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

Abstract

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.

Sperm‐associated antigen 9 is a biomarker for early cervical carcinoma

“…26 Furthermore, our earlier MAPK interaction studies of SPAG9 with the JNK signaling pathway demonstrated that SPAG9 functions as a scaffolding protein. 16 The JNK signaling interaction is well known to play an important regulatory role in cell proliferation, differentiation, apoptosis, cellular transformation, and tumor cell growth, and may be equally important for the outcome of SPAG9 expression in the cervix. 16,27,28 To our knowledge, the mechanism underlying the association between SPAG9 and carcinogenesis is still unclear.…”

“…16 The JNK signaling interaction is well known to play an important regulatory role in cell proliferation, differentiation, apoptosis, cellular transformation, and tumor cell growth, and may be equally important for the outcome of SPAG9 expression in the cervix. 16,27,28 To our knowledge, the mechanism underlying the association between SPAG9 and carcinogenesis is still unclear. Perhaps the overexpression of SPAG9 changes the stability or kinetics of MAPK signaling pathways, leading to facilitation of cell invasion and migration; this warrants further studies.…”

“…cDNA synthesis was performed using FastLane Cell cDNA kits (Qiagen GmbH, Hilden, Germany). 16 Subsequently, cDNA was used as a template for reverse transcriptase-polymerase chain reaction (RT-PCR) using SPAG9 primers 5 0 -GACAGAGATGATTCGGGCATCACGAGAAAA-3 0 (forward) and 5 0 -CTAAGTTGA TGACCCATTATTA-TACCTCGACTG-3 0 (reverse). RT-PCR for b-actin mRNA expression was performed as an internal control.…”

Cancer testis antigens in newly diagnosed and relapse multiple myeloma: prognostic markers and potential targets for immunotherapy

“…SPAG9 is present in all but one sample in this study, and has been described to mediate JNK signaling. [34][35] For immunotherapy, expression in normal tissues raises concerns of generating autoimmune responses following vaccination. Still, some TS antigens, like MAGEA3, are targeted in current immunotherapeutic protocols.…”

JSAP1/JIP3 and JLP regulate kinesin-1-dependent axonal transport to prevent neuronal degeneration

“…9 Splice variants of JLP are known as JIP4 (Kelkar et al 2005) or SPAG9. 11 JIP proteins were first identified as scaffold proteins for specific JNK and p38 mitogen-activated protein kinase (MAPK) signaling modules. These proteins were also isolated as KLC-binding proteins and suggested to function as adaptor proteins linking cargoes to kinesin-1.…”