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Original Paper	Pdf file on Synergy OPEN omments
Acta Biochim Biophys Sin 2008, 40: 855-863
doi:10.1111/j.1745-7270.2008.00468.x

New member of the guanosine triphosphatase activating protein family in the human� epididymis

Xiangqi Li^1,2#, Qiang Liu^2#, Shigui Liu¹, Jinsong Zhang², and Yonglian Zhang^2,3*

¹ College of Life Science, Sichuan University, Chengdu 610064, China

² Shanghai Key Laboratory for Molecular Andrology, State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China

³ Shanghai Institute of Planned Parenthood Research, Shanghai 200032, China

Received: April 14, 2008�������

Accepted: June 10, 2008

This work was supported by the grants from the National Natural Science� Foundation of China (30230190 and 30570684), the National Basic Science Research and Development Project (2006CB504002 and 2006CB944002) and the Chinese Academy of Sciences Knowledge Innovation� Program (KSCX1-YW-R-54)

^# These authors contributed equally to this work

*Corresponding author: Tel/Fax, 86-21-54921163; E-mail, [email protected]

The effect of the guanosine triphosphatase activating proteins� (GAPs) on spermatogenesis has been studied for years, though no GAPs have been explored in epididymis, an essential� organ for sperm maturation. In this study, a new GAP member, designated as MacGAP, was cloned in human epididymis. The MacGAP gene encodes a protein of 618 amino acids with a putative size of 70 kDa and harbors the conserved� RhoGAP domain. The N-terminal and C-terminal peptides of MacGAP were expressed and their corresponding antisera were prepared. The antisera against N-terminal peptide could detect antigen as low as 0.3 ng, and its specificity was also confirmed. However, the antisera against C-terminal peptide failed to detect its antigen because of its low sensitivity. Immuno�histochemistry showed that the MacGAP protein was dependent on epididymis and had a region-specific expression� pattern, with high expression in the epithelial cells basal section in the caput region. The results have created a foundation� for further interpretation of the biological effects of GAPs in sperm maturation.

Keywords������� epididymis; MacGAP; RhoGAP; signal transduction

It has been well documented that the epididymis, which was recognized as a useless organ several years ago, is divided into three major regions, caput, corpus and cauda. Each region expresses different specific proteins and has distinct functions, providing an essentially regionalized environment for sperm maturation (ie motility development in caput, zona binding in corpus and oolemma fusion in cauda) after being produced by testis [14]. Clinically, the importance of epididymal maturation for sperm is highlighted by the high percentage of male infertility that results from the malfunction of epididymis [5]. Theoretically, the epididymis may be an ideal target organ for male contraception by virtue of its many advantages such as no hormone side effect and no genetic side effect and so on �[6]. Accordingly, many epididymal proteins have been identified and characterized recently and only a few of them [1], such as Bin1b [7,8], ESP13.2 [9-11], HE6 [12], and Crisp-1 [13,14], have been confirmed to be involved in sperm maturation. However, our understanding of the involvement of epididymal proteins in sperm maturation is far from perfect.

Previously, we reported that 25 novel expressed sequence� tag (EST) clones were isolated and sequenced from a subtracted Macaca mulatta epididymis-specific complementary DNA (cDNA) library [15]. Among them, ESC7, the 550 bp EST, revealed no homology to known protein. Later, we found that the ESC7 sequence had high homology to the MacGAP gene in humans, which had just been cloned in the HMC-1 cell line (mast cells). Its protein was predicted to be a member of the family of guanosine triphosphatase (GTPase) activating proteins (GAPs).

GAPs belong to a large family of signaling molecules that stimulate endogenous guanosine triphosphate hydrolysis� to GDP by GTPase; it is in the same family as the Ras, Rho, Arf, Rab and Ran GAP [16-18]. Remarkably, approximately 0.5% of genes have been predicted to encode� the proteins related to GAPs in human and Drosophila� [18]. Considering their abundance, GAPs will undoubtedly play important roles in many biological processes. GAPs have already been implicated in almost all aspects of intracellular vital processes, including proliferation, differentiation, cytoskeletal organization, vesicle trafficking, nucleocytoplasmic transport and gene expression by promoting GTPase inactivation through the enhancement of the weak intrinsic GTP-hydrolysis activity� up to five orders of magnitude [16]. Moreover, they are also associated with various genetic disorders, including cancer [18]. Recently, many investigations on GAPs have been carried out [16-18]. However, most GAPs have yet to be characterized since the first was discovered 20 years ago [16], and quantities of GAP candidates remain unknown.

The role of GAP during spermatogenesis in testis has been investigated [19,20], but there is still no information available concerning GAP during sperm maturation in epididymis. Based on the EST fragment of ESC7 in monkey� epididymis and the MacGAP sequence in the HMC-1 cell line, we cloned the MacGAP gene in human epididymis (named RhoGAP18 and called hESC7 in our laboratory), prepared the antisera and then evaluated its expression features� at the protein level as to better understand its potential� biological importance in epididymal sperm maturation.

Materials and Methods

Animals and human tissue sources

Healthy male New Zealand rabbits (body weight approximately 2.5 kg) were purchased from the Animal Center of the Chinese Academy of Sciences (Shanghai, China) and housed for 6-7 d so that they would adapt to the new circumstance before manipulation. Experiments were carried� out according to the protocol approved by the Ethics Committee of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China).

The testes and epididymis were obtained from a healthy 25-year-old donor who died in a car accident. Artificial circulation was maintained to preserve the organs assigned for transplantation (Shanghai Huashan Hospital, Shanghai, China). This procedure was approved by the Ethics Committee� of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

cDNA and protein sequence analysis

The tissue samples from the human epididymis were frozen� immediately in liquid nitrogen. Total RNA was extracted with Trizol (Invitrogen, Carlsbad, USA) according to the manufacturer's protocol and reversely transcribed by SuperScript reverse transcriptase (Gibco BRL, Grand Island, USA) according to the manufacturer's recommendations. Based on the sequence of MacGAP in the HMC-1 cell line (GenBank accession No. AB053293), a 2032-bp fragment with open reading frame (ORF) was amplified by polymerase� chain reaction at denaturing, annealing, and extension temperatures of 94 �C for 30 s, 61 �C for 30 s, and 72 �C for 150 s, respectively, with LA-Taq (TaKaRa, Dalian, China). The primers were as follows: 5'-TACACT�A�TGAA�CCAGGAAAGCACCA-3' (forward) and 5'-ATTC�TGGCAGCAGCACAAATCTATG-3' (reverse). The amplified� fragment was verified by sequencing (Invitrogen).

The signal peptide cleavage sites, N-glycosylation sites and phosphorylation sites were analyzed by SiganlP 3.0 server (http://www.cbs.dtu.dk/services/SignalP), NetNGlyc 1.0 server (http://www.cbs.dtu.dk/services/NetNGlyc/) and NetPhos 2.0 server (http://www.cbs.dtu.dk/services/NetPhos/). The conserved domains were predicted� using http://www.expasy.ch/prosite/. The alignment� of amino acid sequences was carried out using the Clustal W method of DNAStar software (DNASTAR Inc., Madison, USA).

Preparation of polyclonal antiserum

Because the MacGAP protein was too large for us to prepare� its antibody, the N-terminal 295 amino acid (aa) and the C-terminal 313 aa were expressed to produce antigen. The cDNA fragment encoding N-terminal 295 aa of MacGAP was amplified by PCR using LA-Taq (TaKaRa) with the primers: 5'-GCTAGCATGAAC�CA�GGAAAG�CA�C�CA-3' (NheI underlined) and 5'-AAGC�T�TCTACATT�CC�T�GGTACTTTCC-3' (HindIII underlined, and stop codon italicized). The cDNA fragment encoding C-terminal 313 aa of MacGAP was amplified with two primers: 5'-GCTAGCTCTCGAATTGAAGAGAG-3' (NheI underlined) and 5'-AAGCTTC�TACAATGGCTTTGACTT-3' (HindIII underlined). The amplified fragments were inserted into T-easy vector, verified by sequencing (Invitrogen) and then transferred into the vector pET-28a(+) using NheI/HindIII sites. The recombinant proteins were induced by 1 mM isopropyl -D-thiogalactopy�ranoside in Escherichia coli BL21(DE3) and purified [21]. The polyclonal antisera against the purified� recombinant peptides were prepared with minor modifications [22]. For each manipulation, 600 mg antigen� was used per rabbit.

Protein extracts and Western blot analysis

Total protein extracts of the human epididymis and testis were prepared [23]. The supernatant proteins were assayed� for their concentrations and stored at -20 �C for further use. Supernatant protein extracts for each sample were separated on 12% sodium dodecylsulfate-polyacrylamide gel electrophoresis and semi-dry blotted to polyvinylidene difluoride membranes (Amersham Pharmacia Biotech, Piscataway, USA). Immunodetection of proteins was carried� out by standard procedures using a 1:5000 dilution of primary antibody; however, the secondary antibody was diluted 1:5000 in normal human serum. The primary antibody� was the polyclonal antisera against recombinant N-terminal peptide, and the secondary antibody was the goat anti-rabbit immunoglobulin G-horseradish peroxidase (Calbiochem, San Diego, USA). The peroxidase activity was detected with a chemiluminescent substrate (Western blot chemiluminescence reagent plus; Amersham Pharmacia Biotech).

Immunohistochemical staining

Immunohistochemical staining was carried out [23], except� that the secondary antibody was diluted in normal human serum. The dilutions of the primary antibody and the secondary� antibody were 1:50 and 1:400, respectively. The ABC (avidin-biotin-peroxidase) staining� kit (Sino-American� Biotechnology Company, Luoyang, China) was used according� to the manufacturer's instructions for detecting� staining signals. The manipulations were carried out at 37 �C.

Tissue chip analysis

Human tissue chip was ordered from Shaanxi Chaoying Biotechnology Company (Xi'an, China). Positive signals were examined following the manufacturer's instructions. Immunostaining were observed under an Olympus BX-52 microscope (Tokyo, Japan).

Results

Cloning of MacGAP cDNA in human epididymis

The 550 bp EST sequence of ESC7 in monkey corresponds� to the 21422691 bp fragment of MacGAP cDNA (total 3305 bp) [Fig. 1(A)]. A 2032 bp sequence (17-2048 bp of MacGAP cDNA) with ORF was amplified� in epididymis� [Fig. 1(A)]. The entire 1857 bp ORF encodes� a protein of 618 amino acids with a putative size of 70 kDa. No signal peptide was predicted by SiganlP 3.0 server. Moreover, no N-glycosylation site was predicted by NetNGlyc 1.0 server. Thirty-four potential phosphorylation sites were predicted in this sequence: 20 serine, seven threonine and seven tyrosine. The encoded MacGAP protein harbors one conserved GAP domain (RhoGAP domain, 279-478 aa) of GAP family. However, most of the other members examined� in the RhoGAP subfamily generally have several domains [Fig. 1(B)]. Based on the alignment of the RhoGAP domain of MacGAP with other reported RhoGAP members with identified functions in humans, they appeared� to have several conserved key amino acid residues� that are part of the GTPase interaction� sites R, K and P. This new RhoGAP protein shared 21%-25% identity in amino acids sequence with other RhoGAP members, such as RhoGAP7, 9, 10, 13, 19, 21, 24 and 26 [Fig. 1(C)].

Preparation of polyclonal antibody against human MacGAP

The N-terminal fragments of MacGAP were subcloned into expression vector pET-28a(+). The recombinant peptide� (about 43 kDa) with a histidine-tag in the N-terminus� was expressed in the inclusion bodies [Fig. 2(A)], which was confirmed by Western blot analysis with monoclonal antibodies of histidine-tag [Fig. 2(B)]. The recombinant protein was purified and used as the antigen to immunize rabbits. The sensitivity of the prepared antisera� was examined, suggesting that the polyclonal antisera at a dilution of 1:10,000 by Western blot analysis could detect as little as 0.3 ng antigen [Fig. 2(C)]. The C-terminal recombinant� peptide of MacGAP was also successfully expressed and used as an antigen to immunize rabbit. However, the antisera failed to detect specific bands in total protein extracts (data not shown). Therefore, the antisera against N-terminal MacGAP were not used in subsequent� studies.

Region-specific expression of MacGAP native protein in human epididymis

To determine region-specific expression for MacGAP, total proteins from three epididymal regions, the caput, corpus and cauda, were analyzed by Western blot analysis. A band of approximately 70 kDa, which matched the predicted protein size, was detected in the caput region of epididymis, but not in the testis or the corpus and cauda regions of epididymis [Fig. 3(A)]. However, a non-specific band at 55 kDa in both preimmune serum and anti-MacGAP antiserum was visualized, which was much stronger than the MacGAP band [Fig. 3(A)]. This non-specific band was produced by the second antibody, as it was also observed when the second antibody was used alone [Fig. 3(B)]. When the secondary antibody was diluted with normal human serum, only one band was observed in the caput region of epididymis [Fig. 3(C)], further confirming the specificity of the antisera prepared here.

To further determine cell localization of MacGAP in human epididymis, immunohistochemistry analysis was carried out (Fig. 4). Results showed that positive signals presented in the epithelial cells in the distal caput region [Fig. 4(B), 2], especially at the cytoplasm near the basal membrane. A very weak signal was also observed in the proximal corpus region. There were no signals in the lumen� of any of the regions.

Epididymis-specific expression of MacGAP native protein� in human tissues

To confirm epididymis-specific expression for MacGAP, its tissue distribution was analyzed by tissue chip technology� (Fig. 5). It was shown that positive signals selectively presented in epididymis but not in other tissues� examined. Furthermore, positive signals were detected in the distal caput and proximal corpus region, which was consistent with results of immunohistochemistry analysis.

Discussion

In this study, the MacGAP in epididymis was cloned and predicted to encode protein harboring the conserved RhoGAP domain of RhoGAP family. This domain, spanning� approximately 100-200 residues, is composed of seven a-helices and is sufficient for productive interaction with Rho targets [18,24]. RhoGAP are usually large proteins possessing several additional functional domains implicated in regulation, membrane targeting and localization [16,25]. Here, most of RhoGAP members examined have other domains besides RhoGAP domain. MacGAP has only a RhoGAP domain of approximately 200 amino acids, including� conserved key residues (arginine, lysine, and proline) in the formation of RhoGAP/RhoGTPase complexes� [Fig. 1(C)], which suggests it might be active as a RhoGAP protein by stimulating the intrinsic GTP-hydrolysis activity of RhoGTPase. However, its biochemical� activity remains unclear. Thirty-four phosphorylation� sites were predicted in the MacGAP sequence. Phosphorylation of RhoGAP proteins is known to be involved in regulating RhoGAP activity, subcellular localization and interaction with other proteins [24,26]. The function of MacGAP protein might also possibly be regulated by phosphorylation. However, the function and the regulation of MacGAP in epididymis remains undetermined.

GAPs have been found to be associated with the production� of the spermatozoa [27,28], and six GAPs have been identified and characterized in the testis [20]. There is a pressing need to understand whether GAP can contribute� to sperm maturation in epididymis. In this study, expression features of the novel GAP member MacGAP were successfully analyzed with its N-terminal antibody. The selective expression in epididymis and the enriched localization in the caput region for MacGAP suggest that it has a special physiological role in epididymal sperm maturation. The localization of MacGAP at the cytoplasm near the basal membrane of the duct in epididymis suggested� that the MacGAP protein might modulate the expression of functional genes in sperm maturation by mediating the signal from outside the basal membrane, perhaps from blood. Signal transduction is a very hot topic in basic science� and clinical applications, and some potential signal molecules have been found in the male reproductive system� [29,30]. As the first GAP protein found in epididymis, MacGAP's role in signal transduction in the physiology of epididymal epithelium is well worth further investigation. Experimental studies in humans are largely precluded for obvious ethical and practical reasons, such as difficulty in obtaining materials; thus, further investigations should be conducted using murine animals.

The preparation of antibody is not an easy task, and many steps are required to produce a higher titer antibody [31]. In this study, only the antibody against N-terminus of MacGAP succeeded, which suggested that it was important� to select a suitable fragment to produce a polyclonal antibody. However, with this high titer antibody, we did not achieve the desired results because of strong non-specific reactions (Fig. 3), and also because MacGAP was a very low-abundance protein in epididymis based on a cDNA array (data not shown). Conventionally, goat serum� is the best reagent to block this non-specific reaction, but it did not work well. Although milk could block this non-specific signal, human serum, used here, also works well. So human-serum provides another option� besides milk for blocking strong non-specific signals in investigating low abundant protein in human tissue.�

This study identified a region-specific putative RhoGAP family member, MacGAP, in epididymis that may have roles in RhoGTPase regulation through its RhoGAP domain. This, in turn, might affect sperm maturation by influencing� signaling events.

Acknowledgements

We would like to thank Shanghai Huashan Hospital (Shanghai, China) for the preparation of human tissues. We are also grateful to our colleagues for their fruitful discussions.

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