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In jawed vertebrates, B cell receptors (BCR) are primary pathogen detectors that activate downstream signaling pathways to express adaptive immune effectors. In jawless vertebrates, the variable lymphocyte receptors (VLR) B positive lymphocytes can express and secrete specific VLRB molecules in an analogous manner to that of immunoglobulins by B cells in jawed vertebrates. Our study is the first to demonstrate the possibility of incubation of fertilized eggs and artificial breeding of Lampetra morii larvae throughout their life cycle under laboratory condition. We also found that VLRB, lamprey B-cell linker (L-BLNK), and lamprey nuclear factor-kappa B (L-NF-κB) play key roles in early larval development. Aeromonas hydrophila was found to be a lethal pathogen of L. morii larvae causing rapid infection at a concentration of 107 cfu/mL qRT-PCR results revealed that gene expression levels of VLRB, L-BLNK, and L-NF-κB were up-regulated significantly. Ten-day infection trials showed that VLRB, L-BLNK, and L-NF-κB are crucial for lamprey immune response. Furthermore, the expression levels of L-BLNK and L-NF-κB were down-regulated drastically both at mRNA and protein levels after bacterial infection than in the naive group of VLRB morphants. A similar expression pattern of VLRB and L-BLNK was found in L-NF-κB morphants post bacterial infection. The results were strikingly different in the other two morphants. The VLRB and L-NF-κB expression levels were found to be down-regulated at mRNA and protein levels by less than 30% and 45%, respectively, in L-BLNK morphants compared to those in the naive group. These results indicate that L-BLNK and L-NF-κB might participate in VLRB-mediated immune response. Additionally, in VLRB morphants, the mRNA expression levels of some genes, especially the ones expressed in VLRB+ lymphocytes but not in VLRA+ lymphocytes, were found to be affected. Therefore, these findings of B-like lymphocytes in lamprey offer key evidence with regard to the evolution of adaptive immunity. • We report that L. morii has been cultured in laboratory through the complete life cycle, and VLRB, L-BLNK and L-NF-κB have been proven to be critical for its early development. • We provide evidences that L-BLNK and L-NF-κB seem to be key signaling molecules in VLRB signaling pathway in immune responses of VLRB+ lymphocytes. • These findings of B-like lymphocytes in lampreys offers key evidences into the evolution of adaptive immunity. [ABSTRACT FROM AUTHOR]

Studies of lamprey early development have produced major insights into primitive developmental patterns and the evolution of vertebrates. In this study, the effectiveness of the morphological and histological characteristics of Lampetra japonica during embryological, yolk‐sac larval and ammocoete stages were examined. Based on the external and histological morphology, the embryological phase was divided into eight stages, including the zygote, cleavage, blastula, gastrula, neural plate and groove, head protrusion, prehatching and hatching stages. A set of key morphological and histological transformations occurred through the yolk‐sac larval stage, reflecting the larva‐ammocoete transition. In the ammocoete stage, all organ systems were differentiated. Except for the melanophores, the lamprey did not develop any further morphological complexity beyond the ammocoete stage. The total length and body weight of larva and ammocoete generally increased over time. We hope that this study might form the basis for future studies of the phylogenetic development of this interesting group of animals. [ABSTRACT FROM AUTHOR]

At present, there is a research gap concerning the specific functions and mechanisms of the Notch gene family and its signaling pathway in jawless vertebrates. In this study, we identified a Notch1 homologue (Lr. Notch1) in the Lethenteron reissneri database. Through bioinformatics analysis, we identified Lr. Notch1 as the likely common ancestor gene of the Notch gene family in higher vertebrates, indicating a high degree of conservation in the Notch gene family and its signaling pathways. To validate the biological function of Lr. Notch1, we conducted targeted silencing of Lr. Notch1 in L. reissneri and analyzed the resultant gene expression profile before and after silencing using transcriptome analysis. Our findings revealed that the silencing of Lr. Notch1 resulted in differential expression of pathways and genes associated with signal transduction, immune regulation, and metabolic regulation, mirroring the biological function of the Notch signaling pathway in higher vertebrates. This article systematically elucidated the origin and evolution of the Notch gene family while also validating the biological function of Lr. Notch1. These insights offer valuable clues for understanding the evolution of the Notch signaling pathway and establish a foundation for future research on the origin of the Notch signaling pathway, as well as its implications in human diseases and immunomodulation. [ABSTRACT FROM AUTHOR]

Human B cell adaptor for phosphoinositide 3-kinase (BCAP) is identified as an adaptor protein expressed in B cells and plays a critical immunomodulatory role in B cell receptor signaling and humoral immune response. In the current study, a homolog of BCAP (Lja-BCAP) was identified in Lampetra japonica. The open reading frame of Lja-BCAP contains 2181bp nucleotides and encodes a protein of 726 amino acids. After being stimulated by mixed bacteria, the mRNA and protein expression levels of Lja-BCAP and the activation levels of tyrosine kinases increased significantly in peripheral blood lymphocytes, gills and supraneural myeloid bodies, respectively. However, after the knockdown of Lja-BCAP by RNAi in vivo, the activation of tyrosine kinases was inhibited in the above tissues, which indicated that Lja-BCAP participated in the anti-bacterial immune response of lampreys. After lipopolysaccharide (LPS) stimulation, the expression of Lja-BCAP in peripheral blood lymphocytes, gills and supraneural myeloid bodies were significantly up-regulated 2.5, 2.2, and 11.1 times (p < 0.05) compared to the control group, respectively; while after phytohemagglutinin (PHA) stimulation, the up-regulation of Lja-BCAP was only detected in peripheral blood lymphocytes. The above results show that Lja-BCAP mainly participates in the LPS-mediated immune response of lampreys. [ABSTRACT FROM AUTHOR]

The voltage-dependent anion channel 2 (VDAC2) is the abundant protein in the outer mitochondrial membrane. Opening VDAC2 pores leads to the induction of mitochondrial energy and material transport, facilitating interaction with various mitochondrial proteins implicated in essential processes such as cell apoptosis and proliferation. To investigate the VDAC2 in lower vertebrates, we identified Lr-VDAC2, a homologue of VDAC2 found in lamprey (Lethenteron reissneri), sharing a sequence identity of greater than 50 % with its counterparts. Phylogenetic analysis revealed that the position of Lr-VDAC2 aligns with the lamprey phylogeny, indicating its evolutionary relationship within the species. The Lr-VDAC2 protein was primarily located in the mitochondria of lamprey cells. The expression of the Lr-VDAC2 protein was elevated in high energy-demanding tissues, such as the gills, muscles, and myocardial tissue in normal lampreys. Lr-VDAC2 suppressed H 2 O 2 (hydrogen peroxide)-induced 293 T cell apoptosis by reducing the expression levels of Caspase 3, Caspase 9, and Cyt C (cytochrome c). Further research into the mechanism indicated that the Lr-VDAC2 protein inhibited the pro-apoptotic activity of BAK (Bcl-2 antagonist/killer) protein by downregulating its expression at the protein translational level, thus exerting an anti-apoptotic function similar to the role of VDAC2 in humans. • The lamprey VDAC2 protein is highly conserved in evolution and distributed across various critical lamprey tissues. • Lamprey VDAC2 suppresses H 2 O 2 -induced 293 T cell apoptosis by downregulating BAK expression. • A VDAC2 homologue from the Chinese northeast lamprey was cloned and characterized for the first time. [ABSTRACT FROM AUTHOR]

Abstract: Nuclear factor of kappa B (NF-κB) is a stimuli-activated transcription factor, regulates the expression of a diverse array of genes. Inhibitor of kappa B-epsilon (IκB-ɛ) is an inhibitor of NF-κB, which retains NF-κB in an inactive state in the cytoplasm. Lampreys (Lampetra japonica) belong to the lowest class of vertebrates with little information about its IκBs. We have identified a cDNA sequence IκB-ɛ-like in the lamprey and the deduced amino acid sequence of IκB-ɛ-like. It contains a conserved DSGxxS motif and six consecutive ankyrin repeats, which are necessary for signal-induced degradation of the molecule. Phylogenetic analysis indicated it had high sequence homology with IκB-ɛs from other vertebrates. FACS analysis showed that IκB-ɛ-like located in cytoplasm of leukocytes. The degradation of IκB-ɛ-like could be observed in leukocytes of L. japonica stimulated with lipopolysaccharide. These results indicate that IκB-ɛ proteins are conserved across vertebrates and the NF-κB-like signaling pathway may exist in the oldest agnatha. [Copyright &y& Elsevier]

The reissner lamprey Lethenteron reissneri, belonging to the class Cyclostomata, serves as a bridge between invertebrates and jawed vertebrates, and is considered the sister group of jawed vertebrates. However, despite this evolutionary significance, the genetic mechanisms underlying the adaptive evolution of the lamprey lineage remain unclear. Here, we assembled a 1.06 Gb chromosome‐level draft genome of L. reissneri, with 72 chromosomes (ranging in length from 4.5 Mb to 25.9 Mb) and a scaffold N50 length of 13.23 Mb. Genome quality comparisons revealed that the reissner lamprey genome has higher completeness and contiguity than the previously published sea lamprey and Japanese lamprey genomes. Moreover, reissner lamprey, sea lamprey, and Japanese lamprey species share similar transposable element profiles and Hox gene cluster compositions, suggesting that a burst of transposable element activity and whole genome duplication occurred before their divergence. Additionally, the Lip gene copy numbers, which have been studied for their functions in the host defence system, were found to be expanded uniquely in lamprey lineages, suggesting key roles for these genes in lamprey evolution and adaptation. We also identified two neural‐related genes, Nrn1 and Unc13a, with copy number expansions in jawed vertebrates, which may be functionally relevant to the origin of lamprey brains. Hence, this study not only provides the first chromosome‐level reference genome for Cyclostomata, but also highlights features of the unique biology and adaptive evolution of the lamprey lineage. [ABSTRACT FROM AUTHOR]

The complement component 3 of the lamprey, a jawless vertebrate, functions as an opsonin during the phagocytosis of rabbit red cells. Furthermore, lamprey C3 may be activated and cleaved into C3b, which is attached to the surface of target cells in the cytolytic process. However, the mechanism mediating the biological function of C3 in the lamprey is unknown. To our knowledge, this study is the first to show that variable lymphocyte receptors (VLRs) expression were significantly affected by complement C3 knockdown morphants in Lampetra morii. We identified the C3 gene in the lamprey genome based on its orthologs, conserved synteny, functional domains, phylogenetic tree, and conserved motifs. Additionally, we determined the optimal infection concentration of Aeromonas hydrophila to perform immune stimulation experiments in the lamprey larvae. The quantitative real-time polymerase chain reaction and immunofluorescence analyses revealed that the expression of Lampetra morii C3 (lmC3) was significantly upregulated in the larvae infected with 107 CFU/mL of A. hydrophila. The lmC3 morphants (lmC3 MO) of lamprey larvae were generated by morpholino-mediated knockdown. The lmC3 MO larvae were highly susceptible to A. hydrophila infection, which indicated that lmC3 is critical in lamprey immune response. The expression of a selected panel of orthologous genes was comparatively analyzed in the infected wild type, infected lmC3 MO, infected control MO, uninfected wild type and uninfected lmC3 MO one-month-old ammocoete larvae. The knockdown of lmC3 strongly affected the expression of VLRA+/VLRB+/VLRC+-associated genes, which was also confirmed by immunohistochemical analysis. Thus, VLR expression were significantly affected by complement C3 knockdown morphants in Lampetra morii. • We conducted an analysis of multiple C3 gene family in vertebrates. • We report that A. hydrophila has been characterized as a rapidly lethal pathogen for L.morii. • VLRs expression were significantly affected by complement C3 knockdown morphants in Lampetra morii. [ABSTRACT FROM AUTHOR]

The clinical management of bladder cancer (BCa) is hindered by the lack of reliable biomarkers. We aimed to investigate the potential of lamprey immunity protein (LIP), a lectin that specifically binds to multi-antennary sialylated N -glycolylneuraminic acid (Neu5Gc) structures on UMOD glycoproteins in the urine of BCa patients. Primary BCa patients had higher levels of LIP-bound Neu5Gc in urine than healthy participants and patients receiving postoperative treatment did. In addition, lectin chip assay and mass spectrometry were used to analyze the glycan chain structure, which can recognize the UMOD glycoprotein decorated with multi-antennary sialylated Neu5Gc structures. Furthermore, compared with urine samples from healthy patients (N = 2821, T/C = 0.12 ± 0.09) or benign patients (N = 360, T/C = 0.11 ± 0.08), the range of the urine T/C ratio detected using LIP test paper was 1.97 ± 0.32 in patients with bladder cancer (N = 518) with significant difference (P < 0.0001). Our results indicate that LIP may be a tool for early BCa identification, diagnosis, and monitoring. Neu5Gc-modified UMOD glycoproteins in urine and Neu5Gc-modified N -glycochains and sialyltransferases may function as potential markers in clinical trials. • To establish a new ELISA method for LIP protein for diagnosis of bladder cancer • Identify LIP protein to identify sialic acid modified sugar chain in urine • Based on the principle of TRFIA, the LIP protein strip kit is established [ABSTRACT FROM AUTHOR]

Intelectins play an important role in innate immune response. In a previous study, lamprey inteletins purified by galactose-Sepharose were inactive and insoluble. Herein, we provided a simple and effective method to purify natural intelectins from the serum of lamprey (Lethenteron japonicum) using proteinG agarose. SDS-PAGE, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), and mass spectrometry (MS) were used to analyze the purified proteins. The purified proteins were identified to be lamprey serum lectin and intelectinB. The activity analysis results indicated that the proteins had certain extent agglutination activity. The effective method will be useful to study their immune functions and molecular mechanisms. [ABSTRACT FROM PUBLISHER]

Cathepsin D (EC 3.4.23.5) is a lysosomal aspartic proteinase of the pepsin superfamily which participates in various digestive processes within the cell. In the present study, the full length cDNA of a novel cathepsin D homologue was cloned from the buccal glands of lampreys ( Lampetra japonica ) for the first time, including a 124-bp 5′ terminal untranslated region (5′-UTR), a 1194-bp open reading frame encoding 397 amino acids, and a 472-bp 3′-UTR. Lamprey cathepsin D is composed of a signal peptide (Met 1-Ala 20), a propeptide domain (Leu 21-Ala 48) and a mature domain (Glu 76-Val 397), and has a conserved bilobal structure. Cathepsin D was widely distributed in the buccal glands, immune bodies, hearts, intestines, kidneys, livers, and gills of lampreys. After challenging with Escherichia coli or Staphylococcus aureus , the expression level of lamprey cathepsin D in the buccal gland was 8.5-fold or 6.5-fold higher than that in the PBS group. In addition, lamprey cathepsin D stimulated with Escherichia coli was also up-regulated in the hearts, kidneys, and intestines. As for the Staphylococcus aureus challenged group, the expression level of lamprey cathepsin D was found increased in the intestines. The above results revealed that lamprey cathepsin D may play key roles in immune response to exogenous pathogen and could serve as a potential antibacterial agent in the near future. In addition, lamprey cathepsin D was subcloned into pcDNA 3.1 vector and expressed in the human embryonic kidney 293 cells. The recombinant lamprey cathepsin D could degrade hemoglobin, fibrinogen, and serum albumin which are the major components in the blood, suggested that lamprey cathepsin D may also act as a digestive enzyme during the adaptation to a blood-feeding lifestyle. [ABSTRACT FROM AUTHOR]

Highlights: [•] The first to report a novel protein G-binding protein (NPGBP) contained in the lamprey liver. [•] Cloned the full-length cDNA of NPGBP gene and production of the recombinant protein and the polyclonal antiserum. [•] NPGBP and mammalian Ig molecules combination with protein G in competitive mechanism. [•] NPGBP showed agglutination activity against the pathogenic microorganism and promote endothelial cell mitosis. [Copyright &y& Elsevier]

Abstract: Lamprey as a “living fossil” of immunological origin and “rich treasure” of biological pharmaceutical development has caused attention of scholars. The cDNA library construction and EST sequencing of blood had been done previously in our lab, and bioinformatics analysis provided a gene fragment which is highly homologous with natterin family, named natterin-like. To elucidate the characterization and phylogeny of natterin-like genes in early evolution, we cloned the full-length cDNA of natterin-like gene from the blood of Lampetra japonica. The open reading frame of this sequence contained 942bp and encoded 313 amino acids, including a lectin-like domain and a pore-forming toxin-like domain. Using reverse transcription PCR, natterin-like mRNA was also detected in lamprey blood, kidney, heart, liver, medullary, gonad, but absent in lamprey intestine and gill. Our results suggested that in lampreys and most of other species, there might be only one natterin-like gene, which was fused by certain sequences during evolution and encoded proteins with more functions. It is similar between C terminal of natterin-like protein and Aerolysin in space structure and the lectin-like domain of natterin-like equivalent to glycoprotein binding motif of Aerolysin in function. We also propose that the defense mechanism against specific predators in historical evolution of lamprey. Our findings may provide insights into the function and characterization of natterin-like genes as well as other gene families in vertebrates and provide a foundation for identification and structural, functional, and evolutionary analyses of more natterin-like genes and other gene families. [Copyright &y& Elsevier]

Abstract: Lampreys, the surviving representative of jawless vertebrates, have been a focal point in the search for the evolutionary origin of adaptive immunity. They have independently evolved the variable lymphocyte receptor (VLR)-based adaptive immune system that protects themselves from infection by a variable of microorganisms. The standard immunization schedule for Japanese lamprey (Lampetra japonica) was established to prepare antisera by injection of Escherichia coli, Bacillus proteus, Staphylococcus aureus, Mycobacterium smegmatis, RRBCs, SRBCs, NB4 cells and Hela cells. In this study, we demonstrated the activities of lamprey antisera, which might be helpful to research the collaboration between VLR-based adaptive immune system and complement system in jawless vertebrates. [Copyright &y& Elsevier]

Abstract: Bruton''s tyrosine kinase (BTK) plays an important role in the development, differentiation and signal transduction of B-lymphocyte. Lymphocyte-like cells and unique variable lymphocyte receptors have been found in the Japanese lamprey Lethenteron japonicum, an agnatha known to be lacking T or B lymphocyte, and the oldest agantha in existence. In this study a BTK-like gene from L. japonicum was identified and characterized. It contained a 1992-bp open reading frame (ORF), a 157-bp 5′-untranslated region (UTR) and a 567-bp 3′-UTR. Phylogenetic analysis indicated it had high sequence homology with BTKs from other vertebrates. Real-time PCR analysis showed that the transcript of the L. japonicum BTK-like gene was distributed in various immune associated tissues, and the level of which was upregulated in leukocytes after the animal was stimulated with LPS. This observation was further confirmed by FACS and western blot analyses. These results indicated that the BTK-like gene identified in L. japonicum may play an important role in the immune response of lamprey. [Copyright &y& Elsevier]

• Conjugation with inulin enhanced storage and hypersaline stability of DhaA. • Conjugation with inulin enhanced the thermal and pH stability of DhaA. • Conjugation with inulin largely preserved the catalytic activity of DhaA. • Conjugation with inulin slightly altered the structure of DhaA. Haloalkane dehalogenase DhaA catalyzes the hydrolytic cleavage of carbon-halogen bonds and produces alcohol, a proton and a halide. However, DhaA suffers from the poor environmental stability, such as sensitivity to high temperature, low pH, hypersaline and organic solvent. In order to improve the environmental stability of DhaA, DhaA was covalently conjugated with inulin, a hydrophilic polysaccharide in the present study. Each DhaA was averagely conjugated with 7∼8 inulin molecules. The conjugated inulin could form a hydration layer around DhaA, which increased the conformational rigidity and decreased the entropy of the enzyme. Conjugation of inulin maintained 75.5 % of the enzymatic activity of DhaA and slightly altered the structure of DhaA. As compared with DhaA, the conjugate (inu-DhaA) showed slightly different kinetic parameters (K m of 2.9 μmol/L and K cat of 1.0 s−1). Inulin conjugation could delay the structural unfolding and/or slow the protonation process of DhaA under undesirable environment, including the long-term storage, low pH, hypersaline and organic solvent stability. As a result, the environmental stability of DhaA was markedly increased upon conjugation with inulin. Thus, inulin conjugation was an effective approach to enhance the environmental stability of DhaA. [ABSTRACT FROM AUTHOR]

In jawless vertebrates, the lamprey complement component C1q (LC1q) acts as a lectin and activates lamprey complement component C3 (LC3) in association with mannose-binding lectin (MBL)-associated serine protease (MASP) via the lectin pathway. Furthermore, LC1q may interact with variable lymphocyte receptor B (VLRB) in a complex with antigens and mediate the activation of LC3, leading to cytolysis. In the present study, we found, for the first time, that LC1q plays a critical role in VLRA/VLRC-mediated immune response. Escherichia coli , Shigella flexneri , Aeromonas hydrophila , Pseudomonas plecoglossicida , Aeromonas allosaccharophila , P. luteola , Brevundimonas diminuta , and Bacillus cereus were isolated from infected Lampetra morii in our laboratory and identified using the 16s rRNA method. A. hydrophila was confirmed as a rapidly spreading lethal pathogen in the larvae of L. morii and was used in subsequent immune stimulation experiments. The results of real-time quantitative polymerase chain reaction (Q-PCR) and immunofluorescence analyses indicated that the RNA and protein expression levels of LC1q were upregulated following exposure to 107 cfu/mL of A. hydrophila, compared to the levels of the naïve group. We obtained LC1q morphants (LC1q MO) of lamprey larvae by morpholino-mediated knockdowns. We found that LC1q played key roles in the embryonic development of lamprey. The median lethal time (LT50) of LC1q MO larvae was 2 d after being exposed to the pathogens, whereas the LT50 of control MO was 5 d. The drastic decrease in LT50 values after LC1q knockdown implies that LC1q plays a critical role in lamprey immune response. Gene expression profiles of LC1q-deficient A. hydrophila , control MO A. hydrophila, wild type A. hydrophila , and naive 1-month-old ammocoetes larvae were compared by examining the expression levels of a selected panel of orthologous genes. It is worth mentioning that LC1q MO affected the VLRA+/VLRC + population genes but did not affect the VLRB + populations. Immunohistochemical data indicated that LC1q deficiency also affected VLRA and VLRC but not VLRB. Thus, LC1q plays a critical role in VLRA/VLRC-mediated immune response in lamprey. • We report that LC1q have been proven to be critical for L.morii early development. • LC1q play important roles in resisting the A. hydrophila invasion in its proammocoete larvae stage. • LC1q plays a critical role in VLRA/VLRC mediated immune response in the lamprey. [ABSTRACT FROM AUTHOR]

Background: In previous research, we found that lamprey immune protein (LIP) possessed cytocidal activity against tumor cells, but the mechanism of the selective recognition and killing of tumor cells by LIP was not identified. Methods: Superresolution microscopy, crystallographic structural analysis, glycan chip assay, SPR experiments, FACS assays, computational studies and mass spectrometric analysis firmly establish the mode of action of LIP, which involves dual selective recognition and efficient binding. Results: We determined the overall crystallographic structure of LIP at a resolution of 2.25 Å. LIP exhibits an elongated structure with dimensions of 105 Å × 30 Å × 30 Å containing an N-terminal lectin module and a C-terminal aerolysin module. Moreover, the Phe209-Gly232 region is predicted to insert into the lipid bilayer to form a transmembrane β-barrel, in which the hydrophobic residues face the lipid bilayer, and the polar residues constitute the hydrophilic lumen of the pore. We found that LIP is able to kill various human cancer cells with minimal effects on normal cells. Notably, by coupling biochemical and computational studies, we propose a hypothetical mechanism that involves dual selective recognition and efficient binding dependent on both N-linked glycans on GPI-anchored proteins (GPI-APs) and sphingomyelin (SM) in lipid rafts. Furthermore, specific binding of the lectin module with biantennary bisialylated nonfucosylated N-glycan or sialyl Lewis X-containing glycan structures on GPI-APs triggers substantial conformational changes in the aerolysin module, which interacts with SM, ultimately resulting in the formation of a membrane-bound oligomer in lipid rafts. Conclusions: LIP holds great potential for the application of a marine protein towards targeted cancer therapy and early diagnosis in humans. [ABSTRACT FROM AUTHOR]