Biomedical subjects

Anil Suri

Publications and source records attributed to Anil Suri.

11 recordsLinked to original sources

PubMed2006

Immunogenicity study of recombinant human sperm-associated antigen 9 in bonnet macaque (Macaca radiata).

BACKGROUND: The human sperm-associated antigen 9 (hSPAG9) is of special interest attributing to the findings indicating that SPAG9 is an acrosomal molecule. SPAG9 is not only restricted to acrosomal compartment but also persists in equatorial segment post-acrosome reaction, which is a key location in sperm-egg interaction. METHODS AND RESULTS: Immunogenicity studies in macaques were carried out with recombinant hSPAG9 (rhSPAG9) adsorbed on alum, which resulted in high titres of anti-rhSPAG9 antibodies as determined by enzyme-linked immunosorbent assay (ELISA). Immunoblotting analysis employing anti-rhSPAG9 antibodies generated in monkeys indicated that antibodies specifically reacted with native SPAG9 from macaque and human sperm and rhSPAG9 protein. Furthermore, indirect immunofluorescence experiments demonstrated SPAG9 localization in the acrosomal compartment of macaque and human sperm. In addition, monkey antibodies against rhSPAG9 significantly inhibited the human spermatozoa adherence or penetration in zona-free hamster oocytes. CONCLUSION: These results demonstrate that rhSPAG9 adsorbed on alum is highly immunogenic in subhuman primate model and therefore represents a suitable sperm-based vaccine immunogen for fertility trials in macaque.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Cancer testis antigens--their importance in immunotherapy and in the early detection of cancer.

The development of successful immunotherapeutic strategies requires the identification and characterisation of immunogenic cancer antigens that will be recognised by the host immune system, leading to tumour rejection. The concept of immunotherapy is based on the assumption that antigenic structures expressed in tumours can be used for therapeutic approaches employing the autologous immune system or by the application of immunotherapeutic reagents. Based on this concept, there is a great need to gain profound knowledge of the actual protein/antigen expression and its distribution pattern within normal tissues and cancerous tissues. Cancer testis (CT) antigens represent a unique class of tumour antigens, which are expressed in a variety of cancerous tissues and are silent in normal tissues, except for the testis. Owing to their restricted gene expression in the testis and various malignancies, CT antigens represent potential defined targets for antigen-based vaccination and antigen-directed immunotherapy to control cancer growth. Moreover, the analysis of humoral and cellular immune responses to CT antigens has proved useful for identifying novel cancer serum biomarkers with potential implications in early diagnosis of cancer.

Animals↗

PubMed2005

Small interference RNA-mediated knockdown of sperm associated antigen 9 having structural homology with c-Jun N-terminal kinase-interacting protein.

Recently, we reported a novel testis-specific sperm associated antigen 9 (SPAG9) protein, a new member of the JNK-interacting protein family, having a functional role in sperm-egg fusion [N. Jagadish, R. Rana, R. Selvi, D. Mishra, M. Garg, S. Yadav, J.C. Herr, K. Okumura, A. Hasegawa, K. Koyama, A. Suri, Biochem. J. 389 (2005) 73-82]. NCBI Blast searches revealed SPAG9 nucleotide sequence similarities with ESTs of various cancerous tissues. In the present study, we compared the efficiency of two independent SPAG9 specific small interfering RNA (siRNA) constructs, BS/U6/spag9 and BS/U6/spag9-I, to ablate the SPAG9 expression in mammalian cells. A positive correlation between the ratio of target gene versus siRNA and the suppression of SPAG9 expression was observed. Further, the cotransfection of BS/U6/spag9 with pcDNA-SPAG9 and pFlag-CMV2-JNK-3 resulted in specific suppression of SPAG9 without affecting JNK-3 expression. The present investigation will eventually extend the application of SPAG9 siRNA in in vivo targeting experiments that aim to define the SPAG9 functional genomics in tumor and reproductive biology.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Sperm-based contraceptive vaccines: current status, merits and development.

At the present growth rate, the world population is estimated to reach a phenomenal 8.9 billion people by the year 2050, posing a great risk of overpopulation. Therefore, new strategies of contraception are required. A novel contraceptive strategy that is receiving considerable attention is that of immunocontraception. Spermatozoa have proteins that are unique, cell specific, immunogenic and accessible to antibodies. The targeting of antibodies to gamete-specific antigens implicated in sperm function, sperm-egg binding and fertilisation could block sperm binding and thus fertilisation. The present review highlights the current status, relative merits and future directions for various sperm-based candidate antigens with regard to the development of a contraceptive vaccine.

Animals↗

PubMed2005

Immunogenicity and contraceptive potential of recombinant human sperm associated antigen (SPAG9).

Human sperm-associated antigen 9 (hSPAG9) is a potential target for sperm-based contraceptive vaccine in lieu of its location on the sperm acrosomal compartment and its implication in sperm-egg interaction. SPAG9 is an acrosomal molecule which is not only restricted to a specific region (domain) of the acrosome but also undergoes relocation to the equatorial region in a stage-specific manner during acrosome reaction, demonstrating its potential role in sperm-egg binding. Human SPAG9 nucleotide sequence revealed 94% identity with macaque SPAG9 and 96.8% with baboon SPAG9 over the entire sequence. The amino acid sequence comparison of human SPAG9 with macaque and baboon revealed an overall homology of 84.9% and 90.6%, respectively. The presence of a high level of homology at the amino acid and nucleotide levels indicates that SPAG9 is conserved in macaque, baboon and human sharing common function and common origin in the biological past. Immunogenicity studies were carried in rats, which demonstrated that recombinant hSPAG9 protein adsorbed on alum is highly immunogenic. Antibodies thus generated after immunization reacted with recombinant human SPAG9 (rhSPAG9) and native SPAG9 protein from human sperm in Western blot analysis. In an in vitro assay, anti-rhSPAG9 antibodies inhibited sperm adherence to or penetration in zona-free hamster egg penetration test. Further, anti-SPAG9 antibodies inhibited the binding of human sperm to intact human oocyte as well as to matched hemi-zonae, indicating that the recombinant protein is a suitable contraceptive vaccinogen. Together these results demonstrate that the rhSPAG9 adsorbed on alum is immunogenic in nature, which is a permissible adjuvant for immunogenicity and fertility trials in non-human primates.

Acrosome Reaction↗

PubMed2005

Characterization of immune response in mice to plasmid DNA encoding human sperm associated antigen 9 (SPAG9).

To examine the immunogenicity of the plasmid DNA encoding human sperm associated antigen 9 (hSPAG9), the cDNA corresponding to hSPAG9 was cloned in mammalian expression vector pcDNA 3.1 down stream of cytomegalovirus promoter. Immunization of female BALB/cJ mice with pcDNA-hSPAG9 plasmid DNA in saline by intramuscular (i.m.), by adsorbing onto gold microcarriers (delivered by gene gun) and by recombinant hSPAG9 (r-hSPAG9) protein generated antibody response against Escherichia coli expressed r-hSPAG9 protein and native SPAG9 in human sperm. Although mice immunized with r-hSPAG9 protein exhibited highest antibodies titres (P<0.001), the difference in the antibody titres seen by the two modes of plasmid DNA delivery were not significant (P>0.05). A dominant IgG1 isotype response was observed in mice immunized with pcDNA-hSPAG9 plasmid DNA delivered by gene gun as compared to a mixed IgG1-IgG2a isotype response in mice immunized with r-hSPAG9 protein and pcDNA-hSPAG9 plasmid DNA delivered by i.m. Further, antibodies generated by pcDNA-hSPAG9 plasmid DNA localized acrosomal compartment of human sperm and inhibited sperm adherence to or penetration in zona-free hamster egg penetration test. These studies for the first time, demonstrate the feasibility of generating an immune response to sperm specific hSPAG9 protein by DNA vaccine and that antibodies thus generated recognize native SPAG9 in human sperm.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Characterization of a novel human sperm-associated antigen 9 (SPAG9) having structural homology with c-Jun N-terminal kinase-interacting protein.

We report a novel SPAG9 (sperm-associated antigen 9) protein having structural homology with JNK (c-Jun N-terminal kinase)-interacting protein 3. SPAG9, a single copy gene mapped to the human chromosome 17q21.33 syntenic with location of mouse chromosome 11, was earlier shown to be expressed exclusively in testis [Shankar, Mohapatra and Suri (1998) Biochem. Biophys. Res. Commun. 243, 561-565]. The SPAG9 amino acid sequence analysis revealed identity with the JNK-binding domain and predicted coiled-coil, leucine zipper and transmembrane domains. The secondary structure analysis predicted an alpha-helical structure for SPAG9 that was confirmed by CD spectra. Microsequencing of higher-order aggregates of recombinant SPAG9 by tandem MS confirmed the amino acid sequence and mono atomic mass of 83.9 kDa. Transient expression of SPAG9 and its deletion mutants revealed that both leucine zipper with extended coiled-coil domains and transmembrane domain of SPAG9 were essential for dimerization and proper localization. Studies of MAPK (mitogenactivated protein kinase) interactions demonstrated that SPAG9 interacted with higher binding affinity to JNK3 and JNK2 compared with JNK1. No interaction was observed with p38alpha or extracellular-signal-regulated kinase pathways. Polyclonal antibodies raised against recombinant SPAG9 recognized native protein in human sperm extracts and localized specifically on the acrosomal compartment of intact human spermatozoa. Acrosome-reacted spermatozoa demonstrated SPAG9 immunofluorescence, indicating its retention on the equatorial segment after the acrosome reaction. Further, anti-SPAG9 antibodies inhibited the binding of human spermatozoa to intact human oocytes as well as to matched hemizona. This is the first report of sperm-associated JNK-binding protein that may have a role in spermatozoa-egg interaction.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Contraceptive vaccines targeting sperm.

Overpopulation is a global problem of significant magnitude, with grave implications for the future. Development of new contraceptives is necessary, as existing forms of birth control are unavailable, impractical and/or too expensive for many individuals due to sociological, financial or educational limitations. Immunocontraception and, in particular, the targeting of antibodies to sperm-specific antigens implicated in sperm-egg binding and fertilisation offers an attractive approach to control fertility. Sperm-specific antibodies may impair fertility by inhibiting sperm motility, by reducing penetration of the cervical mucus by sperm, or by interfering in sperm capacitation or the acrosome reaction; alternatively, antisperm antibodies may invoke the complement cascade, resulting in sperm lysis. The antibodies raised against sperm-specific antigens have proved to be extremely effective at reducing sperm-egg interactions in vitro; fertility trials in subhuman primates will eventually be needed to prove the effectiveness of the sperm antigens in terms of contraceptive efficacy before trials in humans can be justified. In addition, existing and emerging strategies (such as sperm proteomics, the determination of molecular and structural details of sperm proteins, and the modelling of protein-ligand interactions using X-ray and/or NMR structures to name a few) are expected to provide the experimental foundation for the design of small molecule inhibitors with antifertility effects. The technology underpinning vaccine development is constantly being developed and the introduction of DNA/RNA vaccines is certain to impact upon the field of immunocontraception.

Animals↗

PubMed2005

Sperm associated antigen 9 (SPAG9): a new member of c-Jun NH2 -terminal kinase (JNK) interacting protein exclusively expressed in testis.

Previously, we cloned and sequenced a novel human sperm associated antigen 9 (SPAG9). Northern blot analysis and RNA in situ hybridization experiments revealed testis- and stage-specific expression of SPAG9 mRNA, mainly confined to round spermatid suggesting haploid germ cell expression Studies on the human and non-human primates (macaque and baboon) have shown a homology of 84.9% and 90.6% at amino acid level and 94% and 96.8% at DNA level, respectively. The presence of high level of homology at amino acid and DNA level indicates that SPAG9 is conserved in human, baboon and macaque sharing common function and common origin in the biological past. In addition, SPAG9 protein revealed structural homology with c-Jun NH2-terminal kinase (JNK) interacting protein (JIP). The amino acid sequence analysis of SPAG9 predicted coiled coil, leucine zipper and transmembrane domain, speculating the involvement of SPAG9 mediated signal transduction pathways in reproductive processes.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Sperm specific proteins-potential candidate molecules for fertility control.

The increase in population growth rate warrants the development of additional contraceptive methods that are widely acceptable, free from side effects and less expensive. Immunocontraception, and in particular the targeting of antibodies to gamete-specific antigens implicated in sperm egg binding and fertilization, offers an attractive approach to control fertility. The development of a contraceptive vaccine based on sperm antigen represents a promising approach to contraception. In mammals, fertilization is completed by the direct interaction of sperm and egg, a process mediated primarily by sperm surface proteins. Sperm have proteins that are unique, cell specific, immunogenic and accessible to antibodies. A few of the sperm specific proteins have been isolated and characterized. The antibodies raised against the sperm specific antigens have proved to be extremely effective at reducing sperm-egg interaction in vitro; fertility trials in sub-human primates would eventually prove the effectiveness of the sperm antigens in terms of contraceptive efficacy.

Animals↗

PubMed2004

Molecular cloning, expression of testicular transcript abundant in germ cells and immunobiological effects of the recombinant protein.

PROBLEM: It has been well documented that antisperm antibodies can be causative factors for infertility. In this report we have identified a protein on human sperm referred as human sperm-associated protein (HSAP) using serum of an immunoinfertile woman; it is thus a sperm-specific protein--a candidate molecule for control of fertility. METHOD OF STUDY: An immunoinfertile woman serum showing head-head sperm agglutination and acrosomal localization, reacted with human sperm protein of apparent molecular weight of 48 kDa on Western blot. Anti-48 kDa antiserum was raised in rabbit by eluting 48 kDa protein and was used to screen the human testis cDNA expression library. A putative positive hsap cDNA clone was obtained, sequenced and subjected to tissue specificities studies by Northern blotting. The cell type-specific expression was done using in situ RNA hybridization studies. To obtain recombinant HSAP (r-HSAP), hsap cDNA was cloned in pET 22b(+) expression vector. r-HSAP was expressed as polyhistidine fusion protein in Escherichia coli and purified. Rabbits were immunized with the purified r-HSAP, which led to generation of antibodies. In order to evaluate in vitro immunocontraceptive potential, the anti-r-HSAP antibodies were characterized by agglutination assay, zona-free hamster egg penetration assay, indirect immunofluorescence (IIF) assay, and by flow cytometry analysis. RESULTS: We have cloned a human testis gene encoding a protein (HSAP) of 328 amino acids. Antibodies against the purified recombinant protein specifically recognized approximately 40 kDa r-HSAP, and a cognate 48 kDa protein band in human sperm extract in Western blot procedure. The anti-r-HSAP antibodies localized acrosomal compartment, inhibited sperm binding/attachment in zona-free hamster penetration assay and revealed surface binding with human live sperm by flow cytometry. The cDNA sequence has been submitted to EMBL and has been given the accession number Y16676. CONCLUSION: This study has put in evidence that novel sperm-specific r-HSAP has role in sperm function and may have application in the development of a contraceptive vaccine. The availability of the recombinant protein will facilitate studies on the assessment of its potential as a contraceptive immunogen.

Amino Acid Sequence↗