title: Three low molecular weight cysteine proteinase inhibitors of human seminal fluid: purification and enzyme kinetic properties.
abstract: The cystatins form a superfamily of structurally related proteins with highly conserved structural folds. They are all potent, reversible, competitive inhibitors of cysteine proteinases (CPs). Proteins from this group present differences in proteinase inhibition despite their high level of structural similarities. In this study, three cysteine proteinase inhibitors (CPIs) of low molecular weight were isolated from human seminal fluid (HSF) by affinity chromatography on carboxymethyl (CM)-papain-Sepharose column, purified using various chromatographic procedures and checked for purity on sodium-dodecyl PAGE (SDS-PAGE). Matrix-assisted laser desorption-ionization-time-of flight-mass spectrometry (MALDI-TOF-MS) identified these proteins as cystatin 9, cystatin SN, and SAP-1 (an N-terminal truncated form of cystatin S). All three CPIs suppressed the activity of papain potentially and showed remarkable heat stability. Interestingly SAP-1 also inhibits the activity of trypsin, chymotrypsin, pepsin, and PSA (prostate specific antigen) and acts as a cross-class protease inhibitor in in vitro studies. Using Surface Plasmon Resonance, we have also observed that SAP-1 shows a significant binding with all these proteases. These studies suggest that SAP-1 is a cross-class inhibitor that may regulate activity of various classes of proteases within the reproductive systems. To our knowledge, this is the first report about purification of CPIs from HSF; the identification of such proteins could provide better insights into the physiological processes and offer intimation for further research.
authors.affiliation: Department of Biophysics, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, India.
authors.forename: Vikash Kumar
authors.initials: VK
authors.lastname: Yadav
authors.forename: Nirmal
authors.initials: N
authors.lastname: Chhikara
authors.forename: Kamaldeep
authors.initials: K
authors.lastname: Gill
authors.forename: Sharmistha
authors.initials: S
authors.lastname: Dey
authors.forename: Sarman
authors.initials: S
authors.lastname: Singh
authors.forename: Savita
authors.initials: S
authors.lastname: Yadav
mesh_terms: D002846:Chromatography, Affinity; D015891:Cystatins / Q000737:chemistry / Q000235:genetics; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D006801:Humans; D007700:Kinetics; D008297:Male; D008969:Molecular Sequence Data; D008970:Molecular Weight; D055550:Protein Stability; D029589:Seminal Plasma Proteins / Q000737:chemistry* / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature
chemical_list: D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D029589:Seminal Plasma Proteins