Existence and functions of hypothalamic kisspeptin neuropeptide signaling system in a non-chordate deuterostome species

The kisspeptin (Kp) system is a central modulator of the hypothalamic-pituitary-gonadal axis in vertebrates. Its existence outside the vertebrate lineage remains largely unknown. Here we report the identification and characterization of Kp system in the sea cucumber Apostichopus japonicus. The gene encoding the Kp precursor, generates two mature neuropeptides, AjKiss1a and AjKiss1b. The Kp receptors, AjKissR1 and AjKissR2, are strongly activated by synthetic A. japonicus and vertebrate Kps, triggering a rapid intracellular mobilization of Ca2+, followed by receptor internalization. AjKissR1 and AjKissR2 share similar intracellular signaling pathways via Gαq/PLC/PKC/MAPK cascade, when activated by C-terminal decapeptide (AjKiss1b-10). The A. japonicus Kp system functions in mutiple tissues which are closely related to reproduction and metabolism. Overall, our findings uncover for the first time, to our knowledge, the existence and function of the Kp system in a non-chordate species and provide new evidence to support the ancient origin of the hypothalamic neurosecretory system.


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Kp receptors (Fig. 1E).  To verify the exact expression and localization of the putative A. japonicus Kp receptors,156 AjKissR1 and 2 with an N-terminal FLAG-tag or with enhanced green fluorescent protein 157 (EGFP) fused to the C-terminal end, were constructed and stably or transiently expressed in 158 human embryonic kidney 293 (HEK293) cells. As shown in Fig. 2A, confocal microscopy 159 revealed that AjKissR1 and 2 were predominantly expressed and localized to the cell surface, 160 with some intracellular accumulation, in the absence of the ligand in HEK293 cells. Next, to 161 examine whether AjKissR1 and AjKissR2 are activated by synthetic Kps, the calcium probe 162 fura-2-based Ca 2+ mobilization assay was performed. As shown in Fig. 2B, both AjKiss1a and 163 AjKiss1b elicited a rapid increase of intracellular Ca 2+ , in a concentration-dependent manner, 164 in HEK293 cells transfected with AjKissR1 and AjKissR2, respectively. However, AjKissR1 165 was preferentially activated by AjKiss1b, with an EC50 value of 8.06 nM (Fig. 2B2), whereas 166 AjKissR2 was more specifically activated by AjKiss1a, with an EC50 value of 1.98 nM (Fig.  167

2B1). 168
Agonist-mediated internalization from the cell surface to the cytoplasm has been recognized 169 as a key mechanism in regulating the strength and duration of GPCR-mediated cell signaling 170 and to directly reflect the activation of the receptor [32,33]. In this study, C-terminal fusion 171 expression of AjKissR1 and 2 with EGFP was used to track the internalization and trafficking 172 of receptors. As shown in Fig. 2C, AjKissR1 and 2 were activated by AjKiss1b and AjKiss1a, 9 respectively, to undergo significant internalization from the plasma membrane to the 174 cytoplasm. These data provide clear evidence that AjKissR1 and 2 are functional receptors 175 that are specific for neuropeptides AjKiss1b and AjKiss1a, respectively.  To examine the cross-reactivity of A. japonicas and vertebrate Kp receptors, A. japonicus, 189 human, frog, and zebrafish Kps (hKiss1-10, XtKiss1b-10, zfKiss1-10, and zfKiss2-10) were 190 10 used to detect their potential in triggering intracellular Ca 2+ mobilization. As indicated in Fig.  191 2, for AjKissR1, hKiss1-10 and XtKiss1b-10 exhibited higher potency, however, both 192 zfKiss1-10 and zfKiss2-10 showed much lower potency in eliciting Ca 2+ mobilization (Fig. 193 3A), while for the activation of AjKissR2, XtKiss1b-10, zfKiss1-10, and zfKiss2-10 had a 194 higher potency than hKiss1-10 (Fig. 3B). However, human neuropeptide S (NPS) showed no 195 potency for the activation of both AjKissR1 and AjKissR2 ( Fig. 3C and D). Further analysis 196 demonstrated that both AjKiss1a and AjKiss1b could activate hKiss1R, zfKiss1Ra, and 197 zfKiss1Rb with different potency (Fig. 3E, F and G).  AjKiss1b, a combination of functional assays, with different inhibitors, was performed. As 5B and C). Moreover, we determined that PKCα, PKCβI, and PKCβII are involved in the 252 activation of the MAPK pathway, using a PKC subtype recruitment assay ( Fig. 5D and E). 253 Overall, these results suggest that AjKissR1 and AjKissR2, once activated by ligand, can 254 activate the MAPK cascade, particularly ERK1/2, via the G αq /PLC/PKC signaling pathway 255 (Fig. 5F).        provides valuable infromation for further investigation (Fig. 7). In 2013, Kp-type receptors 389 were first annotated in the genome of the acorn worm (S. kowalevskii) and purple sea urchin Moreover, the presence of Kps in extracts of radial nerve cords was confirmed by proteomic 395 mass spectrometry in the crown-of-thorns starfish A. planci [49]. Recently, a 180-residue 396 protein comprising two putative Kp-type peptides has been predicted and a C-terminally 397 amidated peptide GRQPNRNAHYRTLPF-NH2 was confirmed by mass spectrometric 398 analysis of centrol nerve ring extracts [25]. These advances provide a basis for experimental 399 studies on the Kp/KpR system in echinoderms. 400 In the present study, we cloned the full length of Kiss cDNA sequence from the nerve ring,

Cross interaction between A. japonicus and the Kp/KpR systems of vertebrates 436 confirmed the existence of Kp signaling systems in Echinoderm 437
In the mammalian genome, a single Kiss1 gene produces a mature 54-amino acid peptide, 438 Kp-54, which is further proteolytically truncated to 14 and 13 amino acid carboxyl-terminal 439 peptides, Kp-14 and Kp-13, with a common C-terminal decapeptide (Kp-10) core [53,54] In this study, our data showed that upon synthetic peptide stimulation, both AjKissR1 and 475 AjKissR2 induced a rapid and transient rise of intracellular Ca 2+ , in a dose-dependent manner, 476 via the G αq -coupled signaling pathway. Further investigation of AjKissR1 and AjKissR2 477 mediated cell signaling indicated that AjKissR1 and AjKissR2 share similar intracellular 478 signaling pathways, via G αq /PLC/PKC and ERK1/2 phosphorylation. Our results showed no 479 significant accumulation of cAMP, as detected by ELISA, indicating that G αs -dependent PKA 480 signaling was not activated by the Kp receptors of A. japonicus. Since the G αq -coupled PKC 481 signaling pathway, mediated by identified Kp systems, is conserved in all chordate species 482 and A. japonicus, and the G αs -dependent PKA signaling was conserved in only a few teleost 483 Kp receptors (mainly from the KpR3 subfamily), we propose that G αq -coupled signaling 484 25 activation originally evolved in this hypothalamic neuropeptide system. 485 expression plasmids, RT-PCR was performed using total RNA extracted from A. japonicus 547 ovaries, to synthesize template cDNA. PCR amplification for coding sequences of AjKissR1/2 548 was performed using specific primers, with restriction sites (Supplementary Table 2). The 549 corresponding PCR products were then cloned to pCMV-FLAG and pEGFP-N1 vectors, 550 28 respectively, using restriction enzymes and Rapid DNA Ligation Kit (Beyotime, China). 551 FLAG-hKiss1R plasmid was constructed using total synthesized DNA (Wuhan Transduction 552 Bio) with specific primers containing restriction sites (Supplementary Table 2). All constructs 553 were sequenced to verify the correct sequences, orientations, and reading frames. were washed three times with PBS and then fixed with 4% paraformaldehyde in PBS for 10 576 min at room temperature. Finally, the cells were mounted in mounting reagent 577 (DTT/PBS/glycerol,1:8:2) and visualized by fluorescence microscopy on a Zeiss laser 578 scanning confocal microscope, which was attached to a Zeiss Axiovert 200 microscope and 579 linked to a LSM5 computer system. 580

loading. 614
Immunoreactive bands were detected with an enhanced chemiluminescent substrate 615 (Beyotime), and the membrane was scanned by using a Tanon 5200 Chemiluminescent 616

Supplementary information 936
Existence and functions of hypothalamic kisspeptin neuropeptide signaling system in

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The signal peptide, predicted by online SignalP-5.0 Server, is labeled in box with full lines; the 969 cleavage sites, predicted based on previously known consensus cleavage motifs by using the NeuroPred