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.
Jagadish
Nirmala
N
Genes and Proteins Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India.
Rana
Ritu
R
Selvi
Ramasamy
R
Mishra
Deepshikha
D
Garg
Manoj
M
Yadav
Shikha
S
Herr
John C
JC
Okumura
Katsuzumi
K
Hasegawa
Akiko
A
Koyama
Koji
K
Suri
Anil
A
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D002874:Chromosome Mapping; D005306:Fertilization / Q000502:physiology; D005786:Gene Expression Regulation; D006801:Humans; D007150:Immunohistochemistry; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D009865:Oocytes / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D013094:Spermatozoa / Q000378:metabolism*; D040681:Structural Homology, Protein; D015044:Zona Pellucida / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; C485750:MAPK8IP3 protein, human; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; C111047:SPAG9 protein, human; D048031:JNK Mitogen-Activated Protein Kinases