Proteases and protease inhibitors.

Approximately 18% of the proteins in the Drosophila melanogaster ejaculate are predicted proteases or protease inhibitors. LaFlamme et al. found that proteases in the seminal fluid can destroy, activate or otherwise modulate the function of other proteins [4]. Several seminal proteins in this organism are activated or degraded after mating, and proteolysis is an effective way to accomplish this process since these proteins act outside of the cell where most other regulatory processes cannot be used. Proteases and protease inhibitors are known to be major constituents of the male seminal fluid, and RNAs encoding 120 predicted proteases and 8 protease inhibitors were identified in our previous AG transcriptome analysis in E. sinensis [6]. During epididymal transit, sperm bind new proteins, and proteins already present in the membrane are modified [22]. Enzymes in addition to proteases also likely play key roles in membrane transformations, and several of these were detected in the present study, including cathepsin A, B, protein-disulfide isomerase, inorganic pyrophosphatase, AICAR transformylase, cystathionine-beta-synthase and cytoplasmic manganese superoxide dismutase.

In the mammalian cauda epididyma, protease activity is modulated by protease inhibitors. We identified several predicted protease inhibitors among E. sinensis AG proteins. These included predicted SERPINs (serine protease inhibitors), glycoprotein, pacifastin and serine collagenase 1 precursor. We propose that such components of the seminal fluid are likely important for regulating proteolysis for essential processes or to preserve sperm and tissue integrity [23]. Proteins in mammal seminal fluid include serpin 6 and HongrES1, which functions as a decapacitation factor (DF) for sperm, preventing capacitation. HongrES1 is specifically expressed in the cauda epididymis of rat and guinea pig [24,25] where it is important for fertility. Given our findings of predicted serpins in Chinese mitten crab AGs, we used antibodies to HongrES1 to test if there is similar protein associated with sperm of Chinese mitten crab. We further examined the amount of this Es-serpin on sperm before and after the AR. The results (Fig 4) showed that after the occurrence of AR, Es-serpin cross reactivity on the sperm surface gradually decreased. This is consistent with the variation of DF in sperm, and suggests that we may have identified a DF in E. sinensis.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 4. Changes in immunofluorescence staining patterns of antibodies against human Es-serpin in E. sinensis sperm during the acrosome reaction.

Sperm were incubated in eggs water for 40min, and the reaction was terminated by 4% paraformaldehyde respectively at 10min, 20min, 30min, 40min.

https://doi.org/10.1371/journal.pone.0136266.g004

Other protease inhibitors that we detected included Pacifastin, which is a serine peptidase inhibitor [26], that is found in insects and arthropods. Some studies have shown that crustacean pacifastin plays a key role in the immune response, whereas insect pacifastin-like peptides have multiple regulatory functions including ones related to immunity, reproduction and phase transition [27]. There are five additional members (SGPI-1–5) of this family from ovarian tissue of the desert locust Schistocerca gregaria. All of these peptides possess the cysteine pattern that is characteristic of the pacifastin light chain domains (PLD 1–9) and the locust inhibitors PMP-D2, PMP-C and HI. Further, there is evidence that transcripts of these protease inhibitors are differentially regulated during the molting and reproductive cycles of the locust, suggesting that the encoded peptides have distinct physiological roles in a variety of physiological processes [28]. Previously the mRNA expression of the pacifastin gene of E. sinensis was analyzed in different tissues of the crab [29]. Here, we identified the pacifastin protein in extracts from the AG, implying that it may also have a reproductive function in E. sinensis. Further analyses using mutants or RNAi will provide insights into the function and physiological roles of these proteins in crustacean reproduction.

Glycolytic enzymes detected in the present study include predicted glutamate dehydrogenase, alpha glucosidase 2, fructose 1, 6-bisphosphatase, fructose-6-phosphate aminotransferase and UDP-glucose 4-epimerase. These enzymes may hydrolyze sugar moieties present on membrane glycoproteins during epididymal sperm maturation and thus potentially mediate sperm/oocyte interactions [30], they could also hydrolyze sugar moieties on Sfps or even on female reproductive tract proteins, thus potentially playing roles in sperm maturation and physiological regulation of fertilization in crustaceans.

Protein folding and stress response proteins.

We identified some chaperone molecules such as predicted heat shock protein in the AG proteome. Most of these types of proteins function in protein folding or stress response reactions. Glucose-regulated protein 78 (GRP78), a member of the heat shock protein 70 family, is an ER-resident protein responsible for protein folding and assembly [31]. The 94-kDa glucose-regulated protein (GRP94), also known as endoplasmin, 96-kDa glycoprotein (GP96) or HSP108, belongs to the HSP90 family, is an ER chaperone and was first identified in a screen for proteins influenced by glucose starvation. GRP94 mainly localizes to the ER and is thought to be required for the folding and maturation of secretory and membrane proteins [32]. Therefore, the presence of these molecular chaperones among AG proteins suggests that they play roles in aiding the correct folding and assembly of proteins in the AG, and protecting them from stress responses. Endoplasmic reticulum protein 29 (Erp29) plays an important role in the processing of secretory proteins within the ER, possibly by participating in the folding of proteins [33]. HSP70–3 is a BiP homolog involved in assembling protein complexes inside the ER [34]. Indeed, recent studies have shown that cell-surface chaperone proteins can have important roles in reproduction by functioning as intercellular signaling molecules that facilitate sperm-oocyte interactions [35].

Two other interesting proteins that we found were a predicted catalase and a predicted cyclophilin. These are interesting to find in AG secretions. Maturation of sperm involves membrane remodeling and changes in sperm metabolism. For example, when sperms are stored in the cauda segment of the epididymis in mammalian, they must be protected against oxidative/peroxidative reactions, immune attack and damage caused by substances released by dying cells [10]. Catalase serves to protect against toxic effects of hydrogen peroxide by promoting growth of cells, including T-cells, B-cells, myeloid leukemia cells, melanoma cells, mastocytoma cells and normal and transformed fibroblast cells [36]. Here, we identified a predicted catalase in the AG proteome from E. sinensis.

Cyclophilins possess a peptidyl-prolyl cis-trans isomerase (PPIase) activity that rotates the polypeptide chain around a proline between a cis and a trans configuration. They have multiple roles in the folding, assembly and trafficking of cellular proteins [37]. Although considered to be primarily intracellular proteins, some mammalian cells secrete cyclophilins into the extracellular environment, where they have an important role in mediating cellular responses to oxidative stress and in modulating immunity. We also identified cyclophilin A protein in our dataset.

Proteins associated with crustacean molting protein.

Crustaceans possess a calcified exoskeleton that is replaced during growth and development by molting, which occurs periodically under the control of neuropeptide hormones and ecdysteroids. We previously identified 64 unigenes related to hormones in our transcriptome analysis of the testis and AG [6]. Some crustacean molting related proteins in the AG proteome were also identified and will be discussed below.

Cryptocyanin, a crustacean molting protein, has an evolutionary link with arthropod hemocyanins and insect hexamerins [38]. In the Dungeness crab, Cancer magister, hemocyanin reversibly binds oxygen at its highly conserved copper-oxygen binding sites and supplies the crab’s tissues with oxygen. Expression analysis of hemocyanin subunits from the megalopa to adult crab have indicated that there are dramatic changes in subunit composition and oxygen affinity that are correlated with developmental changes in salinity tolerance and ion regulation[39,40]. Cryptocyanin closely resembles hemocyanin in sequence, but lacks several critical copper-binding amino acids and has lost the ability to bind oxygen. Its presence in high concentrations, particularly at specific times in the molt cycle, suggests it has been exploited to carry out new functions. Studying the dynamics of synthesis of hemocyanin and cryptocyanin at the molecular, tissue and organismal levels, including differential responses to hormonal regulation will be an interest area in crustaceans. In this study we identified two cryptocyanin proteins and one hemocyanin protein among secreted AG proteins of male E. sinensis.

Marine invertebrates demonstrate a rich diversity in form and function of their oxygen transport proteins, reflecting how the evolution of new proteins resulting from gene duplications and subsequent mutations is an important process in the adaptive ability of organisms. Phenoloxidase, a third member of the oxygen-transport protein family, functions in sclerotization of the new exoskeleton after molting as well as in immune defense. Its copper-oxygen binding sites are similar to those of hemocyanin, but it is an intracellular protein that is found in circulating hemocytes [41]. Hemocyanin itself has phenoloxidase activity, along with potential anti-microbial activity, which suggests additional functions in response to environmental stressors. The quaternary structure of hemocyanin affects its reactivity as phenoloxidase. It was suggested that prophenoloxidase is released from hemocytes and moves across the epidermis into new exoskeleton during the pre-molting stage and is activated in the early post-molting stage [42]. In crustaceans and insects, 1,3-P-glucan binding proteins (PGBPs) have been purified from plasma [43] and are also found in crustacean blood. These proteins enhance the glucan-mediated activation of the prophenoloxidase activating system (proPO-system) [44]. Vitellogenin may serve one of two functions, as a yolk protein precursor in its classical role and potentially to shuttle iron to developing germ cells.

Functions of signaling pathway factors.

Signaling molecules like phospholipase A, calcium calmodulin, phosphatidylinositol 3-kinase, MAPK signaling pathways and Wnt signaling pathways have been associated with cell proliferation and differentiation as well as with metabolic processes in mammalian granulosa and Sertoli cells [45]. Here, we identified three MAPK signaling pathway proteins (Rap1, FLNA and HSP72) and two Wnt signaling pathway proteins (PP2A and Rho A) among AG proteins of E. sinensis. ERKs and p38 have been demonstrated to be involved in the acrosome reaction and function in spermatogenesis. We also validation the function of p38 in acorosome reaction in sperm of E. sinensis [46]. Gpr48 (G protein-coupled receptor 48) participates in the development of the male epididymis and efferent ducts through regulation of ERα expression via the cAMP/PKA signaling pathway [47].

The 14–3–3 protein positively regulates Ras-mediated pathways and acts downstream or parallel to Raf, but upstream of nuclear factors in Ras signaling [48]. The fibrillarin protein, involved in pre-rRNA processing, is a component of several ribosomal [49] and nucleolar protein complexes and interacts with DDX5 (belonging to the DEAD box protein family) [50]. In mammals, past-1 is highly expressed in the testis and acts in early endocytic membrane fusion and membrane trafficking of recycling endosomes [51]. A recent report demonstrated that the single Drosophila EHD ortholog Past1 is expressed ubiquitously during early embryogenesis, exhibits both plasma membrane associated and punctate cytosolic staining, and is capable of binding in vitro to the adaptor protein Numb [52]. Numb is a conserved membrane-associated protein that antagonizes Notch signaling, a pathway which carry out important functions during oogenesis as well as spermatogenesis [53].

ADP-ribosylation factors (ARFs) are small GTP-binding proteins that play essential roles in intracellular trafficking and organelle structure. ARFs were originally identified as a protein cofactor involved in membrane transport, maintenance of organelle structures and remodeling of the cytoskeleton [54]. Although all ARFs have the ability to regulate the budding and formation of vesicles in the endocytic and exocytic pathways, different ARFs seem to possess different intracellular localizations and functions depending on the specific membrane to which it binds by virtue of recruitment of diverse proteins. Recently, Ran, Rab and Rho proteins, small GTPases in shrimp, were reported to be involved in host defense responses [55]. ARF GTPases are well known to play critical roles in intracellular vesicular trafficking, signal transduction, phagocytosis, endocytosis and remodeling of the cytoskeleton [56]. Here, we identified an ADP ribosylation factor 4 in the E. sinensis AG proteomic analysis. The ecto-ADP-ribosyltransferase 5 (ART 5) protein identified in the AG belongs to a class of enzymes that catalyze the transfer of an ADP-ribose group from NAD to arginine residues on target proteins, such as G proteins, rho, actin, CD44 and integrins [57]. ADP-ribosyltransferases (ARTs) are normally expressed in immune cells and ART5, in the testis [58].

Structural and ubiquitin system proteins.

The heparan sulfate proteoglycan core protein is an integral component of the glomerular basement membrane (GBM), which is responsible for the fixed negative electrostatic membrane charge and provides a barrier that is both size- and charge-selective. It serves as an attachment substrate for cells playing essential roles in vascularization, such as normal heart development, regulating the vascular response to injury and avascular cartilage development [59]. We identified a related protein (Basement membrane-specific heparan sulfate proteoglycan core) in our proteomic analysis, but further analysis is required to determine the function of this protein in AG.

Actin is involved in the initiation of sperm motility, and during epididymal storage in mammals it forms a barrier preventing a premature acrosome reaction [60]. Profilin binds to actin and affects the structure of the cytoskeleton. At high concentrations, profilin prevents the polymerization of actin, whereas it enhances it at low concentrations. By binding to PIP2 (Plasma membrane intrinsic protein 2), profilin inhibits the formation of IP3 (inositol 1,4,5-triphosphate) and DG (diacylglycel). Profilin also can inhibit the androgen receptor (AR) and huntingtin aggregation, and binding of G-actin is essential for its inhibition of AR [61].

In Drosophila reproduction, lectin and cysteine-rich secretory proteins (CRISP) and lipases may play roles in providing energy to the sperm by producing free fatty acids for β-oxidation [62]. Our proteomic analysis detected a predicted lectin in E. sinensis. The laminin receptor can bind to cells via a high affinity receptor, and laminin is thought to mediate the attachment, migration and organization of cells into tissues during embryonic development by interacting with other extracellular matrix components. Fragments of laminin chains can act as biologically active peptides to perturb the blood–testis barrier (BTB) permeability function by accelerating protein endocytosis (e.g., occludin) at the site, thereby destabilizing the BTB integrity to facilitate the transit of preleptotene spermatocytes [63]. Our identification of a predicted laminin receptor in the E. sinensis AG proteome suggests that laminin may be involved in such a process in this crab.

The coatomer is a cytosolic protein complex that binds to dilysine motifs and reversibly associates with Golgi non-clathrin-coated vesicles, which further mediate biosynthetic protein transport from the ER, via the Golgi up to the trans Golgi network [64]. The coatomer protein complex (COP) is required for budding from Golgi membranes and is essential for the retrograde Golgi-to-ER transport of dilysine-tagged proteins. The complex also influences the Golgi structural integrity, as well as the processing, activity and endocytic recycling of LDL (low-density lipoprotein) receptors [66]. It is involved in the Golgi disassembly and reassembly processes during the cell cycle and in autophagy by playing a role in an early endosome function [65]. Kitazawa et al. suggested that COP plays an important role in Drosophila male meiosis, not only through vesicle transport to the cleavage furrow region, but also through the formation of ER-based structures [67]. Required for limiting storage in lipid droplets, COP is involved in lipid homeostasis by regulating the presence of perilipin family members PLIN2 and PLIN3 at the lipid droplet surface. Furthermore, it promotes the association of adipocyte triglyceride lipase (PNPLA2) with the lipid droplet surface to mediate lipolysis, suggesting a relevant role in providing energy for reproduction. Interestingly, we identified four coatomer subunits in the E. sinensis AG proteome.

We also identified components of the ubiquitin proteasome system in the AG proteomic analysis, including 24 proteasome pathway proteins and 2 ubiquitin mediated proteolysis proteins (UBE1 and UBE2D_E). The omnipresent ubiquitin–proteasome system (UPS) is an ATP-dependent enzymatic machinery that targets substrate proteins for degradation by the 26S proteasome by tagging them with an isopeptide chain composed of covalently linked molecules of ubiquitin, a small chaperone protein. UPS has been suggested to play a role in fertilization in humans and some other animals during the process of sperm penetration through the egg’s vitelline coat (VC) [68]. In ascidians, spermatozoa release ubiquitin-activating and conjugating enzymes, proteasomes and unconjugated ubiquitin to first ubiquitinate and then degrade the sperm receptor on the VC. In echinoderms and mammals, the VC [zona pellucida (ZP) in mammals] is ubiquitinated during oogenesis and its sperm receptor is degraded or inactivated during fertilization. Various proteasome subunits and associated enzymes have been detected in spermatozoa and localized to sperm acrosome and other sperm structures [69]. Fertilization is the process whereby individual gametes from the female (egg) and male (sperm) unite to produce offspring. Interaction with the egg is restricted to the sperm head, which contains a large secretory vesicle, the acrosome, overlying the sperm nucleus. Exocytosis of the acrosome (during the AR) is a terminal morphological alteration that must occur prior to penetration of the extracellular coat of the egg (ZP). As is the case with regulated exocytosis in somatic cells, Ca²⁺ is an essential mediator of the AR [70].