Purification, primary structure, and biological activity of the high-mannose N-glycan-specific lectin from cultivated Eucheuma denticulatum

Abstract

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 highmannose 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 highmannose 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.

Biological activity, cDNA cloning and primary structure of lectin KSA-2 from the cultivated red alga Kappaphycus striatum (Schmitz) Doty ex Silva

“…In the present study, we have determined the primary structure of KSA-2, and confirmed that it strongly resembles those of lower organisms, such as OAA from O. agardhii Sato and Hori, 2009), ESA-2 from E. serra , EDA-2 from E. denticulatum (Hung et al, 2015), MBHA from M. xanthus (Romeo et al, 1986;Koharudin et al, 2012), BOA from B. oklahomensis (Whitley et al, 2013) and PFL from P. fluorescens Pf0-1 (Sato et al, 2012) (Fig. 4), including its molecular size and the presence of tandemly repeated motifs.…”

“…The species-specific antibacterial activities have been reported for the lectins from the red algae, E. serra and Galaxaura marginata, in which both lectins displayed antibacterial activity toward the fish pathogen Vibrio vulnificus,but not toward Vibrio neresis and Vibrio pelagius (Liao et al, 2003), and lectins from E. denticulatum toward the shimp pathogen V. alginolyticus, but not toward V. parahaemolyticus and V. harveyi (Hung et al, 2015). Although antibacterial activities have been reported for lectins from various biological sources (Santi-Gadelha et al, 2006;Charungchitrak et al, 2011;Riera et al, 2003), there seems to be little reported for the growth-inhibiting activity against shrimp and fish pathogens, except the high-mannose N-glycan specific lectins from carrageenophytes mentioned above.…”

“…The lectins from these algae have been isolated and showed binding specificity for high-mannose Nglycans Hung et al, 2009;Sato et al, 2011a;Hung et al, 2011Hung et al, , 2015. However, little information is known about genes encoding lectins from these algae, except for the gene encoding lectin EDA-2 from E. denticulatum (GenBank accession no.…”

High-Mannose Specific Lectin and Its Recombinants from a Carrageenophyta Kappaphycus alvarezii Represent a Potent Anti-HIV Activity Through High-Affinity Binding to the Viral Envelope Glycoprotein gp120

“…The recognition mode of KAAs is absolutely different from the other high-mannose specific antiviral lectins including CV-N, GRFT, SVN, MVL, and AH . We also reported the same oligosaccharide binding properties in ESA-2 ) and OAA , and recently in KSA from K. striatum (Hung et al 2011) and EDA from E. denticulatum (Hung et al 2015). A lectin PFL from Pseudomonas fluorescens Pf0-1 belonging to this lectin family also tends to preferentially recognize highmannose glycans with α1-3 Man exposed in the D2 arm but not to other sugar types including complex N-glycans and Oglycans, as revealed by screening with a glycan array having 611 glycan targets (Sato et al 2012).…”

“…A lectin ESA-2 from a red alga Eucheuma serra (Kawakubo et al 1997) was recently found to possess the unique binding specificity with the preferential affinity for high-mannose N-glycans bearing the nonreducing terminal α1-3 Man in D2 arm ). Since then, several lectins with similar binding specificity were isolated from some lower organisms, including red algae (Kawakubo et al 1999;Sato et al 2011a;Hung et al 2011Hung et al , 2015, a cyanobacterium (Sato et al 2000, and bacteria (Cumsky and Zusman 1979;Sato et al 2012;Koharudin et al 2012;Whitley et al 2013). Interestingly, these lectins commonly had tandem-repeated structures of a similar domain composed of about 67 amino acids, and grouped into two types of four and two repeats Sato et al 2007;Sato and Hori 2009) as exemplified with ESA-2 (four repeats) and a lectin OAA (two repeats) from a freshwater cyanobacterium Oscillatoria agardhii.…”