Nirmal Chhikara

Publications (5) View all

• Article: Three Low Molecular Weight Cysteine Proteinase Inhibitors of Human Seminal Fluid: Purification and Enzyme Kinetic Properties.

  Vikash Kumar Yadav, Nirmal Chhikara, Kamaldeep Gill, Sharmistha Dey, Sarman Singh, Savita Yadav
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  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.
  Biochimie 04/2013; · 3.02 Impact Factor
• Article: Heparin and Heparin Binding Proteins: Potential relevance to reproductive physiology.

  Vikash Kumar Yadav, Mayank Saraswat, Nirmal Chhikara, Sarman Singh, Savita Yadav
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  ABSTRACT: Glycosaminoglycans (GAGs) have crucial roles in cell-cell interaction and communication. The communication between sperm and egg during fertilization is the finest example of intercellular communication involving a protein-carbohydrate recognition system. GAGs, especially heparin, are implicated in various processes, such as capacitation, acrosome reaction (AR), and sperm nuclei decondensation by interacting with a wide range of proteins, leading to fertilization. Seminal plasma (SP) comprises of multiple proteins that bind to heparin and related GAGs. Heparin binding proteins (HBPs) originating from secretions of the male accessory sex glands are known to play a vital role during fertilization events. They interact with GAGs present in the female genital tract and enhance the subsequent zona pellucida-induced AR, and thus have been correlated with fertility in some species. Several carbohydrate and zona pellucida-binding proteins, many of which belong to the spermadhesin family, are identified as HBPs. Many studies have been documented about the potential physiological role of some HBPs in various steps of fertilization. However, there is insufficient knowledge about functions executed by various HBPs and their exact mechanism and pathways. An in-depth knowledge of HBPs and their role in fertilization is of fundamental importance to resolve biological pathways and protein interactions at the molecular level. This review surveys some of the relevant findings supporting the potential role of heparin and HBPs in reproduction. It also describes consensus heparin binding sites emerging from known literature on HBPs that interact with heparin.
  Current Protein and Peptide Science 02/2013; · 2.89 Impact Factor
• Article: Human Epididymis Protein-4 (HE-4): A Novel Cross-Class Protease Inhibitor.

  Nirmal Chhikara, Mayank Saraswat, Anil Kumar Tomar, Sharmistha Dey, Sarman Singh, Savita Yadav
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  ABSTRACT: Epididymal proteins represent the factors necessary for maturation of sperm and play a crucial role in sperm maturation. HE-4, an epididymal protein, is a member of whey acidic protein four-disulfide core (WFDC) family with no known function. A WFDC protein has a conserved WFDC domain of 50 amino acids with eight conserved cystine residue. HE-4 is a 124 amino acid long polypeptide with two WFDC domains. Here, we show that HE-4 is secreted in the human seminal fluid as a disulfide-bonded homo-trimer and is a cross-class protease inhibitor inhibits some of the serine, aspartyl and cysteine proteases tested using hemoglobin as a substrate. Using SPR we have also observed that HE-4 shows a significant binding with all these proteases. Disulfide linkages are essential for this activity. Moreover, HE-4 is N-glycosylated and highly stable on a wide range of pH and temperature. Taken together this suggests that HE-4 is a cross-class protease inhibitor which might confer protection against microbial virulence factors of proteolytic nature.
  PLoS ONE 01/2012; 7(11):e47672. · 4.09 Impact Factor
• Article: Purification and characterization of a native zinc‐binding high molecular weight multiprotein complex from human seminal plasma

  Vikash Kumar Yadav, Vijay Kumar, Nirmal Chhikara, Sanjay Kumar, Pallavi Manral, Tara Kashav, Savita Saini, A. Srinivasan, Sarman Singh, Tej P. Singh, Savita Yadav
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  ABSTRACT: The seminal plasma comprises secretions from various accessory sex glands. During fertilization spermatozoa undergo complex sequences of precisely timed events that are regulated by the activation of different intracellular signaling pathways. The precision and efficacy of these pathways are often influenced by the assembly and interactions of multiprotein complexes, thereby directing the flow of regulatory information. Our knowledge about these protein complexes present in human seminal plasma (HuSP) is limited. Here we report the identification and characterization of a native high molecular weight zinc-binding multiprotein complex from HuSP by utilizing 2-DE followed by MS. Twenty-six proteins representing isoforms and/or fragments of 11 different proteins were found to be assembled in this complex. Prostate-specific antigen, zinc α2-glycoprotein, prostatic acid phosphatase, and prolactin inducible protein were the major proteins of this complex. Dynamic light scattering experiments revealed changes in aggregation pattern accompanied with deviation from physiological pH and in presence of SDS. However, no significant changes were observed in the presence of physiological ligands such as zinc and fructose. The present study will be useful and contribute to guide the future studies performed for elucidation of biological significance of this native complex in HuSP.
  Journal of Separation Science 03/2011; 34(9):1076 - 1083. · 2.73 Impact Factor
• Article: Differential proteomics of sperm: insights, challenges and future prospects.

  Anil Kumar Tomar, Mayank Saraswat, Nirmal Chhikara, Sanjay Kumar, Vikash Kumar Yadav, Balwinder Singh Sooch, Tej P Singh, Savita Yadav
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  ABSTRACT: Male factors account for 40% of infertility cases and most are caused by low sperm count, poor sperm quality or both. Defects in sperm are directly linked to reproductive malfunctions, and these defects may be caused by genetic mutations, environmental factors and exposure to free radicals, for example. Almost half of the male infertility cases have no known cause, indicating the lack of sensitive tests for the diagnosis of infertility. Proteomics has evolved as a major research field in biology and medicine, to identify and validate potent targets, at the molecular level, for development of more sensitive diagnostic tools. The recent advances in this field focus on the identification of differentially expressed proteins and analyzing their functional aspects for better understanding of the biological pathways. It not only provides a platform to discover biomarkers of infertility, but may also help in the design of effective male contraceptives. This article discusses various insights of proteomics for exploring biomarkers of male infertility in sperm. It also discusses the enhanced understanding of reproductive physiology offered by data produced by proteomic studies of spermatozoa.
  Biomarkers in Medicine 12/2010; 4(6):905-10. · 0.86 Impact Factor