ABSTRACT
Background & objectives: Several host defense proteins known to possess antimicrobial activities are present on mucosal surfaces and are consequently found in body fluids of vertebrates. Naturally occurring protease inhibitors like cystatins, especially cystatin C (cys C), are abundantly present in human seminal plasma. Although its antiviral activity against herpes simplex virus (HSV) has been demonstrated, the role of this protein against HIV is not well studied. Therefore, the aim of the present study was to evaluate the anti-HIV activities of cys C, which is present innately in the male reproductive tract. Methods: Protein-protein interaction of cys C with various HIV proteins was studied using a commercially available HIV blot and specific interaction with HIV protease was studied by dot-blot technique using commercially available cys C. to purify biologically active cys C from human seminal plasma to be used for subsequent experiments, gel-permeation chromatography followed by affinity chromatography was used. The HIV infectivity inhibition activity of the purified cystatin C was tested in TZM-bl cells. To study its activity on HIV protease, time-course enzyme kinetics studies were performed using spectrometric assay. Results: Cystatin C reacted with some HIV proteins including HIV protease. Biologically active cys C was purified using gel permeation chromatography followed by affinity chromatography. When tested in TZM-bl cells, purified cystatin C demonstrated HIV-infectivity inhibitory activity (IC50: 0.28 μM). enzyme kinetic studies demonstrated that it abrogated the action of HIV protease on its substrate. Interpretation & conclusions: The present data demonstrate that cystatin C possesses anti-HIV activities. molecular models need to be designed with this protein which would assist towards prevention/ therapeutics against HIV.
ABSTRACT
Human immunodeficiency virus (HIV) is genetically extremely variable due to the poor proof reading activity of its reverse transcriptase enzyme. Human immunodeficiency virus isolates are highly variable over time, and exhibit changes in biological phenotype during the course of infection. Different HIV variants exist in different tissues, cells and secretions; including genital secretions and cells of human males and females. Virus present in the urogenital cells and secretions determines the risk for sexual transmission of HIV. The precise association of viral variants from genital secretions and cells in the sexual transmission of HIV to the partner is not fully understood. The presence of viral variants may influence affinity to different host cell receptors which may affect the transmission, infectivity, cellular immunity and pathogenesis of HIV. Delineation of the role of host and pathogenic variation will lead to a better understanding of the process of sexual transmission of HIV. Furthermore, it will also help in designing the strategies for development of preventive or therapeutics vaccines and microbicides for control and management of HIV/AIDS.
ABSTRACT
Mammalian alkaline phosphatase (ALP) is attached to the plasma membrane by a unique glycosylphosphatidylinositol (GPI) anchor. The influence of such a complex anchoring device on the enzyme function is not fully understood. Here, we report the effect of cleavage of the GPI anchor on the activity of goat liver plasma membrane ALP (GLPM-ALP). Phosphatidylinositol-specific phospholipase C (PI-PLC) purified from Bacillus cereus was used for the cleavage of the GPI anchor (delipidation) and hence for release of ALP from the membrane. Detergents — octyl-β-D-glucopyranoside (OG) and triton X100 (TX100) were also used for solubilization of ALP from the membrane. Resistance to solubilization by TX100 suggested the association of GPI-ALP with lipid rafts. Solubilization of GLPM-ALP with OG had no effect on the enzyme activity; however, delipidation with PI-PLC resulted in enhanced ALP activity. Kinetic analysis showed catalytic activation of PI-PLC-treated GLPM-ALP with an increase in Vmax (35%) without a significant change in Km. Moreover, this change in Vmax was observed to be independent of pH and buffer. The results suggested the implication of GPI anchor in modulating the catalytic property of GLPM-ALP, thus indicating the role of this special anchoring structure in the enzyme regulation.