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.

Figure 1. SPAG9 is a new member of the JIP proteins

(A) Structure of the SPAG9 and deleted mutants are shown schematically. The native SPAG9 is 766 amino acids in length and is shown on the top. The C-terminal deletion mutant SPAG9ΔT and the truncated mutant SPAG9ΔLZΔT are shown below. (B) Schematic illustration of the domain structure of the JIP proteins. JBD, JNK-binding domain; Coil, predicted coiled coil; LZ, leucine zipper; T, predicted transmembrane domain; SH3, Src homology domain 3; PTB, phosphotyrosine binding. (C) Multiple amino acid sequence alignment of SPAG9 and other members of the JNK-binding protein family. The conserved JBD is shown in a box; sequence identity of SPAG9 with JIP3 protein is shown as shaded amino acids. Numbers on the side refer to amino acid positions relative to the first residue. (D) Binding of SPAG9 to mammalian MAPKs. COS-1 cells were co-transfected with 0.5 μg of FLAG–JNK1, FLAG–JNK2, FLAG–JNK3, FLAG–p38α and FLAG–ERK2 along with 1.5 μg of either pcDNA 3.1-Myc-His-empty vector or SPAG9. Cell lysates were immunoprecipitated using anti-SPAG9 antibody and immunoblotted with anti-FLAG monoclonal antibody to show specific interaction of JNKs. Control panels for total cell lysates are shown below. Arrow indicates the position of SPAG9 approx. 170 kDa. (E) Interaction of SPAG9ΔLZΔT and JNKs was investigated as described above which revealed no association. Arrow shows the position of SPAG9ΔLZΔT ∼43 kDa.

Figure 2. Chromosomal localization of the SPAG9 gene

Mapping of SPAG9 in human and mouse chromosome by fluorescence in situ hybridization. Human and mouse metaphase spread hybridized with SPAG9 cDNA. The signals are visible on both copies of chromosomes. (A) Arrow heads; human chromosome 17 (h17). (C) Arrow heads; mouse 11C (m11). (B, D) DAPI (4,6-diamidino-2-phenylindole) banding of human (arrows h17) and mouse chromosomes (arrows m11). (E–G) Southern blotting of SPAG9 gene after digestion with different restriction endonucleases and probed with different SPAG9 cDNA probes. (E) The 5′-end of SPAG9 cDNA, (F) full-length SPAG9 cDNA and (G) the 3′-end SPAG9 cDNA. Identical band numbers, pattern and the sizes indicate that the human genome contains a single copy SPAG9 gene (arrow indicates the location of faint band). The markers on the left represent a ladder λDNA/HindIII fragments (Gibco BRL, Gaithersburg, MD, U.S.A.). Abbreviations: E, EcoRV; A, ApaI; K, KpnI.

Figure 3. Characterization of SPAG9 protein

Western-blot analysis of rSPAG9 and HSE using denatured SDS/PAGE gel under reducing conditions. (A) Affinity-purified rSPAG9 stained with Coomassie Brilliant Blue is shown in lane 1. Western blotting of rSPAG9 (lane 2) and HSE (lane 4) show a specific band of approx. 170 kDa. Preimmune control revealed no reactivity (lanes 3 and 5). Specificity of HSE is shown in lane 6 by including recombinant SPAG9 (15 μg/ml) in the incubation with primary antibody, which resulted in loss of immunoreactivity with native SPAG9 in HSE proteins. (B) Urea/PAGE of rSPAG9. Coomassie Blue staining (lane 1) and Western blotting of rSPAG9 (lane 2) show a specific band of approx. 170 kDa. Prestained protein molecular-mass standards (BENCHMARK, Gibco BRL). (C) Primary sequencing of SPAG9 showing the peptide stretches (light and dark grey shaded) that matched with published SPAG9 (EMBL Nucleotide Sequence Database accession no. X91879) protein identified by MS. (D) CD of the purified and refolded recombinant SPAG9 protein exhibited minima at 220 and 208 nm.

Figure 4. Localization of SPAG9 in human spermatozoa

Epifluorescent photomicrograph showing the immunofluorescence pattern of the human spermatozoa. (A) Rat anti-SPAG9 antibody reacted strongly with the acrosomal compartment of sperm head. (C) Preimmune or neutralized serum did not react with any of the region of the spermatozoa. The phase-contrast pictures of (A) and (C) are (B) and (D) respectively (original magnification was ×630). (E) Immunoelectron microscopy of human spermatozoa. Longitudinal section of sperm head revealed immunogold labelling of SPAG9 over the acrosomal compartment. The arrows indicate gold particles associated with the plasma membrane (solid arrows), outer acrosomal membrane (dotted arrows), inner acrosomal membrane (solid arrows) and acrosomal matrix (open arrows). PM, plasma membrane; OAM, outer acrosomal membrane; IAM, inner acrosomal membrane; AM, acrosomal matrix (original magnification was ×45000).

Figure 5. Immunolocalization of SPAG9 in acrosome-reacted spermatozoa

Immunolocalization staining showing equatorial localization of SPAG9 in the human sperm head. (A) Indirect immunofluorescence light microscopic image (arrows, original magnification was ×630). (B) Electron micrograph (longitudinal section) (arrows, original magnification was ×67000). Few gold particles are also shown to be associated with the inner acrosomal membrane (arrows). IAM, inner acrosomal membrane; ES, equatorial segment.

Figure 6. Flow cytometric, immunoflourescence microscopy and immunoblot analysis of SPAG9 and its deletion mutants in COS-1 cells

(A–C) Flow cytometric analysis of SPAG9, SPAG9ΔT and SPAG9ΔLZΔT constructs. Peak 1, pcDNA 3.1 vector control. Peak 2 in each construct reveals displacement of fluorescence on the x-axis representing surface binding of anti-SPAG9 antibodies showing surface location of SPAG9. Note that only SPAG9 transfected cells revealed surface localization (A). (D–I) Fluorescence microscopy of cells transfected with SPAG9, SPAG9ΔT and SPAG9ΔLZΔT constructs showing surface localization of SPAG9 in live and fixed (permeablized) cells (original magnification was ×500). Only SPAG9 transfected cells revealed surface localization (D). (J) Immunoblot analysis of SPAG9 and its mutants (SPAG9ΔT, SPAG9ΔLZΔT) in cell lysates (L) and culture medium (M). Note the secretion of SPAG9ΔT in the medium fraction. Lane 5, pcDNA vector cell lysate control (C). It may be noted that the SPAG9 protein (lane 1) and SPAG9ΔT (lanes 3 and 4) move with anamolous mobility corresponding to an apparent molecular mass of approx. 170 and 140 kDa respectively. However, SPAG9ΔLZΔT (lane 6), the deleted mutant lacking JBD, leucine zipper and the transmembrane domain, runs according to its deduced molecular mass of approx. 43 kDa. Prestained protein molecular-mass standards (BENCHMARK, Gibco BRL) were used.

Figure 7. Role of SPAG9 in human spermatozoa binding to human zona pellucida

Intact human oocytes were co-incubated with spermatozoa preincubated with (A) IgG from rat preimmune serum, (B) IgG from rat anti-SPAG9 antibody, (C) IgG from the macaque preimmune serum and (D) IgG from macaque anti-SPAG9 antibody. Note the inhibition in binding of spermatozoa to oocytes (B, D). (E, F) Spermatozoa binding in matched human hemizona. Note the inhibition of binding of spermatozoa in MHZ by rat anti-SPAG9 antibody (F).