Zhenhui Liu

Vitellogenin is a cidal factor capable of killing bacteria via interaction with lipopolysaccharide and lipoteichoic acid

Vitellogenin (Vg) has been shown to be involved in host immune defense. However, the underlying mechanism by which Vg functions is largely unknown, and which component in Vg is essential for the execution of its immune role remains lacking. Here, we demonstrate clearly that fish Vg is capable of killing the whole cells of Gram-negative bacterium Escherichia coli and Gram-positive bacterium Staphylococcus aureus rather than their protoplasts; and that Vg has distinct binding sites specific for lipopolysaccharide (LPS), lipoteichoic acid (LTA) and peptidoglycan (PGN), respectively. Of note, the interaction between Vg and bacterial cells via the different binding sites results in distinct effects: the binding of Vg to E. coli via LPS and to S. aureus via LTA is lethal, whereas the binding of Vg to S. aureus via PGN is not. Moreover, Vg exhibits a lectin-like activity because its antibacterial activities can be suppressed by the carbohydrates like d-mannose, N-acetyl-d-glucosamine and d-fucose. Finally, the polypeptide chain integrity and carbohydrate residues of Vg are indispensable for its antibacterial activity, but the lipidation and phosphorylation are not necessary. Taken together, Vg is a bacteriocidal factor capable of killing E. coli and S. aureus whole cells via interaction with LPS and LTA existing in the bacterial cell walls rather than attacking their plasma membranes.

Molecular and immunochemical demonstration of a novel member of Bf/C2 homolog in amphioxus Branchiostoma belcheri: Implications for involvement of hepatic cecum in acute phase response

A complement system operating via the alternative pathway (AP) similar to that of vertebrates has been demonstrated in the primitive chordate amphioxus. However, the factor B (Bf), a key specific protease in the AP, remains elusive in amphioxus to date. We demonstrate in this study the presence of a factor B-like protein in amphioxus Branchiostoma belcheri by both immunoblotting and molecular cloning. The factor B-like protein was immunohistochemically localized in the hepatic cecum. The B. belcheri factor B-like gene, BbBf/C2, encoded a mosaic protein with three complement control protein (CCP) domains, a von Willebrand factor A (vWFA) domain and a serine protease (SP) domain. Peculiarly, BbBf/C2 had an epidermal growth factor-like domain (EGF_CA) located between CCP1 and CCP2, therefore BbBf/C2 had a modular structure of CCP-EGF_CA-CCP-CCP-vWFA-SP, making it a novel member of Bf/C2 family proteins. Real-time PCR assay revealed that lipopolysaccharide (LPS) challenge resulted in a quick and continuously significant up-regulation of BbBf/C2 expression in the hepatic cecum, while BbBf/C2 was only expressed for a short time in the hind-gut following LPS challenge though the expression level was temporarily higher than that in the hepatic cecum. Similarly, immuno-dot blotting showed that challenge with LPS triggered a significant elevation of BbBf/C2 synthesis in the hepatic cecum and hind-gut, with a higher rise in the former tissue. These results indicate that both hepatic cecum and hind-gut may be involved in the immune response induced by LPS, but the hepatic cecum, like the vertebrate liver, is the primary tissue synthesizing BbBf/C2 in response to LPS challenge, thereby playing a major role in the acute phase response.

Characterization, expression, and response to stress of p8 gene in amphioxus.

The amphioxus Branchiostoma belcheri tsingtaunese homolog of p8, Bbp8, was identified from the gut cDNA library. The full-length Bbp8 cDNA consists of 1032 bp, which is clearly longer than those of p8 in human, mouse, rat, frog, zebrafish and fruit fly. The genomic DNA sequences of amphioxus p8 contain three exons and two introns, which is similar to the exon/intron organization of p8 homologues in vertebrates such as human, mouse and zebrafish, while it is sharply different to the organization of p8 gene in fruit fly, which has only one exon. Sequence alignment and phylogenetic analysis showed that the basic helix-loop-helix (bHLH) region of p8 is well conserved during the long process of evolution, and Bbp8 is more close to its homologous proteins in the invertebrates than to those in the vertebrates. RT-PCR and In situ hybridization histochemistry demonstrated the expression of Bbp8 in all the tissues assayed, with relatively higher expression in the gut, gill and ovaries. Quantitative real-time PCR assay revealed quick up-regulation of Bbp8 transcripts on lipopolysaccharide (LPS) challenge and starvation, implying a stress-related function for Bbp8.

EST analysis of the immune-relevant genes in Chinese amphioxus challenged with lipopolysaccharide

It is generally accepted that the adaptive immune system is only present in vertebrates but not in invertebrates. Amphioxus is the most basal chordate and hence is an important reference to the evolution of the adaptive immune system. Here, a cDNA library of lipopolysaccharide-challenged amphioxus was constructed in order to identify immune genes. A total of 3024 expressed sequence tags (ESTs) were examined and 63 out of 398 annotated genes (16.3%) appeared related to immunity. Most of them encode cell adhesion molecules or signal proteins that are involved in immune responses. Although the key molecules such as TCR, MHC, Ig or VLR involved in the adaptive immune system were not identified in our database, we demonstrated the presence of histocompatibility-relevant genes and lymphocyte immune signaling-relevant genes. These findings support the statement that amphioxus presents some components that may be recruited by adaptive immune processes.

Evolution and expression of gamma-aminobutyric acid type A receptor-associated protein from the amphioxus Branchiostoma belcheri

The cDNA encoding a gamma-aminobutyric acid type A (GABAA) receptor-associated protein (GABARAP) was identified from the gut cDNA library of amphioxus Branchiostoma belcheri. It consisted of 1246 bp with a 354 bp open reading frame coding for a 117 amino acids protein of 13.9 kDa. The phylogenetic tree analysis showed that amphioxus GABARAP clustered with GABARAPs, separating from GABARAP-like proteins including amphioxus GABARAPL2. Amphioxus GABARAP gene had an exon–intron organization similar to human, mouse, zebrafish and sea squirt GABARAP homologs in terms of both exon number and sequence homology of each exon, hinting at the clue that GABARAP gene transcription is regulated similarly in all the chordates. In situ hybridization histochemistry revealed a ubiquitous expression pattern of amphioxus GABARAP gene, although it was temporally expressed specifically in the primitive gut of 2- to 10-day larvae, suggesting a conserved role of GABARAP in amphioxus as well as in mammalian species.

Demonstration of a Functional Kisspeptin/Kisspeptin Receptor System in Amphioxus With Implications for Origin of Neuroendocrine Regulation

Amphioxus belongs to the Cephalochordata, which is the most basal subphylum of the chordates. Despite many studies on the endocrine system of amphioxus, key information about its regulation remains ambiguous. Here we clearly demonstrate the presence of a functional kisspeptin/kisspeptin receptor (Kiss-Kissr) system, which is involved in the regulation of reproduction in amphioxus. Evolutionary analyses revealed large expansion of Kiss and Kissr (gpr54) genes in amphioxus, and they might represent the ancestral type of the Kiss/gpr54 genes in chordates. Amphioxus Kiss was obviously expression at the cerebral vesicle and the Hatschek pit, whereas amphioxus gpr54 messenger RNA (mRNA) was abundantly present in nerve cord, ovary, and testes. Amphioxus GPR54-Like1 (GPR54L-1) was shown to be located on the cell membrane. The synthetic amphioxus Kiss-like (KissL) peptides were capable of activating the amphioxus GPR54L-1 with different potencies, hinting the interaction between Kiss and GPR54. Moreover, the expression of amphioxus gpr54 mRNA was significantly decreased during low or high temperature extremes. Importantly, the injection of amphioxus KissL could cause an elevation of zebrafish blood luteinizing hormone level and induce the expression of amphioxus gpb5, a gene encoding the ancestral type of vertebrate pituitary glycoprotein hormones. Also, the expression levels of BjkissL-2 or Bjgpr54L-1 were downregulated after spermiation or spawning. Collectively, the amphioxus Kiss-Kissr system has a correlation with the regulation of reproduction. Our studies provide insights into the functional roles and evolutionary history of the Kiss-Kissr system, as well as the origin of the vertebrate neuroendocrine axis for controlling reproduction.

Characterization and expression of amphioxus ApoD gene encoding an archetype of vertebrate ApoD proteins.

Here we report a homologue of the apolipoprotein D gene (AmphiApoD) in amphioxus, Branchiostoma belcheri tsingtauense, the first such finding in a basal chordate cephalochordate. The main features of the protein predicted from AmphiApoD are characteristic of the apolipoprotein D. Phylogenetic analysis places AmphiApoD at the base of the phylogenetic tree, suggesting that AmphiApoD is the archetype of the vertebrate ApoD genes. Both whole mount in situ hybridization and Northern blotting and RT—PCR as well as in situ hybridization histochemistry reveal that AmphiApoD is expressed in tissues derived from mesoderm and endoderm including notochord and hind‐gut, which contrasts with the strong expression patterns of ApoD genes in the ectodermal derivatives in mammals and birds. The expression profiles of the ApoD gene may have been changed to be expressed in the endo‐mesodermal derivatives in amphioxus after the vertebrate and cephalochordate lineages diverged; alternatively, the ApoD gene may first have been expressed in the endo‐mesoderm during embryogenesis in the last common ancestor of all chordates, and subsequently came to be expressed in the ectodermal derivatives of vertebrates including mammals and birds.

Characterization and tissue-specific expression of phosphatidylcholine transfer protein gene from amphioxus Branchiostoma belcheri

An amphioxus cDNA, encoding phosphatidylcholine transfer protein (AmphiPCTP), was identified for the first time from the gut cDNA library of Branchiostoma belcheri. It contains a 660-bp open reading frame corresponding to a deduced protein of 219 amino acids. Phylogenetic tree analysis showed that AmphiPCTP clustered with PCTP subgroup of PCTP subfamily containing steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domains. AmphiPCTP had an exon-intron organization similar to that of human and rat PCTP genes in terms of both exon number and sequence homology of each exon, suggesting that PCTP has probably maintained a similar function in both amphioxus and mammalian species. Both in situ hybridization histochemistry and whole-mount in situ hybridization revealed a tissue-specific expression pattern of AmphiPCTP with the high levels in the hepatic caecum and primitive gut, including the region where the hepatic caecum will form later during development. This apparently agrees with the hypothesis that amphioxus hepatic caecum is equivalent to vertebrate liver. These results suggest a conserved role of PCTPs in amphioxus as well as mammalian species.

Characterization and expression profile of AmphiCD63 encoding a novel member of TM4SF proteins from amphioxus Branchiostoma belcheri tsingtauense

The study on CD antigen genes remains lacking in the cephalochordate amphioxus to date. In this report, the cDNA encoding CD63 was identified for the first time from the gut cDNA library of amphioxus Branchiostoma belcheri tsingtauense. Primary structural examination showed that the protein encoded by the cDNA contained four potential transmembrane domains characteristic of transmembrane 4 superfamily (TM4SF) proteins and a conserved CCG motif in the putative major extracellular loop. BLAST search revealed that the cDNA is closely associated with other known CD63 antigen genes, and it was thus designated AmphiCD63. Phylogenetic analysis indicated that AmphiCD63 is extremely close to vertebrate CD63, CD151 and CD53, suggesting they may have been evolved from a common ancestral gene. RT-PCR analysis exhibited that AmphiCD63 mRNA was abundant in muscle, ovary, foregut including hepatic caecum and hindgut, while it was present at considerably lower levels in notochord and gill and absent in testis.

Identification of the ribosomal proteins s15a and L19 from the amphioxus branchiostoma belcheri tsingtauense

Abstract The complete cDNA and deduced amino acid sequences of the ribosomal protein S15a (AmphiS15a) and L19 (AmphiL19) of the amphioxus Branchiostoma belcheri tsingtauense were identified in the present study. The AmphiS15a cDNA is 540 nucleotides in length and encodes a 130 amino acid protein with a calculated molecular mass of 149 kDa. It shares 70-85% sequence identity to its known counterparts in the eukaryotes including human, rat, sea urchin, fruit fly and yeast. The AmphiL19 cDNA comprises 728 nucleotides and encodes a 197 amino acid protein with a calculated molecular mass of 23.4 kDa. It shows 59-79% sequence identity to the eukaryotic L19 proteins from human, rat, mouse, fruit fly, Caenorhabditis elegans and yeast Both AmphiS15a and AmphiL19 are highly basic and contain putative nucleolar localization signals. As house-keeping genes, they may both be useful to normalize gene expression in characterization of various genes from the amphioxus.