Makoto Hirayama

The production of inflammatory cytokines such as tumor necrosis factor (TNF)-α by activated macrophage cells plays an important role in the development of intestinal inflammation. The present study investigated the anti-inflammatory effect of the protein hydrolysates prepared from the jack bean (JBPHs), Canavalia ensiformis (L.) DC, using the enzyme Alcalase, in a murine macrophage model, RAW 264.7 cells, which were stimulated by lipopolysaccharides. JBPHs reduced the TNF-α expression at the protein and mRNA levels through the downregulation of cellular signaling pathways involved in nuclear factor kappa B (NF-κB), extracellular signal-regulated kinase (ERK), and p38. A combination of mass spectrometry and in silico approaches identified 10 potential anti-inflammatory peptides in the JBPHs, including LFLLP and DFFL. Interestingly, while LFLLP targeted the NF-κB pathway, DFFL targeted p38 and ERK to suppress the TNF-α production in the RAW 264.7 cells. In addition, LFLLP and DFFL were localized in the cytosol of the cells. These results demonstrated that LFLLP and DFFL were incorporated by RAW 264.7 cells and, at least in part, contributed to the reduction in TNF-α by JBPHs. These peptides isolated from JBPHs may well be utilized as new alternatives to alleviate intestinal inflammation.

The physiological impacts of polysaccharides and lipids in seaweed on human health are becoming clearer, but the behavior of the protein components, especially lectins, after ingestion remains poorly understood. In this study, we examined the resistance of edible red algae-derived lectins to digestive enzymes. We found that a lectin extracted from Meristotheca papulosa (MPL-1), belonging to the Jacalin-related lectin family, was relatively stable when subjected to both peptic and tryptic digestion and retained its hemagglutination activity after 24 h of digestion. The activity of MPL-1 was also maintained without a large change for 24 h following exposure to enzymes such as papain, Actinase E, and proteinase K, suggesting that MPL-1 possesses a strong resistant property against proteolytic digestion. We examined the anti-proliferation activity of the MPL fraction from the algal body against HT-29, a human colon cancer-derived cell line, and found that it showed a strong inhibitory activity with a half-maximal inhibitory concentration of 4.5 μg/mL. This activity was nullified in the presence of yeast mannan, an inhibitory sugar compound of lectin, demonstrating that MPL expressed its activity through binding to the glycan moieties on HT-29. This study indicates that this proteinase-resistant lectin could play a vital role after ingestion and is expected to have an inhibitory effect against colorectal cancer.

Marine algal lectins specific for high-mannose N-glycans have attracted attention because they strongly inhibit the entry of enveloped viruses, including influenza viruses and SARS-CoV-2, into host cells by binding to high-mannose-type N-glycans on viral surfaces. Here, we report a novel anti-influenza virus lectin (named HBL40), specific for complex-type N-glycans, which was isolated from a marine green alga, Halimeda borneensis. The hemagglutination activity of HBL40 was inhibited with both complex-type N-glycan and O-glycan-linked glycoproteins but not with high-mannose-type N-glycan-linked glycoproteins or any of the monosaccharides examined. In the oligosaccharide-binding experiment using 26 pyridylaminated oligosaccharides, HBL40 only bound to complex-type N-glycans with bi- and triantennary-branched sugar chains. The sialylation, core fucosylation, and the increased number of branched antennae of the N-glycans lowered the binding activity with HBL40. Interestingly, the lectin potently inhibited the infection of influenza virus (A/H3N2/Udorn/72) into NCI-H292 cells at IC₅₀ of 8.02 nM by binding to glycosylated viral hemagglutinin (K_D of 1.21 × 10⁻⁶ M). HBL40 consisted of two isolectins with slightly different molecular masses to each other that could be separated by reverse-phase HPLC. Both isolectins shared the same 16 N-terminal amino acid sequences. Thus, HBL40 could be useful as an antivirus lectin specific for complex-type N-glycans.

We have isolated a novel lectin, named HRL40 from the green alga Halimeda renschii. In hemagglutination-inhibition test and oligosaccharide-binding experiment with 29 pyridylaminated oligosaccharides, HRL40 exhibited a strict binding specificity for high-mannose N-glycans having an exposed (α1-3) mannose residue in the D2 arm of branched mannosides, and did not have an affinity for monosaccharides and other oligosaccharides examined, including complex N-glycans, an N-glycan core pentasaccharide, and oligosaccharides from glycolipids. The carbohydrate binding profile of HRL40 resembled those of Type I high-mannose specific antiviral algal lectins, or the Oscillatoria agardhii agglutinin (OAA) family, which were previously isolated from red algae and a blue-green alga (cyanobacterium). HRL40 potently inhibited the infection of influenza virus (A/H3N2/Udorn/72) into NCI-H292 cells with half-maximal effective dose (ED₅₀) of 2.45 nM through high-affinity binding to a viral envelope hemagglutinin (K_D, 3.69 × 10⁻¹¹ M). HRL40 consisted of two isolectins (HRL40-1 and HRL40-2), which could be separated by reverse-phase HPLC. Both isolectins had the same molecular weight of 46,564 Da and were a disulfide -linked tetrameric protein of a 11,641 Da polypeptide containing at least 13 half-cystines. Thus, HRL40, which is the first Type I high-mannose specific antiviral lectin from the green alga, had the same carbohydrate binding specificity as the OAA family, but a molecular structure distinct from the family.

The red algae, Eucheuma denticulatum and Kappaphycus striatum have been widely cultivated in Vietnam as a source of carrageenophytes for industry. In the past, biochemical properties of lectins isolated from these algae has been characterized and evaluated extensively. However, gene coding for such lectins isn’t studied yet. In this study, their full length cDNA is amfplified using cDNA ends (RACE) methods. Sequence analysis revealed that cDNA of EDA-2 from E. denticulatum consisted of 1,158 bp containing 103 bp of a 5'untranslated region, 248 bp of 3'untranslated region, and 807 bp of an open reading frame; and cDNA of KSA-2 from K. striatum consisted of 1174 bp containing 94 bp of the 5'-untranslated region, 273 bp of 3'untranslated region and 807 bp of the open reading frame. The cDNA of both EDA-2 and KSA-2 encoded for a polypeptide of 269 amino acids including an initiating methionine, but differed in sequences and molecular masses. The deduced amino acid sequences of EDA-2 and KSA-2 composed of four tandem repeated domains with about 67 amino acids each. The primary structure of EDA-2 and KSA-2 is highly similar to those of the high mannose N-glycan specific lectins including OAA from cyanobacterium, BOA, MBHA and PFA from bacteria, and ESA-2, KAA-1, KAA-2 from macro red algae, which showed strong anti-HIV and anti-influenza virus activities. These results indicate that these cultivated algae are becoming promising materials for production of anti-virus reagent or functional food that can prevent virus infection in future.

Lectin sensitivity of the recent pandemic influenza A virus (H1N1-2009) was screened for 12 lectins with various carbohydrate specificity by a neutral red dye uptake assay with MDCK cells. Among them, a high mannose (HM)-binding anti-HIV lectin, ESA-2 from the red alga Eucheuma serra, showed the highest inhibition against infection with an EC₅₀ of 12.4 nM. Moreover, ESA-2 exhibited a wide range of antiviral spectrum against various influenza strains with EC₅₀s of pico molar to low nanomolar levels. Besides ESA-2, HM-binding plant lectin ConA, fucose-binding lectins such as fungal AOL from Aspergillus oryzae and AAL from Aleuria aurantia were active against H1N1-2009, but the potency of inhibition was of less magnitude compared with ESA-2. Direct interaction between ESA-2 and a viral envelope glycoprotein, hemagglutinin (HA), was demonstrated by ELISA assay. This interaction was effectively suppressed by glycoproteins bearing HM-glycans, indicating that ESA-2 binds to the HA of influenza virus through HM-glycans. Upon treatment with ESA-2, no viral antigens were detected in the host cells, indicating that ESA-2 inhibited the initial steps of virus entry into the cells. ESA-2 would thus be useful as a novel microbicide to prevent penetration of viruses such as HIV and influenza viruses to the host cells.

Three isolectins from cultivated Eucheuma denticulatum were isolated. They were commonly monomeric proteins of about 28 kDa with a range of averaged molecular weights from 27,834 to 27,868 Da among the isolectins and shared almost the same 20 N-terminal amino acid sequences. Complementary DNA (cDNA) cloning based on the rapid amplification cDNA ends (RACE) methods elucidated the full-length sequence of EDA-2 which encodes 269 amino acids, including initiating methionine, with four tandemly repeated domains of about 67 amino acids. The primary structure of EDA-2 is highly similar to those of the high-mannose N-glycan specific lectins including Oscillatoria agardhii (OAA) and Burkholderia oklahomensis EO147 (BOA) from cyanobacteria, Myxococcus xanthus (MBHA) and Pseudomonas fluorescens Pf0-1 (PFL) from bacteria, and ESA-2 from a macro red alga. The hemagglutination activities were commonly inhibited by the glycoproteins bearing high-mannose N-glycans, but not by monosaccharides examined, including mannose. In a direct binding experiment with pyridylaminated oligosaccharides, an isolectin EDA-2 exclusively bound to high-mannose type N-glycans, but not to other glycans that include complex types and a core pentasaccharide of N-glycans, indicating that it recognized the branched oligomannoside moiety. Its binding activity was subtly different among the oligomannoside structures examined, showing that the lectin has preference affinity for high-mannose type N-glycans with an exposed (α1-3) mannose residue in the D2 arm. Interestingly, EDAs, the mixture of three isolectins inhibited the growth of shrimp pathogenic bacterium, Vibrio alginolyticus, although it did not affect the growth of V. parahaemolyticus and V. harveyi. Growth inhibition of V. alginolyticus with EDAs was not observed in the presence of yeast mannan bearing high-mannose N-glycans, suggesting that EDAs caused the activity through binding to the target receptor(s) on the surface of V. alginolyticus. These results indicate that cultivated carrageenophyte E. denticulatum is a good source of a lectin(s) that may be useful as a carbohydrate probe and an antibacterial reagent.