REVIEW
Sperm associated antigen 9 (SPAG9): a new member ofc-Jun NH2-terminal kinase (JNK) interacting protein
exclusively expressed in testis
Nirmala Jagadish, Ritu Rana, Deepshikha Mishra, Manoj Kumar and Anil Suri
Genes and Proteins Laboratory, National Institute of Immunology, New Delhi, India(Received for publication on November 22, 2004)(Revised for publication on January 20, 2005)(Accepted for publication on February 17, 2005)

Abstract. 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 ahomology 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 inhuman, 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. (Keio J Med 54 (2): 66–71, June 2005)
Key words: Sperm associated antigen 9 (SPAG9), leucine zipper, JIP, acrosome

ﬁlamentation, osmoregulation and cell wall biosynthesis.4–8 Recently, selective activation of physiologicalprocesses involving JNK pathways was described in
Xenopus laevis during oocyte maturation.9,10
In mammals, MAPKs can be subdivided into threegroups: extracellular signal regulated kinase (ERK), cJun NH2 -terminal kinase (JNK) and p38 MAPK.11–13
The structural organization of MAPK kinases into speciﬁc signaling modules appears to be facilitated byscaffolding proteins such as the JNK interacting protein
JIP1, JIP2 and JIP3 in mammalian cells.14–20 The scaffolding proteins tether various MAPK kinase kinases(MEKKs), MAPK kinases (MKKs) and MAPKs inclose proximity so that successive phosphorylation
events occur efﬁciently, thus conferring speciﬁcity to aparticular combination of kinases. Recently in humansperm, ERK, a member of the MAPK family was
shown to be associated with human spermatozoa withdirect or indirect function in sperm capacitation.21
The mammalian spermatozoon is a terminally differ-

Introduction
Mammalian fertilization requires a series of specialized cell-cell interactions that include gamete recognition, adhesion, signaling and fusion. In mammals, theprocess of sperm-egg fusion is characterized by an increase in intracellular Ca 2þ and pH1 and the tyrosine
phosphorylation of several proteins.2,3 While some ofthese phosphoproteins have been identiﬁed on sperm,
their role is poorly deﬁned. In somatic cells, inductionof tyrosine phosphorylation is associated with activationof down-stream targets including kinases such as
MAPK. The MAPK family of serine/threonine kinases
occupies a focal point in signal transduction and is
involved in the regulation of growth, adhesion, secretion and other physiological events. MAPK signaling
pathways are known to relay, amplify and integratesignals from a diverse range of extracellular stimuli. In
yeast Saccharomyces cervisiae, ﬁve MAPK pathways
have been described, that regulate mating, sporulation,

Presented at the 1378th Meeting of the Keio Medical Society in Tokyo, October 9, 2004
Reprint requests to: Dr. Anil Suri, Genes and Proteins Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi110067, India, e-mail: anil@nii.res.in66Keio J Med 2005; 54 (2): 66–71

67

ently we are trying to speculate the role of SPAG9 as a
JIP protein and its signiﬁcance in human sperm-egg
interaction.

entiated cell. Its surface is covered by a continuous
plasma membrane that is divided into distinct domains
in which functional molecules are distributed. The
acrosome is an internal organelle located at the anterior
head and contains hydrolytic enzymes. The anterior
acrosome participates in the acrosome reaction, which
is an indispensable event during fertilization. Thesesurface domains and the acrosome are formed during
spermiogenesis, during which associated molecules aretransported and organized. Many of the molecules thus
arranged are functionally immature but gradually become mature during epididymal maturation. Some ofthem are further altered and redistributed during thefertilization process and play various roles.22
Our previous ﬁndings have demonstrated an exclusive expression of SPAG9 in haploid round spermatid
cells during spermatogenesis in macaque,23 baboon24
and human.25 By homology search in the genetic database, SPAG9 cDNA was found to be a member ofUnigene cluster Hs. 129872 encoded by chromosome
17. Based on structural homology with JIP3, SPAG9
was earlier deﬁned as JIP3g scaffolding protein16 andhas been recently classiﬁed as JIP4 protein.26 It was
found that SPAG9 is structurally distinct from thepreviously described JIP114 and JIP220 proteins. Pres-A

Domain Structure of SPAG9 Protein
The human SPAG9 open reading frame (ORF)
encodes 766 amino acid residues with a calculated
molecular mass of 84 kDa and an isoelectric point (pI)of 4.9. Running the algorithms provided in Simple
Modular Architecture Tool on the ExPASy server
revealed several features: 1) a characteristic leucine
zipper motif (LZ); 2) two coiled-coil domains (coil) and3) a transmembrane domain (T)25 (Fig. 1A). There are
six putative N-linked glycosylation sites, six putativecAMP-/cGMP-dependent protein kinase phosphorylation sites, six putative protein kinase C phosphorylationsites, eleven putative casein kinase II phosphorylationsites. Deduced amino acid sequence also harboured ten
putative myristoylation sites and a characteristic leucine
zipper (LZ) motif with six leucine repeats from aminoacid 234 to 269. Studies on non-human primates
revealed that the macaque SPAG9 ORF encodes 712
amino acid residues with a calculated molecular mass of
78.19 kDa and an isoelectric point (pI) of 5.44. Further

Coiled coil
Human

Coiled coil

N JBD

T

C

T

C

LZ

N
BaboonJBD

NMacaque

JBD

LZ

T

C

LZ

B
Human 766 amino acids

Hydrophobic

Hydropathy Index

Hydrophilic
Baboon 760 amino acids

Hydrophobic

Hydrophilic
Macaque 712 amino acids

Hydrophobic

Hydrophilic

Fig. 1 (A) Schematic illustration of the domain structure of the SPAG9. JBD, JNK binding domain; Coil, predicted coiled coil; LZ, leucine
zipper; T, predicted transmembrane domain. (B) Hydropathy plot of the deduced amino acid sequence of human, baboon, macaque SPAG9protein.

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Jagadish N, et al: SPAG9 a new member of JNK interacting protein

analysis of macaque SPAG9 protein revealed JNK
binding domain, leucine zipper motif, two coiled coildomain and a transmembrane domain (Fig. 1A). In
addition, six potential N-linked glycosylation sites, ﬁve
putative cAMP- and cGMP-dependent protein kinase
phosphorylation sites, eleven putative protein kinase C
phosphorylation, nine putative casein kinase II phosphorylation sites, one tyrosine sulfation site and ten
putative N-myristoylation sites were found at different
regions.23 Similarly, amino acid analysis of baboon
SPAG9 also revealed JNK binding domain, leucine
zipper motif, two coiled coil domains and a transmembrane domain (Fig. 1A). In addition six potential Nlinked glycosylation sites, ﬁve putative cAMP- andcGMP-dependent protein kinase phosphorylation sites,
seven putative protein kinase C phosphorylation site,eight putative casein kinase II phosphorylation site, one
tyrosine kinase phosphorylation and nine putative Nmyristoylation sites were found at different regions.24
Structural homology analysis with JIP proteins
showed presence of signature domains of LZ motif,
identiﬁed as a structure common to a separate class of
DNA binding proteins including nuclear transcription
factors.27 The classical LZ motif of the DNA binding
proteins includes leucine repeats with an upstream
basic domain called the cluster-spacer-cluster. The LZ
motif of the SPAG9 protein does not have any upstream basic domain and thus differs from the classicalDNA binding proteins. Literature survey shows membrane associated proteins such as the voltage gated Kþ channels,28 glucose transporters of vertebrate cells29
and the fusion (F) glycoprotins of several paramyxoviruses30 with LZ motifs that are not DNA binding proteins. The function of LZ motifs in SPAG9 may
be to aid in dimerization of individual monomers toform a functional dimers for transportation to the cellsurface. As SPAG9 is a membrane-associated protein,
it is quite likely that LZ motif in SPAG9 is involved
in its dimerization and transportation to the sperm
surface.
Hydrophobicity
The hydrophobicity plot generated from deduced
human SPAG9 protein contained a 658 amino acids
extracellular domain, a 20 amino acid transmembrane
helical domain and 88 amino acids cytoplasmic domain
at the COOH terminus.25 Three putative antigenic
determinant sites were identiﬁed based on the threehighest points of average hydrophilicity (Ah ). As theseputative antigenic sites are present within the extracellular domain, it supports the surface localization of
SPAG9, which might play a signiﬁcant role in either
sperm egg interaction or could act as a signal transducer
from the melieu of the female reproductive tract tothe sperm during fertilization. The hydrophobicity plot
generated from deduced macaque and baboon SPAG9
revealed a similar pattern to the predicted human
SPAG9 protein23–25 (Fig. 1B). Both the proteins haveextracelluar domain, a transmembrane helix and acytoplasmic domain at the COOH terminus. This analysis suggests that human SPAG9, macaque SPAG9 andbaboon SPAG9 proteins have similar over all structures
and thus potentially conserved function.
SPAG9 is Evolutionarily Conserved in Human and
Non-human Primates
A new era in the elucidation of genome evolution
has been heralded with the availability of numerous
genome sequences. As more complete genomes aresequenced, conservation of gene order between different organisms is emerging as an informative property of
the genomes. Conservation of gene order has been used
for predicting function and functional interactions ofproteins, as well as for studying the evolutionary relationships between genomes. Conservation of regions
also shows which regions are functionally important.
The higher the degree of conservation the more likelythat a region is involved in the function that is heavily
intertwined with many aspects of cellular function ordevelopment.
Studies have shown that SPAG9 protein from human and non-human primates (baboon and macaque)shares common N-terminal amino acid sequences
which further infer their shared ancestry. Remarkably,
our data from macaque, baboon and human showedthat SPAG9 protein exhibits conserved amino acidsequences and are highly homologous to each other in
the N-terminal region.23–25 However, C-terminal aminoacids sequences of macaque SPAG9 (641–712 aa) is
considerably diverged and has only 18.5% homology
with hSPAG9 at the (689–766 aa). Similarly, baboon
SPAG9 is 90.6% identical to human SPAG9, however,reveals 16.7% homology at the C-terminus of baboon
SPAG9 (689–760 aa) when compared to hSPAG9(689–766 aa) (Fig. 2). C-terminus amino acid difference
has also been reported in non-human primate homologue of human SP17.31 This degree of evolutionarychange in SPAG9 is similar to some other proteins forwhich human and non-human primates sequence havebeen determined. Of the few sperm proteins that havebeen sequenced from human and non-human primates,
most are identical at the amino acid levels; SP-17(97%),31 LDH-C4 (99.3%),32 SP-10 (85%).33 Homology sequence search using SPAG9 also showedhomology with PHET (100%),34 JSAP 1 (JIP 3 – JNK
interacting protein 3) (50%),14 D. melanogaster Sunday
driver (36%),35 C. elegans Coiled coil protein (30%).36

Keio J Med 2005; 54 (2): 66–71

69

Human

1

Baboon1

689

Macaque

1

641

689

31 amino acids
deletion

16 amino acids
deletion

Human 690
Baboon 690
Macaque 641

DKPPEMEAENSEVDENvPTAEEATEATEGNAGSAEDTVDISQTGVYTEHVFTDP 743
LETTIKLIIKDVLVVKVANIDN-NQRNRHNLSLLHRNTHVTAPKRSATHISAGH 742
VETTRNGSRKYEVDENVANSEK-KQLKLQKGMRGQLKTQWTSPKLASTQSMSLQ 694

Human 744
Baboon 743
Macaque 695

LGVQIPEDLSPVYQSRYNNGSSTSRVYGIAIILRPSVSVEVILWEFRSRRPLPSVSVEV

766
760
712

Fig. 2 Amino acids alignment of the human, baboon and macaque SPAG9 protein is shown schematically. The human SPAG9 is 766 aminoacids in length and is shown on the top followed by baboon (760 aa) and macaque (712 aa). A comparison of the deduced SPAG9 amino acidsequences of human, baboon and macaque revealed that the protein exhibits a high degree of homology at N-terminal region. Comparing
C-terminus amino acids of human SPAG9 (690–766) with baboon (690–760) reveals divergence as shown. Comparing C-terminus aminoacids of human SPAG9 (690–766) with macaque (641–712) reveals divergence as shown. Macaque amino acid sequence also revealed inframedeletions of 31 and 16 amino acids.

Structural Homology with c-Jun NH2 -terminal Kinase
Interacting Protein
The amino acid sequence analysis of SPAG9
revealed the primary sequence identity to JNK binding
domain of JNK interacting protein (JIP). Though many
JIP proteins are related in structure and/or function, thegenes that encode these proteins are not closely linked
in the genome. The human JIP1, and JIP2, which are
related in function, are located on human chromosome
11 (11p 11.2-p12, 12 exons) and human chromosome
22 (22q13, 12 exons) respectively,37,38 whereas human
JIP3 gene is located on human chromosome 16(16p13.3, 30 exons). Sperm speciﬁc SPAG9 protein isclassiﬁed as JIP4,26 which is structurally related to JIP3.
The members of JIP3 group include the JIP3a andJIP3b proteins, which appear to represent the protein
products of alternatively spliced transcripts derived
from the JIP3 gene. The other JIP3 group of proteins
includes JIP3b (accession no. AB11088), which is also
structurally related to JIP3.16,39 Both JIP3 and JIP3b14
are expressed in the brain, while SPAG9 (JIP4) was
shown to be expressed in testis.23–25,34 Further, it wasdemonstrated that UNC-16, the C. elegans having 30%
similarity with SPAG9, interacts with JNK1 and JNKK1 kinases that have the highest sequence similarity tomammalian JNK3 and MKK7, respectively.36 Recently,
protein highly expressed in testis (PHET), a novel testicular antigen, has been identiﬁed and reported to be
ectopically over expressed in systemic sclerosis (SSc)dermal ﬁbroblasts. By homology search in the genetic
database, PHET cDNA was found to be a member of
the UniGene cluster Hs.129872 encoded by chromosome 17q21, but had a unique exon composition and acharacteristic mRNA expression proﬁle restricted tothe testis. PHET was shown to have 100% homology
with SPAG9 and was speculated to be involved in JNK
signal pathway.34
Localization of SPAG9
Haploid germ cell expression has been a uniquefeature of the testis speciﬁc genes. RNA in situ hybridization studies in human, macaque and baboon testis
section using antisense SPAG9 riboprobe identiﬁed the
SPAG9 transcript on round spermatids of the seminiferous cycle and not on any other cell type.23–25 The
absence of the SPAG9 transcript in spermatogonia andspermatocytes suggests that the SPAG9 is transcribed
post meiotically in human and non-human primates.
In indirect immunoﬂuorescence studies, anti-SPAG9
antibodies revealed a selective acrosomal localizationof SPAG9 in macaque23 and baboon24 sperm.
Conclusion
In conclusion, testis speciﬁc SPAG9 is classiﬁed as a
new member of JNK interacting protein based on its
structural homology with JIP proteins. The amino acidsequence analysis revealed conservation of SPAG9protein in human and non-human primates predicting
functional interactions of proteins, as well as the evolutionary relationships between genomes. Primary sequence analysis of SPAG9 revealed identity to JNK
binding domain and predicted coiled coil, leucine zipper
and transmembrane domain. Therefore, it is suggested
that SPAG9 may function as scaffold protein in JNK
cascade. As a JIP family member SPAG9 merits further
evaluation as JNK scaffolding protein in upstream of
MAPK module, speculating the involvement of SPAG9

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Jagadish N, et al: SPAG9 a new member of JNK interacting protein

mediated signal transduction pathways in reproductive
processes.
Acknowledgement: We thank Professor S.K. Basu, Director, National
Institute of Immunology for constant encouragement for this work.
This work was supported by grants from the Department of Biotechnology, Government of India, Mellon foundation and CONRAD,
USA, Indo-US programme on CRHR.

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