Unique properties of prothrombin in the bullhead shark Heterodontus japonicus: the first report of purification and characterization of a blood coagulation factor in Chondrichthyes.

Prothrombin is a serine protease precursor of the blood coagulation system. In this study, the primary structure of prothrombin of a cartilaginous fish, bullhead shark (Heterodontus japonicus), was determined using RNA-Seq and the protein was purified from the blood plasma. Bullhead shark prothrombin was found to be comprised of four domains, as in the case of reported mammalian homologues. Two arginine residues that should be cleaved by activated factor X were found in the amino acid sequence of the shark prothrombin, but only one of the two cleavage sites for thrombin or meizothrombin was conserved. The apparent molecular mass of the shark prothrombin on SDS-PAGE was 110 kDa, whereas that of its amino acid sequence was 65 kDa. Potential N-glycosylation sites were found at 79th, 108th, 121st, 179th, 199th, 507th, and 527th asparagine residues in the shark prothrombin, and treatment with N-glycosidase reduced the molecular mass to 65 kDa. This indicates that, in contrast to human prothrombin, which has only 7-kDa N-glycans, the prothrombin of the shark is highly N-glycosylated. This study is the first to report on the purification and characterization of blood coagulation factors in a cartilaginous fish.

Expression profile of kalliklectin, a soluble-type mannose receptor, during embryogenesis and early larval development in fugu (Takifugu rubripes).

Kalliklectin is a unique fish-specific lectin, whose sequence is similar to the heavy chain of mammalian plasma kallikrein and coagulation factor XI. In this study, we aimed to evaluate dynamic expression profiles of the lectin gene, during early developmental stages, in fugu, Takifugu rubripes. Reverse transcription-polymerase chain reaction (RT-PCR) showed that the kalliklectin gene was not expressed until 14 h post-fertilization (hpf), while the mRNA was detected after 30 hpf. In real-time quantitative PCR (qPCR), the gene was first expressed at 10.5 hpf; then, the expression level increased with a peak at 30 hpf and then gradually decreased. On the other hand, western blotting with specific antibody detected the lectin protein at all tested stages, including the unfertilized egg, which suggests that the lectin detected in the early stages was a maternal factor. Immunohistochemistry demonstrated that kalliklectin was localized at the basement membranes of the newly hatched larvae, while the lectin was widely detected in epidermal cells in larva at 5 dph. A 40-kDa lectin was partially purified from unfertilized eggs using mannose-affinity chromatography, and the lectin was determined as kalliklectin by liquid chromatography with quadrupole time-of-flight tandem mass spectrometry (LC/Q-TOF-MS) analysis, which indicated that the lectin is functional in the eggs. The egg lectin can bind to Gram-positive bacterial pathogens of fish, such as Lactococcus garvieae and Streptococcus iniae. We conclude that fugu kalliklectin might be an important immunocomponent, transferred from mother to offspring.

Serum amyloid P-component/C-reactive proteins in fugu (Takifugu rubripes) egg with binding ability to disease-causing bacteria by carbohydrate-recognition.

Two galactose-binding proteins were purified from the eggs of Takifugu rubripes by affinity chromatography. These proteins were detected at 26 and 23 kDa under reducing and at 40 and 45 kDa under non-reducing conditions at SDS-PAGE. The peptide sequences from both proteins matched to short-type pentraxin. The 26-kDa lectin was glycosylated, while the other one was not, indicating that these could be glycoforms of pentraxin. Messenger RNA of pentraxin was detected in eggs and embryos at 1-cell stage, was undetectable till blastula, and finally detected again after gastrula, suggesting that the mRNAs in eggs and 1-cell embryos were maternal in origin, and autologous transcription of the gene occurred after blastula. Since they bind to pathogenic bacteria, egg pentraxins may have immunological functions during embryogenesis. This is the first study to show the presence of short-type pentraxin in fish eggs and the diversity of fish egg lectins.

D-mannose-specific Immunoglobulin M in Grass Puffer (Takifugu niphobles), a Nonhost Fish of a Monogenean Ectoparasite Heterobothrium okamotoi, Can Act as a Trigger for its Parasitism.

Heterobothrium okamotoi, a monogenean gill parasite, exhibits high host specificity for the tiger puffer, Takifugu rubripes, and it has been experimentally verified that the parasite cannot colonize either closely related species such as the grass puffer Takifugu niphobles or distantly related fish such as the red seabream Pagrus major. Previously, we demonstrated in T. rubripes that immunoglobulin M (IgM) with d-mannose affinity induced deciliation of the oncomiracidia, the first step of parasitism, indicating that the parasite utilizes the molecule as a receptor for infection. In the present study, we purified mannose-specific IgM from 2 nonhost species, T. niphobles and P. major, by affinity and gel-filtration chromatography techniques and compared their deciliation-inducing activity against H. okamotoi oncomiracidia. The IgM of the former showed activity, whereas the latter had no effect, suggesting that in addition to d-mannose-binding ability, the crystallizable fragment domain of IgM, which is not part of the antigen-binding domain, plays an important role in host recognition by the oncomiracidia, such as direct binding to the parasites. It also suggests that the host specificity of H. okamotoi is relatively low upon initial recognition, and the specificity is established by exclusion in nonhosts during a later stage.

Arabidopsis PsbP-Like Protein 1 Facilitates the Assembly of the Photosystem II Supercomplexes and Optimizes Plant Fitness under Fluctuating Light.

In green plants, photosystem II (PSII) forms multisubunit supercomplexes (SCs) containing a dimeric core and light-harvesting complexes (LHCs). In this study, we show that Arabidopsis thaliana PsbP-like protein 1 (PPL1) is involved in the assembly of the PSII SCs and is required for adaptation to changing light intensity. PPL1 is a homolog of PsbP protein that optimizes the water-oxidizing reaction of PSII in green plants and is required for the efficient repair of photodamaged PSII; however, its exact function has been unknown. PPL1 was enriched in stroma lamellae and grana margins and associated with PSII subcomplexes including PSII monomers and PSII dimers, and several LHCII assemblies, while PPL1 was not detected in PSII-LHCII SCs. In a PPL1 null mutant (ppl1-2), assembly of CP43, PsbR and PsbW was affected, resulting in a reduced accumulation of PSII SCs even under moderate light intensity. This caused the abnormal association of LHCII in ppl1-2, as indicated by lower maximal quantum efficiency of PSII (Fv/Fm) and accelerated State 1 to State 2 transitions. These differences would lower the capability of plants to adapt to changing light environments, thereby leading to reduced growth under natural fluctuating light environments. Phylogenetic and structural analyses suggest that PPL1 is closely related to its cyanobacterial homolog CyanoP, which functions as an assembly factor in the early stage of PSII biogenesis. Our results suggest that PPL1 has a similar function, but the data also indicate that it could aid the association of LHCII with PSII.