title: Disulphide bond reduction and S-carboxamidomethylation of PSP94 affects its conformation but not the ability to bind immunoglobulin.
abstract: 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 beta-microseminoprotein (beta-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.
authors.affiliation: Division of Structural Biology, National Institute for Research in Reproductive Health (Indian Council of Medical Research), Jehangir Merwanji Street, Parel, Mumbai-400012, India.
authors.forename: Dhanashree D
authors.initials: DD
authors.lastname: Jagtap
authors.forename: Akkaladevi
authors.initials: A
authors.lastname: Narahari
authors.forename: Musti J
authors.initials: MJ
authors.lastname: Swamy
authors.forename: Smita D
authors.initials: SD
authors.lastname: Mahale
mesh_terms: D000577:Amides / Q000737:chemistry* / Q000378:metabolism; D002264:Carboxylic Acids / Q000737:chemistry* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007136:Immunoglobulins / Q000276:immunology*; D008745:Methylation; D010084:Oxidation-Reduction; D029584:Prostatic Secretory Proteins / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D000577:Amides; D002264:Carboxylic Acids; D004220:Disulfides; D007136:Immunoglobulins; D029584:Prostatic Secretory Proteins; C043076:beta-microseminoprotein