Satisfying QTPP of Erythropoietin Biosimilar by QbD through DoE-Derived Downstream Process Engineering.

Well-characterized and scalable downstream processes for the purification of biologics are extremely demanding for delivering quality therapeutics to patients at a reasonable price. Erythropoietin (EPO) is a blockbuster biologic with diverse clinical applications, but its application is limited to financially well-off societies due to its high price. The high price of EPO is associated with the technical difficulties related to the purification challenge to obtain qualified products with a cost-effective defined process. Though there are reports for the purification of EPO there is no report of a well-characterized downstream process with critical process parameters (CPPs) that can deliver EPO consistently satisfying the quality target product profile (QTPP), which is a critical regulatory requirement. To advance the field, we applied the quality by design (QbD) principle and design of experiment (DoE) protocol to establish an effective process, which is scalable up to 100× batch size satisfying QTPP. We have successfully transformed the process from static mode to dynamic mode and validated it. Insignificant variation (p > 0.05) within and between 1×, 10×, and 100× batches showed that the process is reproducible and seamlessly scalable. The biochemical analysis along with the biofunctionality data ensures that the products from different scale batches were indifferent and comparable to a reference product. Our study thereby established a robust and scalable downstream process of EPO biosimilar satisfying QTPP. The technological scheme presented here can speed up the production of not only EPO but also many other life-saving biologics and make them available to the mass population at a reduced cost.

DoE-derived continuous and robust process for manufacturing of pharmaceutical-grade wide-range LNPs for RNA-vaccine/drug delivery.

Lipid nanoparticle (LNP) technology has become extremely demanding for delivering RNA-products and other drugs. However, there is no platform to manufacture pharmaceutical-grade LNPs with desired particle size from a wide range in continuous mode. We have developed a unique platform to obtain any specific size-range of LNPs from 60 to 180 nm satisfying pharmaceutical regulatory requirements for polydispersity index, sterility, dose uniformity and bio-functionality. We applied design of experiment (DoE) methodology and identified the critical process parameters to establish the process for global application. Cross-point validation within the response map of DoE confirmed that the platform is robust to produce specific size (± 10 nm) of LNPs within the design-range. The technology is successfully transformed to production scale and validated. Products from R&D, pilot and production batches for a candidate SARS-CoV-2 mRNA-vaccine generated equivalent biological responses. The data collectively established the robustness and bio-uniformity of doses for global RNA-vaccine/drug formulation.

An mRNA-based vaccine candidate against SARS-CoV-2 elicits stable immuno-response with single dose.

D614G genotype of SARS-CoV-2 virus is highly infectious and responsible for almost all infection for 2nd wave. However, there are currently no reports with D614G as vaccine candidate. Here we report the development of an mRNA-LNP vaccine with D614G variant and characterization in animal model. We have used special mRNA-architecture and formulation that provides suitable response of the product. The surface plasmon resonance (SPR) data with spike protein (S) revealed that immunization generated specific antibody pools against the whole extracellular domain (RBD and S2) of the spike protein. The anti-sera and purified IgGs from immunized mice neutralized SARS-CoV-2-pseudoviruses in ACE2-expressing HEK293 cells in a dose dependent manner. Importantly, single-dose immunization protected mice-lungs from homotypic-pseudovirus entry and cytopathy. The immunologic responses have been implicated by a balanced and stable population of CD4+ cells with a Th1 bias. The data suggested great promise for immediate translation of the technology to the clinic.

A novel knowledge-derived data potentizing method revealed unique liver cancer-associated genetic variants.

BACKGROUND: Next-generation sequencing (NGS) has been advancing the progress of detection of disease-associated genetic variants and genome-wide profiling of expressed sequences over the past decade. NGS enables the analyses of multiple regions of a genome in a single reaction format and has been shown to be a cost-effective and efficient tool for root-cause analysis of disease and optimization of treatment. NGS has been leading global efforts to device personalized and precision medicine (PM) in clinical practice. The effectiveness of NGS for the aforementioned applications has been proven unequivocal for multifactorial diseases like cancer. However, definitive prediction of cancer markers for all types of diseases and for global populations still remains highly rewarding because of the diversity of cancer types and genetic variants in human. RESULTS: We performed exome sequencing of four samples in quest of critical genetic factor/s associated with liver cancer. By imposing knowledge-based filter chains, we have revealed a panel of genetic variants, which are unrecognized by current major genomics data repositories. Total 20 MNV-induced, 5 INDEL-induced, and 31 SNV-induced neoplasm-exclusive genes were revealed through NGS data acquisition followed by data curing with the application of quality filter chains. Liver-specific expression profile of the identified gene pool is directed to the selection of 17 genes which could be the as likely causative genetic factors for liver cancer. Further study on expression level and relevant functional significance enables us to identify and conclude the following four novel variants, viz., c.416T>C (p.Phe139Ser) in SORD, c.1048_1049delGCinsCG (p.Ala350Arg) in KRT6A, c.1159G>T (p.Gly387Cys) in SVEP1, and c.430G>C (p.Gly144Arg) in MRPL38 as a critical genetic factor for liver cancer. CONCLUSION: By applying a novel data prioritizing rationale, we explored a panel of previously unaddressed liver cancer-associated variants. These findings may have an opportunity for early prediction of neoplasm/cancer in liver and designing of relevant personalized/precision liver cancer therapeutics in clinical practice. Since NGS protocol is associated with tons of non-specific mutations due to the variation in background genetic makeup of subjects, therefore, our method of data curing could be applicable for more effective screening of global genetic variants related to disease onset, progression, and remission.

Characterization of the zebrafish cx36.7 gene promoter: Its regulation of cardiac-specific expression and skeletal muscle-specific repression.

Zebrafish connexin 36.7 (cx36.7/ecx) has been identified as a key molecule in the early stages of heart development in this species. A defect in cx36.7 causes severe heart malformation due to the downregulation of nkx2.5 expression, a result which resembles congenital heart disease in humans. It has been shown that cx36.7 is expressed specifically in early developing heart cardiomyocytes. However, the regulatory mechanism for the cardiac-restricted expression of cx36.7 remains to be elucidated. In this study we isolated the 5'-flanking promoter region of the cx36.7 gene and characterized its promoter activity in zebrafish embryos. Deletion analysis showed that a 316-bp upstream region is essential for cardiac-restricted expression. This region contains four GATA elements, the proximal two of which are responsible for promoter activation in the embryonic heart and serve as binding sites for gata4. When gata4, gata5 and gata6 were simultaneously knocked down, the promoter activity was significantly decreased. Moreover, the deletion of the region between -316 and -133bp led to EGFP expression in the embryonic trunk muscle. The distal two GATA and A/T-rich elements in this region act as repressors of promoter activity in skeletal muscle. These results suggest that cx36.7 expression is directed by cardiac promoter activation via the two proximal GATA elements as well as by skeletal muscle-specific promoter repression via the two distal GATA elements.

An Engineered N-Cadherin Substrate for Differentiation, Survival, and Selection of Pluripotent Stem Cell-Derived Neural Progenitors.

For stem cell-based treatment of neurodegenerative diseases a better understanding of key developmental signaling pathways and robust techniques for producing neurons with highest homogeneity are required. In this study, we demonstrate a method using N-cadherin-based biomimetic substrate to promote the differentiation of mouse embryonic stem cell (ESC)- and induced pluripotent stem cell (iPSC)-derived neural progenitor cells (NPCs) without exogenous neuro-inductive signals. We showed that substrate-dependent activation of N-cadherin reduces Rho/ROCK activation and ß-catenin expression, leading to the stimulation of neurite outgrowth and conversion into cells expressing neural/glial markers. Besides, plating dissociated cells on N-cadherin substrate can significantly increase the differentiation yield via suppression of dissociation-induced Rho/ROCK-mediated apoptosis. Because undifferentiated ESCs and iPSCs have low affinity to N-cadherin, plating dissociated cells on N-cadherin-coated substrate increase the homogeneity of differentiation by purging ESCs and iPSCs (~30%) from a mixture of undifferentiated cells with NPCs. Using this label-free cell selection approach we enriched differentiated NPCs plated as monolayer without ROCK inhibitor. Therefore, N-cadherin biomimetic substrate provide a powerful tool for basic study of cell-material interaction in a spatially defined and substrate-dependent manner. Collectively, our approach is efficient, robust and cost effective to produce large quantities of differentiated cells with highest homogeneity and applicable to use with other types of cells.

Ligand-induced internalization, recycling, and resensitization of adrenomedullin receptors depend not on CLR or RAMP alone but on the receptor complex as a whole.

Adrenomedullins (AM) is a multifaceted distinct subfamily of peptides that belongs to the calcitonin gene-related peptide (CGRP) superfamily. These peptides exert their functional activities via associations of calcitonin receptor-like receptors (CLRs) and receptor activity-modifying proteins (RAMPs) RAMP2 and RAMP3. Recent studies established that RAMPs and CLRs can modify biochemical properties such as trafficking and glycosylation of each other. However there is very little or no understanding regarding how RAMP or CLR influence ligand-induced events of AM-receptor complex. In this study, using pufferfish homologs of CLR (mfCLR1-3) and RAMP (mfRAMP2 and mfRAMP3), we revealed that all combinations of CLR and RAMP quickly underwent ligand-induced internalization; however, their recycling rates were different as follows: mfCLR1-mfRAMP3>mfCLR2-mfRAMP3>mfCLR3-mfRAMP3. Functional receptor assay confirmed that the recycled receptors were resensitized on the plasma membrane. In contrast, a negligible amount of mfCLR1-mfRAMP2 was recycled and reconstituted. Immunocytochemistry results indicated that the lower recovery rate of mfCLR3-mfRAMP3 and mfCLR1-mfRAMP2 was correlated with higher proportion of lysosomal localization of these receptor complexes compared to the other combinations. Collectively our results indicate, for the first time, that the ligand-induced internalization, recycling, and reconstitution properties of RAMP-CLR receptor complexes depend on the receptor-complex as a whole, and not on individual CLR or RAMP alone.

Antiviral activity of trappin-2 and elafin in vitro and in vivo against genital herpes.

Serine protease inhibitor elafin (E) and its precursor, trappin-2 (Tr), have been associated with mucosal resistance to HIV-1 infection. We recently showed that Tr/E are among principal anti-HIV-1 molecules in cervicovaginal lavage (CVL) fluid, that E is ∼130 times more potent than Tr against HIV-1, and that Tr/E inhibited HIV-1 attachment and transcytosis across human genital epithelial cells (ECs). Since herpes simplex virus 2 (HSV-2) is a major sexually transmitted infection and risk factor for HIV-1 infection and transmission, we assessed Tr/E contribution to defense against HSV-2. Our in vitro studies demonstrated that pretreatment of endometrial (HEC-1A) and endocervical (End1/E6E7) ECs with human Tr-expressing adenovirus (Ad/Tr) or recombinant Tr/E proteins before or after HSV-2 infection resulted in significantly reduced virus titers compared to those of controls. Interestingly, E was ∼7 times more potent against HSV-2 infection than Tr. Conversely, knockdown of endogenous Tr/E by small interfering RNA (siRNA) significantly increased HSV-2 replication in genital ECs. Recombinant Tr and E reduced viral attachment to genital ECs by acting indirectly on cells. Further, lower viral replication was associated with reduced secretion of proinflammatory interleukin 8 (IL-8) and tumor necrosis factor alpha (TNF-α) and decreased NF-κB nuclear translocation. Additionally, protected Ad/Tr-treated ECs demonstrated enhanced interferon regulatory factor 3 (IRF3) nuclear translocation and increased antiviral IFN-ß in response to HSV-2. Lastly, in vivo studies of intravaginal HSV-2 infection in Tr-transgenic mice (Etg) showed that despite similar virus replication in the genital tract, Etg mice had reduced viral load and TNF-α in the central nervous system compared to controls. Collectively, this is the first experimental evidence highlighting anti-HSV-2 activity of Tr/E in female genital mucosa.

Milk matters: soluble Toll-like receptor 2 (sTLR2) in breast milk significantly inhibits HIV-1 infection and inflammation.

The majority of infants who breastfeed from their HIV-positive mothers remain uninfected despite constant and repeated exposure to virus over weeks to years. This phenomenon is not fully understood but has been closely linked to innate factors in breast milk (BM). Most recently we have focused on one such innate factor, soluble Toll-like receptor 2 (sTLR2) for its significant contribution as an inhibitor of inflammation triggered by bacterial and viral antigens. We hypothesized that sTLR2 in BM inhibits immune activation/inflammation and HIV-1 infection. sTLR2 protein profiles were analyzed in HIV-uninfected BM and showed dramatic variability in expression concentration and predominant sTLR2 forms between women. sTLR2 immunodepleted BM, versus mock-depleted BM, incubated with Pam(3)CSK(4) lead to significant increases in IL-8 production in a TLR2-dependant fashion in U937, HEK293-TLR2, and Caco-2. Importantly, TLR2-specific polyclonal and monoclonal antibody addition to BM prior to cell-free R5 HIV-1 addition led to significantly (P<0.01, P<0.001, respectively) increased HIV-1 infection in TZM-bl reporter cells. To confirm these findings, sTLR2-depletion in BM led to significantly (P<0.001) increased HIV-1 infection in TZM-bl cells. Notably, immunodepletion does not allow for the complete removal of sTLR2 from BM, thus functional testing shown here may underestimate the total effect elicited by sTLR2 against HIV-1 and synthetic bacterial ligand. This study provides evidence for the first time that sTLR2 in BM may provide a dual protective role for infants breastfeeding from their HIV-infected mothers by; (1) immunomodulating pro-inflammatory responses to bacterial ligands, and (2) directly inhibiting cell-free HIV-1 infection. Thus, sTLR2 in BM may be critical to infant health and prove beneficial in decreasing vertical HIV-1 transmission to infants.

Trappin-2/elafin modulate innate immune responses of human endometrial epithelial cells to PolyI:C.

BACKGROUND: Upon viral recognition, innate and adaptive antiviral immune responses are initiated by genital epithelial cells (ECs) to eradicate or contain viral infection. Such responses, however, are often accompanied by inflammation that contributes to acquisition and progression of sexually transmitted infections (STIs). Hence, interventions/factors enhancing antiviral protection while reducing inflammation may prove beneficial in controlling the spread of STIs. Serine antiprotease trappin-2 (Tr) and its cleaved form, elafin (E), are alarm antimicrobials secreted by multiple cells, including genital epithelia. METHODOLOGY AND PRINCIPAL FINDINGS: We investigated whether and how each Tr and E (Tr/E) contribute to antiviral defenses against a synthetic mimic of viral dsRNA, polyinosine-polycytidylic acid (polyI:C) and vesicular stomatitis virus. We show that delivery of a replication-deficient adenovector expressing Tr gene (Ad/Tr) to human endometrial epithelial cells, HEC-1A, resulted in secretion of functional Tr, whereas both Tr/E were detected in response to polyI:C. Moreover, Tr/E were found to significantly reduce viral replication by either acting directly on virus or through enhancing polyI:C-driven antiviral protection. The latter was associated with reduced levels of pro-inflammatory factors IL-8, IL-6, TNFα, lowered expression of RIG-I, MDA5 and attenuated NF-κB activation. Interestingly, enhanced polyI:C-driven antiviral protection of HEC-Ad/Tr cells was partially mediated through IRF3 activation, but not associated with higher induction of IFNß, suggesting multiple antiviral mechanisms of Tr/E and the involvement of alternative factors or pathways. CONCLUSIONS AND SIGNIFICANCE: This is the first evidence of both Tr/E altering viral binding/entry, innate recognition and mounting of antiviral and inflammatory responses in genital ECs that could have significant implications for homeostasis of the female genital tract.

Anti-HIV-1 activity of elafin is more potent than its precursor's, trappin-2, in genital epithelial cells.

Cervicovaginal lavage fluid (CVL) is a natural source of anti-HIV-1 factors; however, molecular characterization of the anti-HIV-1 activity of CVL remains elusive. In this study, we confirmed that CVLs from HIV-1-resistant (HIV-R) compared to HIV-1-susceptible (HIV-S) commercial sex workers (CSWs) contain significantly larger amounts of serine antiprotease trappin-2 (Tr) and its processed form, elafin (E). We assessed anti-HIV-1 activity of CVLs of CSWs and recombinant E and Tr on genital epithelial cells (ECs) that possess (TZM-bl) or lack (HEC-1A) canonical HIV-1 receptors. Our results showed that immunodepletion of 30% of Tr/E from CVL accounted for up to 60% of total anti-HIV-1 activity of CVL. Knockdown of endogenous Tr/E in HEC-1A cells resulted in significantly increased shedding of infectious R5 and X4 HIV-1. Pretreatment of R5, but not X4 HIV-1, with either Tr or E led to inhibition of HIV-1 infection of TZM-bl cells. Interestingly, when either HIV-1 or cells lacking canonical HIV-1 receptors were pretreated with Tr or E, HIV-1 attachment and transcytosis were significantly reduced, and decreased attachment was not associated with altered expression of syndecan-1 or CXCR4. Determination of 50% inhibitory concentrations (IC(50)) of Tr and E anti-HIV-1 activity indicated that E is ∼130 times more potent than its precursor, Tr, despite their equipotent antiprotease activities. This study provides the first experimental evidence that (i) Tr and E are among the principal anti-HIV-1 molecules of CVL; (ii) Tr and E affect cell attachment and transcytosis of HIV-1; (iii) E is more efficient than Tr regarding anti-HIV-1 activity; and (iv) the anti-HIV-1 effect of Tr and E is contextual.

Calcitonin receptor-like receptor (CLR) influences posttranslational events of receptor activity-modifying proteins (RAMPs).

Adrenomedullins (AM) form a multifunctional subfamily of the calcitonin gene-related peptide (CGRP) superfamily, the members of which exert their physiological roles through a 1:1 combination of calcitonin receptor-like receptors (CLRs) and receptor activity-modifying proteins (RAMPs). It has been shown that RAMPs can modify the biochemical properties of CLRs; for example, RAMP escorts CLR to the plasma membrane, affects glycosylation state of CLR, and transforms the ligand selectivity of CLR, but on the other hand the effects of CLRs on the biochemical and functional properties of the partner RAMPs are not well established. In this study, using pufferfish (mefugu, mf) homolog, we revealed that mfCLR1 could affect the post-translational modification and trafficking pathway of mfRAMP1. In addition, mfCLRs boosted mfRAMP1, mfRAMP2b, and mfRAMP3 translocation to cell surface. We further revealed that mfRAMPs, except mfRAMP1 and mfRAMP3, could be expressed as multimers on the plasma membrane. However, only monomeric form of mfRAMP2a, mfRAMP4, and mfRAMP5 could heteromerize with mfCLR1 but not with mfCLR2 or mfCLR3, which was consistent with their abilities to induce cAMP response. Collectively our results indicate that the glycosylation, subcellular trafficking, and pharmacological properties of the components of RAMP-CLR receptor complexes are regulated in an interdependent manner.

Anti-HIV-1 activity of elafin depends on its nuclear localization and altered innate immune activation in female genital epithelial cells.

Elafin (E) and its precursor trappin-2 (Tr) are alarm antiproteases with antimicrobial and immunomodulatory activities. Tr and E (Tr/E) have been associated with HIV-1 resistance. We recently showed that Tr/E reduced IL-8 secretion and NF-κB activation in response to a mimic of viral dsRNA and contributed to anti-HIV activity of cervicovaginal lavage fluid (CVL) of HIV-resistant (HIV-R) commercial sex workers (CSWs). Additionally, Tr, and more so E, were found to inhibit attachment/entry and transcytosis of HIV-1 in human endometrial HEC-1A cells, acting through virus or cells. Given their immunomodulatory activity, we hypothesized that Tr/E could exert anti-HIV-1 activity at multiple levels. Here, using tagged and untagged Tr/E proteins, we comparatively evaluated their protease inhibitory, anti-HIV-1, and immunomodulatory activities, and cellular distribution. E appeared to function as an autocrine/paracrine factor in HEC-1A cells, and anti-HIV-1 activity of E depended on its unmodified N-terminus and altered cellular innate activation, but not its antiprotease activity. Specifically, exogenously added N-terminus-unmodified E was able to enter the nucleus and to reduce viral attachment/entry and transcytosis, preferentially affecting R5-HIV-1(ADA), but not X4-HIV-1(IIIB). Further, anti-HIV-1 activity of E was associated with significantly decreased HIV-1-triggered IL-8 release, attenuated NF-κB/p65 nuclear translocation, and significantly modulated mRNA expression of innate sensors TLR3 and RIG-I in HEC-1A cells. Most importantly, we found that elevated Tr/E in CVLs of HIV-R CSWs were associated with lower mRNA levels of TLRs 2, 3, 4 and RIG-I in the genital ECs from this cohort, suggesting a link between Tr/E, HIV-1 resistance and modulated innate viral recognition in the female genital mucosa. Collectively, our data indicate that unmodified N-terminus is critical for intranuclear localization and anti-HIV-1 activity of E. We also propose that E-mediated altered cellular innate activation most likely contributes to the HIV-R phenotype of these subjects.

Glycosylation regulates pannexin intermixing and cellular localization.

The pannexin family of mammalian proteins, composed of Panx1, Panx2, and Panx3, has been postulated to be a new class of single-membrane channels with functional similarities to connexin gap junction proteins. In this study, immunolabeling and coimmunoprecipitation assays revealed that Panx1 can interact with Panx2 and to a lesser extent, with Panx3 in a glycosylation-dependent manner. Panx2 strongly interacts with the core and high-mannose species of Panx1 but not with Panx3. Biotinylation and dye uptake assays indicated that all three pannexins, as well as the N-glycosylation-defective mutants of Panx1 and Panx3, can traffic to the cell surface and form functional single-membrane channels. Interestingly, Panx2, which is also a glycoprotein and seems to only be glycosylated to a high-mannose form, is more abundant in intracellular compartments, except when coexpressed with Panx1, when its cell surface distribution increases by twofold. Functional assays indicated that the combination of Panx1 and Panx2 results in compromised channel function, whereas coexpressing Panx1 and Panx3 does not affect the incidence of dye uptake in 293T cells. Collectively, these results reveal that the functional state and cellular distribution of mouse pannexins are regulated by their glycosylation status and interactions among pannexin family members.

Zebrafish early cardiac connexin, Cx36.7/Ecx, regulates myofibril orientation and heart morphogenesis by establishing Nkx2.5 expression.

Heart development is a precisely coordinated process of cellular proliferation, migration, differentiation, and integrated morphogenetic interactions, and therefore it is highly susceptible to developmental anomalies such as the congenital heart disease (CHD). One of the major causes of CHD has been shown to be the mutations in key cardiac transcription factors, including nkx2.5. Here, we report the analysis of zebrafish mutant ftk that showed a progressive heart malformation in the later stages of heart morphogenesis. Our analyses revealed that the cardiac muscle maturation and heart morphogenesis in ftk mutants were impaired because of the disorganization of myofibrils. Notably, we found that the expression of nkx2.5 was down-regulated in the ftk heart despite the normal expression of gata4 and tbx5, suggesting a common mechanism for the occurrence of ftk phenotype and CHD. We identified ftk to be a loss-of-function mutation in a connexin gene, cx36.7/early cardiac connexin (ecx), expressed during early heart development. We further showed by a rescue experiment that Nkx2.5 is the downstream mediator of Ecx-mediated signaling. From these results, we propose that the cardiac connexin Ecx and its downstream signaling are crucial for establishing nkx2.5 expression, which in turn promotes unidirectional, parallel alignment of myofibrils and the subsequent proper heart morphogenesis.

Headless splice variant acting as dominant negative calcitonin receptor.

Calcitonin receptor (CTR) is a member of class B G protein-coupled receptor (GPCR) superfamily. We have recently identified the CTR gene and its two transcriptional isoforms, mfCTR and mfCTRDeltaN, in mefugu (mf) (Takifugu obscurus). Here we characterized the mfCTRDeltaN that lacks hormone-binding extracellular N-terminal domain. Strong expression in the liver and weak but broad tissue distribution of its mRNA, revealed by Northern analysis, suggested physiological significance of this headless splice variant. Biochemical and immunocytochemical analyses revealed that it acts as a naturally occurring dominant negative isoform by forming a heterodimer with normal CTR. The headless mfCTRDeltaN characterized here is the first case of N-terminally truncated dominant negative form of GPCR, and immunocytochemistry used for detecting the heterodimer formation may be useful as a novel method for analyzing membrane protein interaction in a living cell.

Fish calcitonin receptor has novel features.

Calcitonin (CT), a 32-amino acid peptide, was initially isolated from fish. Fish CT has higher affinity to mammalian CT receptor (CTR), and has activity on calcium homeostasis. Therefore, fish CT has been used as a drug for the treatment of human bone diseases. However, the physiological roles of CT in fish as well as the characteristics of the fish CTR have not been clarified. Here, we cloned and characterized CTR from mefugu (Takifugu obscurus). Full-length cDNA sequencing revealed that mfCTR (mf, mefugu) consists of N-terminal four tandem putative hormone-binding domains (HBDs). Database mining showed that the multiple HBD-containing CTR is a common feature for some other fishes. Detailed pharmacological studies revealed that mfCTR generated cAMP in response to (1) fish CT, (2) calcitonin gene-related peptide (CGRP) in combinations with receptor activity-modifying proteins (mfRAMPs) 1 and 4, and (3) amylin in combinations with mfRAMPs 1-5. Unlike mammalian CTR, mfCTR showed dual affinity sites. Corresponding EC(50) values of those are in close proximity of the in vivo concentration of CT in fish. Analyses of the deletion mutants of mfCTR demonstrated that only the nearmost HBD to the first transmembrane region is functional to the ligands. Although, fish CT has higher affinity to the human CTR, human CT did not bind to the mfCTR. This is the first report that demonstrates the structure and property of fish receptor for CT, CGRP, and amylin. Fish CTR is the first example that has multiple HBD-like sequences.

Pillar cell and erythrocyte localization of fugu ET(A) receptor and its implication.

Endothelin, a vasoconstrictor peptide, plays important roles not only in the mammalian circulatory system but also in non-mammalian systems, such as the gill lamellar vascular network with complex structural characteristics. Here, we show that (i) the contraction of pillar cells that delimit the lamellar vasculature is controlled by endothelin through the type A endothelin receptor (ET(A)) linked to the intracellular calcium signaling system and (ii) ET(A) receptor is also highly expressed on fugu erythrocytes, a hitherto unexpected finding. Database mining revealed the presence of five endothelin receptor (ETR) sequences in the fugu genome. By Northern blotting, cDNA cloning, and fura-2 monitoring, the branchial ETR subtype was shown to be ET(A) able to induce a Ca(2+) transit. Immunohistochemistry revealed its pillar cell and erythrocyte localization. These results suggest an endothelin/ET(A)-mediated coordinated regulation of the pillar cell shape and erythrocyte membrane flexibility.

Molecular and functional characterization of adrenomedullin receptors in pufferfish.

The receptors for the calcitonin gene-related peptide (CGRP)/adrenomedullin (AM) family peptides were characterized in the mefugu Takifugu obscurus, a euryhaline fugu species very close to Takifugu rubripes, which has as many as five adrenomedullin genes (AM1-5). CGRP and AM share a G protein-coupled core receptor called calcitonin receptor-like receptor (CLR), and the specificity of the CLR is determined by the interaction with receptor activity-modifying proteins (RAMPs). Through database mining, three CLRs (CLR1-3) and five RAMPs (RAMP1-5) were identified, and all of them were cloned by RT-PCR and characterized by functional expression in COS7 cells in every possible combination of CLR-RAMP. The following combinations generated cAMP in response to physiological concentrations of CGRP, AM1 (an ortholog of mammalian AM), AM2, and AM5: CLR1-RAMP1/4 (CGRP), CLR1-RAMP2/3/5 (AM1), CLR2-RAMP2 (AM1), CLR1-RAMP3 (AM2), and CLR1-RAMP3 (AM5). Their expressions were found by Northern blot analysis to be tissue specific and salinity dependent. For example, CLR1-RAMP5 and CLR1-RAMP2 are expressed specifically in the gill and kidney, respectively, suggesting their involvement in osmoregulation. Furthermore, relatively high levels of CLRs and RAMPs were found in the spleen and ovary, suggesting roles in the immune and female reproductive systems. Immunohistochemistry revealed that AM receptors of the following types are expressed in the locations, indicated in brackets, of the mefugu gill and kidney: CLR1-RAMP5 (interlamellar vessels), CLR2-RAMP2 (pillar cells), and CLR1-RAMP2 (apical side of renal proximal tubule cells).

A novel type of urea transporter, UT-C, is highly expressed in proximal tubule of seawater eel kidney.

A new type of urea transporter was identified by a database search and shown to be highly expressed in the renal proximal tubule cells of teleosts; proximal tubule-type urea transporters have not been describe previously. We first identified urea transporter-like sequences in the fugu genome and in an EST database of rainbow trout. Based on these pieces of sequence information, we obtained a full-length cDNA for the eel ortholog, consisting of 378 amino acid residues, and named it eUT-C. Although its sequence similarity to the known urea transporters is low (approximately 35%), its heterologous expression in Xenopus laevis oocytes indicated that it is a facilitative urea transporter sensitive to phloretin. Its activity is not dependent on Na+. Northern blot analysis showed that expression of eUT-C is highly restricted to the kidney, with weak expression in the stomach. In both tissues, eUT-C mRNA was strongly induced when eels were transferred from freshwater to seawater. Immunohistochemistry and in situ hybridization histochemistry revealed proximal tubule cell localization of eUT-C. Taking into account that 1) urea is mainly secreted from the gill where another type of urea transporter (eUT) has been identified and 2) fish excrete a very small volume of urine in seawater, we propose that eUT-C cloned here is a key component working in combination with the gill transporter to achieve an efficient urea excretory system in fish, namely, eUT-C reabsorbs urea from glomerular filtrate and sends it to the gill, through the circulation, for excretion.