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Genes
and proteins expressed specifically in testis |
Principal Investigator : Anil K Suri Project Associates/Assistants
Collaborators The
goal of this project is to investigate testis specific genes expressed in a
restricted, temporal and spatial manner during spermatogenesis. Morphological
and biochemical observations along the molecular biological approaches suggest
that germ cell differentiation is accompanied by selective gene expression.
However, the molecular mechanisms regulating the cell type specifically gene
expression during spermatogenesis are yet to be determined. Identification of
testis specific genes and their characterization would thus enhance our
understanding about the functioning of sperm at the molecular level. This
would possibly highlight the mechanism of sperm development, sperm
functioning, and sperm-egg interaction. Characterization
of A-kinase (PKRA) anchoring protein 4 (earlier called hi; AKAP 4, EMBL
nomenclature) gene product: Mammalian
sperm motility is regulated by a cascade of cAMP-dependent protein
phosphorylation events mediated by protein kinase A. A-kinase anchor proteins
direct protein kinase A activity by tethering the enzyme near its physiologic
substrates. The A-kinase anchor proteins (AKAPs) are a group of structurally
diverse proteins, which have the common function of binding to the regulatory
subunit of protein kinase A (PKA) and confining the holoenzyme to discrete
locations within the cell. AKAP may be involved in the regulation of sperm
motility. In
the course of identifying sperm proteins, we cloned the human hi mRNA from a
testis cDNA expression library. This is referred as AKAP4 [A-kinase (PKRA)
anchoring protein 4, EMBL nomenclature]. Peptides corresponding to target
domain and regulatory domains were synthesized for motility studies. These
peptides were conjugated with stearic acid. Inhibition of sperm motility was
studied using various concentrations of peptides. Characterization
of SPAG9 (Sperm associated sperm antigen 9) Characterization
of the recombinant SPAG9: The
recombinant SPAG9 expressed in E.coli revealed a major band at ~170 kDa
in coomassie blue staining. Since the deduced molecular weight of SPAG9 is
83.9 kDa, the ~170 kDa molecular mass is apparently due to dimerization of
protein. The antibodies to recombinant SPAG9 specifically recognized the
recombinant protein in the western blot procedure, showing a single band of
~170 kDa. Proper folding and secondary structure of SPAG9 protein was
investigated by the CD spectrum analysis. The CD spectra exhibited minima at
220 and 208, which is characteristic of a a-helical
confirmation. The helical content calculated from CD spectra was found to be
40.2%, which was in agreement with the theoretical predictions of 41.38%. Homologues
of human SPAG9 gene: Homologue genes
of SPAG9 were characterized from macaque and baboon. These results will
have implications for understanding the basic biology of SPAG9. Macaque SPAG9
comprises of 2379 bp and has 94% and 92.2% homology with entire nucleotide
sequence of hSPAG9 and baboon SPAG9, respectively. However, ORFs
of macaque SPAG9 and human SPAG9 shared 95.5% identity. The
macaque SPAG9 has inframe deletion of 93 bp starting at 1117 bp and 51
bp inframe deletion starting at 1770 bp as compared to human SPAG9. The
macaque SPAG9 5’ UTR contains 110 nt having 100% identity with hSPAG9
whereas the macaque SPAG9 3’ UTR contains 130 nt as compared to 112
nt in hSAPG9. Sequence comparison of macaque 3’UTR, revealed 18
nucleotides deletion in 3’UTR of the hSPAG9. However, rest of the
3’UTR of macaque showed 100% identity. Significantly, the 5’ UTR and 3’
UTR in macaque SPAG9 has been conserved in the hSPAG9. The
amino acid sequence comparison of macaque SPAG9 with baboon and human SPAG9
revealed an overall homology of 87.0% (712/760 aa) and 84.9% (712/766 aa),
respectively. However, when macaque N-terminal amino acid sequence (1-610 aa)
was compared with baboon and human sequence, 100% identity with conserved
functional domains namely JNK binding domain, leucine zipper motif, two coiled
coil domains and a transmembrane domain was observed. It is noteworthy that
macaque SPAG9 was found to have 31 amino acid deletions starting at 336 and
another 17 amino acid deletion at 553 as compared to baboon and human. The
carboxyl region of macaque SPAG9 expanding from 641 to 712 aa exhibited
the great degree of difference and species conservation in comparison to
baboon and human amino acids. Using genestream, align program, carboxyl region
from 641 to 712 aa of macaque with 693 to 760 aa of baboon and 689 to 766 aa
of human revealed a 29.6% (71/71aa) and 18.5% (71/77 aa) homology
respectively. Domain
structure of SPAG9: The amino acid
sequence analysis of SPAG9 showed the presence of a putative JNK binding
domain (JBD), a characteristic leucine zipper motif (LZ), an extended
coiled-coil domain (coil) and a transmembrane domain/lipoprotein binding
domain site (T). GenBank database search indicated two entries describing
structural homology to SPAG9. One cDNA encodes a predicted protein that has
structural similarity to the C-terminal region and represent a fragment of JNK
interacting protein 3 (JIP3). A second partial human cDNA also encodes a
protein with similarity to C-terminus of SPAG9 but this protein is the product
of distinct gene and may be related member of JIP3 group. SPAG9
interaction with mammalian MAPKs: Structural
homology analysis with JIP protein showed presence of signature domains of JNK
interacting protein 3. Therefore, it was of interest to determine whether
SPAG9 could also interact with MAPKs in a similar manner. The binding
specificity of SPAG9 with various MAPKs was attempted in cotransfection
experiments. Initial studies revealed that SPAG9 interacts with JNK1.
Interaction of SPAG9 with JNK 2, JNK 3, ERK 2 or p38a
in CHO cells is being carried out. Role
of SPAG9-JIP3g
in human sperm-oocyte interaction: The
involvement of SPAG9 protein in fertilization process was further confirmed by
inhibition of sperm-zona interaction by its antibodies. Antibodies raised
against the purified recombinant protein, that specifically recognized the
recombinant as well as cognate SPAG9 protein on the Western blot, completely
blocked sperm binding to zona pellucida of the human oocyte. Besides sperm
zona interaction, it also seems to be involved in acrosome reaction of sperm
that is prerequisite for fertilization. Inhibition of sperm attachment and
tight binding with intact oocyte was observed in sperm treated with SPAG9
antibodies, whereas sperm incubated with preimmune serum did not inhibit the
sperm attachment with zona pellucida. In hemi-zona assays, sperm treated with
SPAG9-JIP3g
antibody also revealed reduction in sperm attachment and tight binding as
compared to sperm treated with preimmune serum. Therefore, SPAG9 sperm
protein, in addition to mediating adherence and fusion, may also be involved
in transducing signals into the oocyte at the time of gamete fusion which
needs to be addressed in future studies. SPAG9
protein and gene expression in panel of infertile men: Panel
of infertile male semen samples was further screened for the presence of SPAG9
protein on sperm. The study revealed that in 75-semen sample, SPAG9 protein
was localized but intensity of localization varied among the semen samples.
However, it was also noted that 46 of the sperm sample did not show reactivity
of SPAG9 indicating the absence of SPAG9 protein. Genomic analysis will be
carried out in the subjects, which did not have SPAG9 protein. This may
eventually explain the infertility related with SPAG9. The semen sample from
fertile donor tested (112 samples) so far demonstrated the presence of SPAG9
protein. SPAG9
gene function: Screening of mouse
genomic library was carried out using SPAG9 cDNA probe. The 29 Kb mouse
genomic clone was isolated successfully. Constructs were prepared deleting the
exons coding for leucine zipper motif. Attempts will also be made to utilize
short hairpin RNA interference (shRNAi) to rapidly determine if SPAG9 molecule
is necessary for either sperm structure or sperm-egg interaction.
Publications Original peer-reviewed articles 1.
Shankar S, Mohapatra B, Verma S, Selvi R, Jagadish N and Suri A (2004)
Isolation and characterization of a haploid germ cell specific sperm
associated antigen 9 (SPAG9) from the baboon. Mol Reprod Dev (in
press).
Reviews/Proceedings 1.
Suri A (2004) Sperm specific proteins-potential candidate molecules for
fertility control. Reprod Biol Endocrinol (in press). |