1. Introduction
The tetraspanin superfamily of proteins, first recognized in 1990, has emerged as the organizer of functionaries of cell-surface proteins because of their ability to cross-talk with certain other signaling and adhesion molecules involved in cell differentiation. They function in the critical role of “molecular facilitators”, assembling larger molecular complexes and providing them with stability assisting in working in a more orderly manner and efficiently. The first protein belonging to the family, ME491/CD63, was characterized in 1988, and the hallmark protein sequence motifs were reported in 1990 [
1,
2]. Tetraspanins were originally identified as tumor antigens and are broadly expressed as integral membrane proteins with as many as 33 members in humans, 37 in insects (
Drosophila), 23 in sea anemones (
Nematostella) and 17 in plants (
Arabidopsis) [
3–
6].
The conserved predicted structure spans four hydrophobic, putative transmembrane domains (TM1–TM4), forming a small and a large extracellular loop (EC1 and EC2) with short intracellular amino and carboxyl tails, and therefore, called TM
4SF proteins. In addition, 4–8 highly conserved extracellular cysteines have been known, of which two are present in a CCG motif located 28–47 residues after third transmembrane domain [
7]. The four cysteine residues generate a defining mushroom like structural signature for tetraspanins large extracellular loop, mediating specific protein–protein interactions in the tetraspanin web [
8,
9]. The tetraspanin superfamily comprises four subfamilies: the CD-non63, CD63, uroplakin and RDS, of which CD63 has the most ancient origin, mostly exerting their function through interaction with integrins at “tetraspanin-enriched micro domains” (TEMs) or “tetraspanin web” at the cell surface [
9–
12]. Tetraspanin proteins have been known to be synthesized in the endoplasmic reticulum (ER) and after palmitoylation are subsequently transported to the cell surface as building blocks of TEMs [
13]. They also have promiscuous presence in liposome-related organelles or secretory lysosomes, fusing with the cell surface, releasing their content into the extracellular environment [
11,
14].
CD63 encoded protein is a cell surface glycoprotein that is known to complex with integrins such as β1 integrins [
15], other tetraspanins e.g., CD81, CD82, CD9, CD151, and CXCR4 [
16], kinases [
15], adaptor proteins [
17] and other proteins including L6 antigen [
18], syntenin-1 [
19], TIMP-1 [
20], HK-ATPase [
21] and MT-1-MMP [
22]. These myriad functions have led to the suggestion that CD63 may function as “adaptor proteins” [
23], which organize the relative position of other cell-surface molecules and modulate their function. CD63 was identified as a platelet-activating antigen, originally known as platelet glycoprotein 40 or melanoma antigen 491 [
24].
CD63 has been found in many types of blood cells and endo/epithelial cells such as dense granules in platelets, α-granules in megakaryocytes, cytotoxic T-cell granules in T-cells, azurophic granules in neutrophils, Weibel-palade bodies in vascular endothelial cells and eosinophilic granules in B cells, dendritic and epithelial cells [
25–
28]. Upon cell activation, CD63 is mobilized to the cell surface via the exocytic pathway or to endosomes via intracellular pathway and gets involved in various immunophysiological processes [
12,
29]. On the other hand, it activates protein-tyrosine kinase (PTK) and enhances the PTK-induced inhibition of ROMK channels [
30]. CD63 also may represent an important new therapeutic target for the development of anti-retroviral drugs as it has been shown that down-regulation of the gene leads to reduced production of HIV protein Tat and also inhibits the production of late protein p24 [
31]. It has also been reported that CD63 is a critical mediator of viral oncogene, latent membrane protein 1 (LMP1), which functions inside and outside infected (tumor) cells by limiting constitutive activation of NF-κB through promotional trafficking in the endosomal-exosomal pathway [
32]. Reduction in CD63 expression, a marker identified in the malignant progression of human melanoma contributes to invasive and metastatic ability of human melanoma cells [
33,
34].
CD63 cDNA has been identified, characterized and studied for its expression in the channel catfish
, Ictalurus punctatus and has shown 52%–55% identity among fish counterparts and only 43%–46% identity among mammalian counterparts with higher expression in intestine and anterior kidney [
35]. Moreover, Liu
et al. [
36] reported the cDNA encoding CD63 from gut cDNA library of amphioxus,
Branchiostoma belcheri tsinglauense and found it to be extremely close to vertebrate CD63 with the transcript found abundantly in muscle, ovary and foregut. CD63 was cloned from the Chinese shrimp,
Fenneropenaeus chinensis and the transcript was found to be expressed in nerves, epidermis and heart with no expression in intestine, muscle and lymphoid organ. The same was also found to be upregulated when challenged by live white spot syndrome virus (WSSV) and heat-inactivated WSSV [
37].
The biological role of tetraspanins in cellular dynamics has been well established, but there are no reports about their functional role in beetle immunity against microbial elicitors.
Tenebrio molitor is a species of darkling beetle and is a resourceful model for studies in biochemistry, immunology and physiology [
38–
40]. It is an efficient laboratory insect because of its larger size, ease of handling and culture throughout the year. In order to gain insight into the innate immune mechanism of insects, we screened immune-related genes in
T. molitor by an expressed sequence tag (EST) study [
41]. We found clones that correspond to the partial sequence of CD63 in
T. molitor. In a larger perspective, we wanted to study the specific interactions of
T. molitor CD63 with microbes and their cell wall components. For the same reason, we made an initial attempt to clone the full-length cDNA (TmCD63), and characterize the sequence in detail using bioinformatics and experimental approaches. In addition, we examined the developmental and tissue-specific expression profiles of the gene at the basal level. We also report the expression profile of the gene after stimulation of
T. molitor larvae with PGN and β-1,3 glucan as well as
Acholeplasma and
Listeria monocytogenes infection.
3. Discussion
Tetraspanins are an evolutionarily conserved family of proteins that have been investigated for their potential functions in regulating cell morphology, motility, invasion, fusion and signaling as organizers of multi-molecular membrane complexes and have been found to be expressed in a wide variety of organisms, encompassing invertebrates and vertebrates [
7,
42]. The exhaustive collation of information on the existence of tetraspanin-family members has been possible due to the recent whole-genome sequencing of various organisms, and this includes 37 family members identified from
Drosophila melanogaster [
4]. The EST information generated from our earlier work on the coleopteran beetle,
Tenebrio molitor [
41], identified a clone that was evaluated to be a homologue of CD63 and is an important candidate for the study of innate immunity in the insect. Molecular cloning and subsequent
in silico analysis of TmCD63 ORF and protein was critical in assessing its sequence characteristics and validating their affinity with other tetraspanin-family members.
The deduced amino acid sequence of TmCD63 was modeled into four TMs (TM I–IV), short
N- and
C-termini, SEL and the LEL containing the “Cys-Cys-Gly” conserved motif, as well as a conserved “Cys188” as is known from other tetraspanin family-members (TM
4SF). It is known that the proteins in this family contain 200–300 amino acid residues that include four TMs, one SEL region containing 13–30 amino acids, a short intracellular sequence, and a variable LEL containing the “Cys-Cys-Gly”, crucial in the determination of functional specificity [
43]. The LEL domain of TmCD63 protein was topologically located between TM3 and TM4 and contained six invariant cysteine residues, although a variation of four to eight cysteine residues allowing the formation of two to four disulphide bridges, have been reported [
43]. It is also interesting to note that the last cysteine (Cys188) in the LEL of TmCD63 is in proximity to helix-3 and TM4 and seems to be completely conserved within the tetraspanin superfamily, suggesting an important conserved role for this residue. In support of this, the 3D structure of CD81, a representative tetraspanin model, defines the LEL into two domains, one conserved and one variable [
44]. The helices a, b and e in LEL seem to be more conserved, whereas helices c and d that traverse the mushroom-like projection in the tetraspanins are considered to be the most evolutionarily divergent sequences; as is the case of CD81 molecules from different species [
45,
46]. TmCD63 LEL seems to fit into group 6a (CCG--CC---C--C), out of the six different amino acid motifs (4a, 6a–c and 8a,b) that have been observed within the tetraspanin superfamily. Out of the six cysteine residues, four of them are bound by two disulfide bridges in TmCD63. Disulfide bridges are responsible in providing a structural scaffold that enables tolerance of wide-variability in the inter-cysteine loops, probably enabling adaptation to diverse protein interactions.
Protein palmitoylation, considered as a modification of juxtamembrane cysteine residues, plays a crucial role in the tetraspanin web due to the formation of thioester-linkages in the protein. All tetraspanin proteins (including CD9, CD63, CD81, CD82, CD151,
etc.) seem to be palmitoylated, as also found in the case of TmCD63 where eight palmitoylated cysteines have been identified. Earlier reports involving site-directed mutagenesis have suggested the utilization of intracellular membrane proximal cysteines (most specifically the cysteines in short
N- and
C-termini) for palmitoylation [
47]. This being conserved in most other tetraspanin family members including TmCD63 (cysteine at positions 8, 222 and 223), can be considered as the most important residues for palmitoylation; although other cysteines proximal to short intracellular loop may be involved as well. The functional relevance of such modifications is in improving the networking and interactions of tetraspanins with other tetraspanins or other proteins, through stabilization of the “tetraspanin web” [
48]. Association of such tetraspanin-enriched microdomains (TEMs), especially the lysosomal tetraspanin CD63, in the intracellular trafficking of type I membrane protein synaptotagmins from Golgi complex to lysosomes and efficient phagocytosis in macrophages have been demonstrated to be palmitoylation-dependent [
49]. Additionally, the presence of an YXXØ [where tyrosine (Y) is succeeded by any two amino acids denoted as “X” and Ø representing a bulky hydrophobic residue] motif in the
C-terminal region is found critical in cell sorting, including lysosomal and basolateral targeting, although the mechanism is not clear [
50]. TmCD63 does contain the tyrosine-based motif as YETV residues in which valine represents a considerably bulky residue with free energy transfer value of 1.70 KCal. The alignment configured for the present study reported the conspicuous presence of the motif in mostly all insect tetraspanin family members, albeit in mammals valine residue was found replaced with methionine. Mammalian CD63, with a
C-terminal YEVM sequence interacts with a PDZ domain in a transmembrane and connector protein syntenin-1 [
19]. The
N-glycosylation sites of TmCD63 and other TM proteins may serve as a function for signal transduction, intercellular stability and subunit folding [
51].
TmCD63 full-length amino acid sequence was further scrutinized by conducting a multiple sequence alignment (MSA) and subsequently the phylogenetic analysis. As expected, the protein showed highest identity with tetraspanin D107 from its close relative, the red flour beetle,
T. castaneum. The phylogenetic tree showed that the beetle tetraspanins formed a separate branch within the insect tetraspanin cluster. The molecular evolution of the superfamily involves rapid amino acid divergence and considerable changes in length in the LEL. The scope of phylogenetic analysis was exhaustive in the present study, to delineate the evolutionary position of TmCD63. Phylogenetic classifications of tetraspanins in a broader set of eukaryotic organisms have been reported [
52,
53]. One important analysis focused on the “Cys-Cys-Gly” motif of the LEL loop that seems to be absent in tetraspanins from Choanoflagellates, Tsp11 class of fungal tetraspanins
Phytopthora, plants as
Selaginella,
Oryza and
Arabidopsis [
54]. The “Cys-Cys-Gly” motif is a characteristic feature in almost every group of fungi and animals and also in protists like unikonts that are more closely related to them. This suggests that the lack of “Cys-Cys-Gly” motif may be an ancestral characteristic of plants and organisms related to plants [
6]. Another pattern that was mapped onto the phylogenetic tree was the cysteine patterns that seem to cluster together with strongly supported clades in the tetraspanin tree [
6]. In this scenario, cysteine pattern 8a is characteristic of TSPAN15L, 6a of CD63L and 6c of the TSPAN13L group of tetraspanins. Insect tetraspanins have been divided into four groups: CD63-like, CD151, TSPAN5, TSPAN7 and TSPAN31, with three families showing high divergence from other insect and non-insect tetraspanins, suggesting specialized roles for these. CD63 is likely to have a more ancient origin as it is associated with a gene expansion in
Drosophila and also has been reported in sponges [
55].
The great biological relevance of tetraspanin family members in terms of varied functionalities prompted us to investigate the spatial and tissue-specific distribution of the CD63 homologue in the model insect,
T. molitor. The expression level of the gene was found to be homogenous in the developmental stages with slightly higher levels in early pupal and adult stages. The TmCD63 mRNA was found constitutively expressed among the larval tissues and was significantly higher in the immune organs such as gut and Malphigian tubules. Most of expression in the adult tissues was observed in the germ cells, especially testis, suggesting its role in germ cell differentiation and maturation. The wide distribution of TmCD63 in immune-related tissues of the larvae led us to further investigate the role of the gene in innate immune response against certain widely used immune elicitors such as the cell wall components of the fungi (β-1,3 glucan), Gram-positive bacteria (Lys-type PGN) and Gram-negative bacteria (DAP-type PGN). The expression of TmCD63 showed an upregulation, more significantly, in the late hours of challenge in case of β-1,3 glucan and Lys-type PGN. Most strikingly, DAP-type PGN showed a significant induction of TmCD63 transcripts within 3 h of injection and was maintained at later stages after challenge. The maintenance of high mRNA levels in the later phases after challenge might be very important in the development of pathological symptoms from an intracellular pathogen,
L. monocytogenes. Earlier report has documented the intracellular expression of CD63 by fluorescence microscopy and suggested that upon infection with the pathogen, endogenous CD63, as well as, CD9 and CD81 were recruited to the bacterial entry site, though finally only CD81 was required for bacterial internalization, identifying for the first time the role of tetraspanins in
L. monocytogenes entry into target cells [
56]. RNA interference data with CD81 have suggested the membrane organizer action required for the integrity of signaling events at
L. monocytogenes entry sites. CD81 also acts as receptor for hepatitis C virus, and neutralizes anti-HCV antibodies, thus inhibiting the binding of virus to LEL of CD81 [
57]. Other reports of recruitment of CD82 to phagosomes in response to pathogenic fungi as
Cryptococcus neoformans,
Candida albicans and
Aspergillus fumigatus and bacteria as
E. coli and
Staphylococcus aureus seems interesting [
58]. In addition, it has been demonstrated that CD63 recruitment to
C. neoformans phagosomes is dependent on phagosomal acidification [
59]. The roles of CD63 with other tetraspanins such as CD9 and CD151 in preventing adherence of
Neisseria meningitides,
Staphylococcus aureus,
Neisseria lactamica,
E. coli and
Streptococcus pneumoniae to human epithelial cells have been observed with the help of tetraspanin antibodies generated against LEL and small interfering RNAs (siRNAs) [
60]. Additionally, development of exciting new therapeutic drugs/vaccines by using the recombinant form of tetraspanins, especially the LEL has already been reported [
61,
62]. These observations support our hypothesis for TmCD63 that it can be implicated as facilitating and improving the immune status of the insect by recruitment and rehabilitation at phagosomes, specifically as the transcript gets expressed towards the later stage of infection.
4. Experimental Section
4.1. Insect Rearing
Larvae of mealworm beetle, T. molitor procured from College of Pharmacy, Pusan National University, Busan, Korea, were reared on wheat bran meal in an environmental chamber at 25 ± 1 °C with 60% ± 5% relative humidity and a 16:8 h light and dark cycle. Only last instar larvae were used for all experiments unless otherwise stated. Tissues such as the gut, Malphigian tubules, fat body, integument, and hemocytes were collected and pooled (n = 3) from larvae and adults (in this case including ovary and testis) for expression analysis. The tissues were snap-frozen in liquid nitrogen, and stored at −80 °C until RNA extraction.
4.2. Chemicals and Strains
All chemicals used for the experiments were of analytical grade, obtained from Sigma Chemical Co. (St. Louis, MO, USA) until otherwise mentioned in the text. The Gram-positive strain, L. monocytogenes (American type culture collection—ATCC 7644) and A. laidlawii (Mycoplasma) strain (Korean Collection of Type Cultures—KCTC 1621) used for immune elicitor challenge studies were procured from Pusan National University, Busan, Korea.
4.3. Construction of cDNA Library of T. molitor Larvae
The total RNA from T. molitor larvae was isolated by TRIzol reagent (Molecular Research Centre, Inc. Cincinnati, OH, USA) after homogenization using TissueLyser (Qiagen, Valencia, CA, USA) and subsequently mRNA was purified using Absolutely mRNA Purification Kit (Stratagene, Santa Clara, CA, USA). The cDNA library was synthesized using Express cDNA Synthesis Kit (Stratagene). The cDNAs of more than 500 bp in length were ligated into pBK-CMV vector and packaged using the ZAP expression cDNA Gigapack® III Gold cloning kit (Stratagene) according to manufacturer’s instructions. Three clones corresponding to the partial fragment of CD63 homologue (TmCD63) with 5′ untranslated region (UTR), were identified by conducting BLASTx and Swissprot analysis (EMBL-EBI, Hinxton, Cambridge, UK).
4.4. Cloning of the Full-Length cDNA of TmCD63
One of the clones (Nor-
Tenebrio-contr-5-4a-PE_P10) having the longest insert size was used as a template to design the 3′ rapid amplification of cDNA ends (RACE) PCR primers (
Table S1). The 3′ end of TmCD63 was cloned using SMARTer™ RACE cDNA amplification kit (Clontech laboratories, Inc. Mountain View, CA, USA) according to manufacturer’s instructions. One microgram of total RNA and 1 μmol·L
−1 of each primer were used to synthesize the 3′-RACE-ready cDNA with an oligo-dT adaptor primer. Because of the terminal transferase activity of the SMARTScribe reverse transcriptase used, the first strand cDNAs possess the adaptor primer sequence with 3′ end. For 3′-RACE, the first PCR was carried out with the universal primer and gene specific forward primer 1 (3′-GSP1), followed by nested gene-specific forward primer 2 (3′-nGSP2). The PCR amplification was done as follows: denaturation at 94 °C for 3 min, annealing at 58 °C for 30 s, extension at 72 °C for 1 min for 25 cycles. The nested PCR products were extracted and separated in 1% agarose gel by using AccuPrep PCR and Gel purification kit (Bioneer Company, Daejon, Korea) and subsequently cloned into TOPO TA cloning vector (Invitrogen Corporation, Carlsbad, CA, USA) and subsequently transformed into competent
E. coli DH5α cells and sequenced. Specific primers and nested primers for amplification have been listed in
Table 1. The full-length cDNA sequence of TmCD63 has been submitted to European Nucleotide Archive-European Molecular Biology Laboratory (ENA-EMBL, Hinxton, Cambridge, UK) with an accession number HG316497.
4.5. TmCD63 Sequence and Phylogenetic Analysis
TmCD63 cDNA sequence was analyzed by UltraEdit-32 Professional Text/HEX editor (version 12.00, IDM Computer Solutions Inc., Hamilton, OH, USA) software package and deduced amino acid sequence was predicted by Open Reading Frame finder at NCBI [
63]. The sequences of CD63 from other representative insects, as well as, the mammalian tetraspanins were obtained from GenBank, and alignments were conducted using Clustal X (version 2.0.12, University of Strasbourg, Strasbourg, France) [
64]. Subsequently, phylogenetic analysis of the full length amino acid sequence was conducted using MEGA version 5.0 software (The Biodesign Institute, Tempe, AZ, USA) [
65]. A phylogenetic tree was constructed using the neighbor joining method [
66]. To evaluate the branch strength of the phylogenetic tree, bootstrap values representing 1000 replicates were taken for analysis. Percentage identities of the full length amino acid sequence of TmCD63 and other representative species were calculated using ClustalW2 (EMBL-European Bioinformatics Institute, Cambridge, UK).
The transmembrane domains in proteins were predicted by TMHMM (Technical University of Denmark, Lyngby, Denmark). The palmitoylation sites at high threshold were predicted by CSS-Palm 3.0 (Cuckoo workgroup, Wuhan, China) [
67]. The prediction of the putative signal peptide sequence was done at the Signal 4.0 server [
68]. The ORF base composition and protein statistics including the theoretical MW and isoelectric point (pI) were performed using the EditSeq and Protean tool of Lasergene 9.0 software (DNASTAR Inc., Madison, WI, USA). The prediction of
N-glycosylation sites was confirmed at NetNGlyc 1.0 server (Technical University of Denmark, Lyngby, Denmark). ProtScale [
69] at ExPASy bioinformatics resource portal was used to predict the hydrophobicity quality of the protein [
70]. ProtParam [
71] at ExPASy bioinformatics resource portal was used for computing various physical and chemical parameters [
72].
4.6. Tissue Distribution and Developmental Expression Profile of TmCD63 by Quantitative Real-Time PCR
Total RNA was extracted from different developmental stages of
T. molitor metamorphosis such as last instar larvae, pre-pupae, pupae (days 1 to 7) and adults (days 1 and 2). In addition, various tissues from gut, Malphigian tubules, fat body, integument as well as ovaries and testis from
T. molitor were dissected and collected to isolate total RNA by using SV Total RNA isolation kit (Promega Corporation, Madison, WI, USA) following manufacturer’s protocol. For hemocytes collection, hemolymph were collected from abdomen using an equal volume of modified Alsevier’s solution (MAS) as anticoagulant [
73] and centrifuged for 10 min at 800 ×
g, 4 °C. RNA concentration and purity of each sample were quantified in NanoDrop 2000 UV-Vis Spectrophotometer (Thermo Scientific, Wilmington, OH, USA; A260/A280 nm ratios >1.8). The RNA integrity was checked by separation on ethidium bromide stained 1% agarose gels. A sample of 2 μg of total RNA was reverse-transcribed using Oligo (dT)
18 primer (Invitrogen, Carlsbad, CA, USA) at 72 °C for 5 min. Quantitative real-time PCR was performed on Exicycler™ 96 real-time quantitative thermal block (Bioneer, Korea) using primers (
Table S1) at an initial denaturation of 94 °C for 3 min, followed by 28 cycles at 94 °C for 40 s, 72 °C for 90 s and 72 °C for 10 min. Each treatment was independently replicated three times. The 2
−ΔΔCt method (where ΔΔ
Ct = Δ
Ct target − Δ
Ct reference) was employed to analyze the expression levels of TmCD63 [
74]. The gene expression levels were normalized to ribosomal protein L27a (accession number X99204), which served as an internal control. The data were presented as relative mRNA expression levels [mean ± standard deviation (SD),
n = 3].
4.7. Immune Elicitors and Challenge Studies
Immune elicitors as the intracellular pathogen, L. monocytogenes, A. laidlawii lysate and live cells, β-1,3 glucan, Lys- and DAP-type peptidoglycan were procured from College of Pharmacy, Pusan National University, Korea. Time course of TmCD63 induction was studied in the whole larvae by injection of 4 μL of immune elicitors into the last instar larvae of mealworm beetle, T. molitor with the Picospritzer III (Parker Hannifin, Hollis, NH, USA). The control group was injected only with phosphate buffer saline (PBS) (0.14 M sodium chloride, 3 mM potassium chloride, 8 mM disodium hydrogen phosphate dodecahydrate, 1.5 mM potassium phosphate monobasic, pH 7.4), as the wounding buffer. Total RNA from the larvae was collected after 3, 6, 12, 18 and 24 h post-injection. All the samples were frozen in liquid nitrogen and then stored at −70 °C until use.
4.8. Time-Course Analysis of TmCD63 by Quantitative Real-Time PCR
Total RNA from
T. molitor was prepared using Total RNA Isolation kit (Promega Corp., Madison, WI, USA) according to the manufacturer’s protocol. Two micrograms of total RNA was reverse-transcribed in a 50 μL reaction mixture with a High capacity cDNA Reverse Transcription Kit (Bioneer, Korea). qRT-PCR was performed on Exicycler™ 96 Real-Time Quantitative Thermal Block (Bioneer, Korea). The 50-μL mixture including 1 μL of cDNA, 10 pmol of each primer (
Table S1) and 25 μL of 1× LightCycler 480 SYBR Green (Takara Bio Inc. Shiga, Japan) was placed in 96 well plates. The PCR program was set with an initial denaturation of 95 °C for 20 s, followed by 40 cycles at 95 °C for 15 s, 60 °C for 1 min and 72 °C for 10 s.
The primers were designed using Primer Quest (Integrated DNA Technologies, Coralville, IA, USA) [
75]. Each treatment was independently replicated three times. The 2
−ΔΔCt method was employed to analyze the expression levels of TmCD63 and the value obtained denoted the
n-fold difference related to the calibrator (uninjected samples). The data were presented as relative mRNA expression levels (means ± S.D.,
n = 3). The data were subjected to a one-way analysis of variance (ANOVA). Significant differences between the treated group and the corresponding control group at each time point were indicated with one asterisk for
p < 0.05 and two asterisks for
p < 0.01.