<p><b>A.</b> PSP94-PAP complex from seminal plasma (100 µg) was co-immunoprecipitated with anti-PAP antibody (lane 1). Protein G beads incubated with seminal plasma in buffer alone served as the negative control (lane 2). 20 µg of seminal plasma was loaded in lane 3 as input and the immunoblot was probed with anti-PSP94 antibody. The immunoreactive band of PSP94 (∼17 kDa) is detected only in lane 1 and not in lane 2. <b>B.</b> PSP94-PAP complex from seminal plasma (100 µg) was co-immunoprecipitated with anti-PSP94 antibody (lane 1). Protein G beads incubated with seminal plasma in buffer alone served as the negative control (lane 2). 20 µg of seminal plasma was loaded in lane 3 as input and the immunoblot was probed with anti-PAP antibody. The immunoreactive band of PAP (∼47 kDa) is detected only in lane 1 and not in lane 2. Molecular weight markers shown are in kDa.</p

Mass spectrometric analysis of spots excised from the 2D gel.

*<p>Note: Discrepancy in the isoelectric point (pI) or molecular weight (MW) of the proteins detected here with that of the protein entries in NCBI database could be probably due to the post-translational modifications or protein processing.</p

Fractionation of human seminal plasma proteins.

<p><b>A.</b> Preparative RP-HPLC profile of seminal plasma proteins (bound fraction from Phenyl Sepharose chromatography) showing three major peaks (fractions I, II and III). <b>B.</b> Immunoblot of fractions I, II and III probed with anti-PSP94 antibody. <b>C.</b> One dimensional SDS-PAGE profile of fractions I, II and III. Molecular weight markers shown are in kDa.</p