Lamprey Protein Research Articles

Lamprey immune protein-1 (LIP-1) from Lampetra japonica induces cell cycle arrest and cell death in HeLa cells

The lamprey (Lampetra japonica), a representative of the jawless vertebrates, is the oldest extant species in the world. LIP-1, which has a jacalin-like domain and an aerolysin pore-forming domain, has previously been identified in Lampetra japonica. However, the structure and function of the LIP-1 protein have not been described. In this study, the LIP-1 gene was overexpressed in HeLa cells and H293T cells. The results showed that the overexpression of LIP-1 in HeLa cells significantly elevated LDH release (P < 0.05), phosphatidylserine exposure and ROS accumulation. The overexpression of LIP-1 also had remarkable effects on the organelles in HeLa cells, while it had no effect on H293T cell organelles. Array data indicated that overexpression of LIP-1 primarily upregulated P53 signaling pathways in HeLa cells. Cell cycle assay results confirmed that LIP-1 caused arrest in the G2/M phase of the cell cycle in HeLa cells. In summary, our findings provide insights into the function and characterization of LIP-1 genes in vertebrates and establish the foundation for further research into the biological function of LIP-1. Our observations suggest that this lamprey protein has the potential for use in new applications in the medical field.

Identification and characterisation of lamprey protein kinase C delta-like gene

Protein kinase C-δ (PKC-δ), a member of the lipid-regulated serine/threonine PKC family, has been implicated in a wide range of important cellular processes, such as cell growth, differentiation, and apoptosis. Lampreys belong to the most primitive class of vertebrates, and there is little information on PKC-δ in these animals. In this study, a PKC-δ-like cDNA sequence and deduced PKC-δ-like amino acid sequence were identified in the Japanese lamprey (Lampetra japonica). The PKC-δ-like gene shared approximately 60% sequence identity with its homologs in jawed vertebrates. The anti-PKC-δ-like polyclonal antibodies were well prepared, and experiments showed that PKC-δ-like was primarily distributed in the supraneural body of the lamprey. Both mRNA and protein levels of PKC-δ-like in supraneural body cells were increased after incubation with cis-diaminedichloroplatinum (CDDP). Moreover, PKC-δ-like protein induced the apoptosis of HEK-293T cells. In addition, the activation of PKC-δ-like resulted in apoptosis. Conversely, the inhibition of PKC-δ-like activity disrupted the CDDP-mediated induction of cellular apoptosis. These results indicate that PKC-δ-like identified in lampreys might play an important role in apoptosis in jawless vertebrates.

Novel neutrophil inhibitory factor homologue in the buccal gland secretion of Lampetra japonica

Abstract To identify the functional gene fragment, a neutrophil inhibitory factor (NIF) like protein was found in the buccal gland of Lampetra japonica, suggesting that this related lamprey protein represents a novel class of integrin receptor antagonists. The recombinant Lampetra japonica-NIF like (rLj-NIF) was identified by SDS-PAGE and purified by using His·Bind affinity chromatography. Effect of rLj-NIF on neutrophil migration suggested that rLj-NIF can act as a neutrophil inhibitory factor. Besides that, oxidative burst activity of neutriphil was tested by flow cytometry using dihydrorhodamine (DHR123) as a fluorogenic substrate, and the data suggested that the mean fluorescence intensity significantly decreased compared with positive controls (p<0.01). All above results indicated that rLj-NIF could also prevent the binding of β2 integrins to the surface of PMN and its FITC-labeled monoclonal antibodies (p<0.05). These data suggest that Lampetra japonica-NIF like protein is secreted by the stage of the parasite at the site of attachment. rLj-NIF plays an essential role in physiological reaction of neutrophil by a novel class of β2 integrin receptor antagonists. The activity of immunosuppressant of L. japonica-NIF could have potential medicinal value in anti-inflammation and therapy of autoimmune diseases.

A short consensus repeat-containing complement regulatory protein of lamprey that participates in cleavage of lamprey complement 3.

The prototype of the short consensus repeat (SCR)-containing C regulatory protein is of interest in view of its evolutionary significance with regard to the origin of the C regulatory system. Lamprey is an agnathan fish that belongs to the lowest class of vertebrates. Because it does not possess lymphocytes, it lacks Ig and consequently the classical C pathway. We identified an SCR-containing C regulatory protein from the lamprey. The primary structure predicted from the cDNA sequence showed that this is a secretary protein consisting of eight SCRs. This framework is similar to the alpha-chain of C4b-binding protein (C4bp). SCR2 and -3 of human C4bp are essential for C4b inactivation, and this region is fairly well conserved in the lamprey protein. However, the other SCRs of this protein are similar to those of other human C regulatory proteins. The lamprey protein binds to the previously reported lamprey C3b/C3bi deposited on yeast and cleaves lamprey C3b-like C3 together with a putative serum protease. The scheme resembles the C regulatory system of mammals, where factor I and its cofactor inactivate C3b. Unlike human cofactors, the lamprey protein requires divalent cations for C3b-like C3 cleavage. Its artificial membrane-anchored form protects host cells from lamprey C attack via the lectin pathway. Thus, the target of this protein appears to be C3b and/or its family. We named this protein Lacrep, the lamprey C regulatory protein. Lacrep is a member of SCR-containing C regulators, the first of its kind identified in the lowest vertebrates.

Identification of an IL-8 homolog in lamprey ( Lampetra fluviatilis ): early evolutionary divergence of chemokines

Subtractive hybridization was used to study river lamprey ( Lampetra fluviatilis ) leukocyte-specific cDNA. A clone representing the most abundant component (12 %) of the leukocyte library subtracted with liver cDNA was isolated and characterized. The cDNA encodes a presumably secreted polypeptide of 101 residues. The 3 ′ untranslated region of the cDNA contains motifs characteristic of the transiently expressing genes. Comparison of the deduced amino acid sequence with known protein sequences revealed its homology to the members of the chemokine superfamily. Designated as LFCA-1, the lamprey protein contains four conserved cysteines, of which the first two are separated by a residue, and a number of other CXC family characteristic residues. LFCA-1 has the highest similarity to the chicken EMF-1 (40 %) and to the mammalian IL-8 (32 – 33 %). However, it lacks the ELR motif essential for the function of the mammalian IL-8-related chemokines. Based on the phylogenetic analysis of the LFCA-1 relationship to the higher vertebrate chemokines, it is concluded that the evolutionary origin of the chemokine superfamily is ancient, and that the divergence of the CXC and CC families most likely occurred at the time or before the first vertebrates emerged.

Identification of an IL-8 homolog in lamprey (Lampetra fluviatilis): early evolutionary divergence of chemokines.

Subtractive hybridization was used to study river lamprey (Lampetra fluviatilis) leukocyte-specific cDNA. A clone representing the most abundant component (12%) of the leukocyte library subtracted with liver cDNA was isolated and characterized. The cDNA encodes a presumably secreted polypeptide of 101 residues. The 3' untranslated region of the cDNA contains motifs characteristic of the transiently expressing genes. Comparison of the deduced amino acid sequence with known protein sequences revealed its homology to the members of the chemokine superfamily. Designated as LFCA-1, the lamprey protein contains four conserved cysteines, of which the first two are separated by a residue, and a number of other CXC family characteristic residues. LFCA-1 has the highest similarity to the chicken EMF-1 (40%) and to the mammalian IL-8 (32-33%). However, it lacks the ELR motif essential for the function of the mammalian IL-8-related chemokines. Based on the phylogenetic analysis of the LFCA-1 relationship to the higher vertebrate chemokines, it is concluded that the evolutionary origin of the chemokine superfamily is ancient, and that the divergence of the CXC and CC families most likely occurred at the time or before the first vertebrates emerged.

The single lamprey neurofilament subunit (NF-180) lacks multiphosphorylation repeats and is expressed selectively in projection neurons.

The lamprey is considered the most primitive living vertebrate and its neurofilaments (NFs) are unique in being homopolymers of a single 180 kDa subunit (NF-180). Previous immunologic studies have suggested that the sidearm of NF-180 is highly phosphorylated selectively in the largest diameter axons. We report in this study the isolation and characterization of cDNA clones encoding the NF-180 lamprey protein. In situ hybridization with digoxigenin-labeled cRNA revealed NF-180 message exclusively in neurons with long axons, such as reticulospinal neurons and cranial motor neurons. The core of NF-180 was similar in structure to those of mammalian neurofilaments, but surprisingly, the carboxy sidearm lacked the multiphosphorylation repeats characteristic of higher vertebrate and invertebrate neurofilaments. Overall there was a paucity of potential phosphorylation sites in the NF-180 carboxy-terminus compared to NF-M and NF-H of mammals, fish and squid. This, along with the highly acidic nature of the NF-180 sidearm, makes it unlikely that phosphorylation of sidearm residues regulates interfilament spacing and axon diameter through global electrostatic repulsion of the carboxy-terminus away from the filament backbone. Furthermore, the expression of a single neurofilament subunit in the lamprey that is most similar to the NF-M of higher vertebrates suggests that all three mammalian neurofilament subunits evolved from a single NF-M-like precursor.

Isolation and properties of a fatty acid-binding protein from the pacific lamprey ( Lampetra tridentata)

1. 1. A survey of 12 vertebrate species showed that palmitate was bound by an albumin-like serum protein in all classes tested except the dogfish and the lamprey. 2. 2. The major palmitate-binding protein of the Pacific lamprey was isolated and found to be of molecular mass 19,000. 3. 3. The amino acid composition of this protein indicates that it is not a member of the albumin superfamily. 4. 4. The 19-kDa lamprey protein binds bilirubin, cortisol and tryptophan only weakly, but binds palmitate with K A = 25 μ M −1 , comparable to the first long-chain fatty acid site of bovine albumin (K A = 34 μ M −1 ) .

Purification of a lamprey complement protein homologous to the third component of the mammalian complement system.

A lamprey protein homologous to the third component of mammalian complement was isolated from lamprey plasma and was tentatively designated lamprey C3. Lamprey C3 is a major protein in lamprey serum with electrophoretic mobility of beta-globulin and with m.w. of 190,000. It consists of three polypeptide chains (84,000-alpha, 74,000-beta, and 32,000-gamma chains) linked by disulfide bonds. The protein retains a unique internal thiolester bond on the alpha-chain that is cleaved on methylamine treatment or on limited proteolysis with trypsin. Lamprey C3 by itself could not bind to zymosan or rabbit red cells, but it could covalently bind to these substances when activated by other factors present in lamprey serum. The binding of lamprey C3 to activating surfaces is mediated by covalent bonds and is accompanied by limited proteolysis of the alpha-chain. A fragment with an m.w. of 35,000 containing the internal thiolester site was isolated from methylamine-treated lamprey C3 bound to activated thiol-Sepharose by extensive tryptic digestion followed by dithiothreitol treatment. Lamprey C3 functions as the essential factor in phagocytosis of rabbit red cells by lamprey phagocytes. However, it is not involved in naturally occurring hemolytic activity in lamprey serum.

A lamprey lens protein related to avian and reptilian delta crystallin.

Delta crystallin has hitherto been considered specific to reptiles and birds. Evidence presented in this paper suggests that a major lens protein in adult river lampreys (Lampetra fluviatilis) is related to chick delta crystallin. Both are of similar size (Mr 45-50,000), and the lamprey protein cross-reacts with antibodies against chick delta crystallin in immunodiffusion, immunoelectrophoretic and immunoblotting tests. However, the charge properties and V8 proteolytic fragment patterns of the lamprey protein are markedly different from those of avian and reptilian delta crystallins. Preliminary evidence on the occurrence of delta-related DNA sequences in the genomes of all vertebrates examined, is also discussed.

The evolutionary sequence and quantities of different antigenic determinants of calf lens alpha crystallin

Immunochemieal cross-reactions between calf lens alpha crystallin and homologous proteins from different vertebrate classes were used for the study of antigenic determinants which, in the evolution of this protein, were derived from common ancestral forms. Determination of the sequence in which the different antigenic determinants evolved was based on estimation of the relative number of antigenic determinants shared by calf alpha crystallin with alpha crystallin from other vertebrate classes. For this estimation, samples of antiserum to calf lens alpha crystallin were absorbed with homologous lens proteins from each of the different vertebrate classes. The unabsorbed antiserum reacted with homologous proteins from all vertebrate classes. Each of the absorbed antisera showed a taxonomically different extent of residual cross-reaction. The species used for absorption were then ordered according to an increasing taxonomic extent of residual cross-reactions of the absorbed anti-calf antiserum, indicating a sequence of species with decreasing numbers of antigenic determinants shared with the mammalian protein. This sequence represents the increasing phylogenetic distances between the species used for absorption and the species used to produce the antiserum. This immunochemical determination showed that the phylogenetic sequence in which antigenic determinants present in the calf alpha crystallin molecule were formed, progressed from the primitive aquatic vertebrates agnatha, elasmobranchii and actinopterygii to the primitive land vertebrates and then to amphibia, reptilia, aves and mammalia. For the estimation of the relative amount (%) of determinants derived from different ancestral forms and transferred in evolution to calf alpha crystallin, 125I bound to a calf alpha crystallin immunoadsorbent after saturation with total 125I anti-calf alpha crystallin Fab fragments was compared to 125I bound to the same amounts of calf alpha crystallin after saturation with 125I Fab fragments of phylogenetically restricted specificity. Of all antigenic determinants in present-day calf alpha crystallin. approximately 42% were found also in the homologous lamprey protein. These determinants originated with the primitive agnatha which started the vertebrate subphylum 450 million years ago. Only about 15% originated with the mammalia, some 200 million years later, rellecting the slow evolutionary change of this protein molecule.