Induction of Recombinant Lectin Expression by an Artificially Constructed Tandem Repeat Structure: A Case Study Using Bryopsis plumosa Mannose-Binding Lectin

Lectin is an important protein in medical and pharmacological applications. Impurities in lectin derived from natural sources and the generation of inactive proteins by recombinant technology are major obstacles for the use of lectins. Expressing recombinant lectin with a tandem repeat structure can potentially overcome these problems, but few studies have systematically examined this possibility. This was investigated in the present study using three distinct forms of recombinant mannose-binding lectin from Bryopsis plumosa (BPL2)—i.e., the monomer (rD1BPL2), as well as the dimer (rD2BPL2), and tetramer (rD4BPL2) arranged as tandem repeats. The concentration of the inducer molecule isopropyl β-D-1-thiogalactopyranoside and the induction time had no effect on the efficiency of the expression of each construct. Of the tested constructs, only rD4BPL2 showed hemagglutination activity towards horse erythrocytes; the activity of towards the former was 64 times higher than that of native BPL2. Recombinant and native BPL2 showed differences in carbohydrate specificity; the activity of rD4BPL2 was inhibited by the glycoprotein fetuin, whereas that of native BPL2 was also inhibited by d-mannose. Our results indicate that expression as tandem repeat sequences can increase the efficiency of lectin production on a large scale using a bacterial expression system.

Functional Expression and Characterization of the Recombinant N-acetyl-glucosamine/N-acetyl-galactosamine-Specific Marine Algal Lectin BPL3

Lectins, characterized by their carbohydrate-binding ability, have an extensive practical application. However, their industrial use is limited by low yields, and few active recombinant lectins have been reported. In this study, the algal lectin BPL-3 (Bryopsis plumosa lectin 3) was successfully produced using a bacterial expression system, BL21(DE3), with an artificial repeated structure (dimeric construct). Recombinant dimeric BPL3 (rD2BPL3) was confirmed by LC-MS/MS spectrometry. Expression efficiency was greater for the construct with the repeat structure (rD2BPL3) than the monomeric form (rD1BPL3). Optimal conditions for expression were 1 mM IPTG at 20 °C. Recombinant lectin was purified under denaturing conditions and refolded by the flash dilution method. Recombinant BPL3 was solubilized in 1× PBS containing 2 M urea. rD2BPL3 showed strong hemagglutination activity using human erythrocytes, similar to that of native BPL3. rD2BPL3 had a similar sugar specificity to that of the native protein, i.e., to N-acetyl-glucosamine (GlcNAc) and N-acetyl-galactosamine (GalNAc). Glycan array results showed that recombinant BPL3 and native BPL3 exhibited different binding properties. Both showed weak binding activity to α-Man-Sp. Native BPL3 showed strong binding specificity to the alpha conformation of amino sugars, and rD2BPL3 had binding activity to the beta conformation. The process developed in this study was suitable for the quality-controlled production of high amounts of soluble recombinant lectins.

Diet of the queen angelfish Holacanthus ciliaris (Pomacanthidae) in São Pedro e São Paulo Archipelago, Brazil

Holacanthus ciliaris is an important benthic-feeding reef fish but the relationship between the composition of its diet and prey availability is still unknown. Here we determined the quantitative composition of the gut contents of H. ciliaris in São Pedro e São Paulo Archipelago, Brazil (SPSPA), and compared it to the abundance of benthic organisms in the area. Holacanthus ciliaris has a relatively diversified diet with more than 30 prey species in SPSPA, especially sponges (13 spp., average 68% of gut contents total weight), algae (12 spp., 25%) and bryozoans (3 spp., 5%). In contrast, the benthic community is composed mainly of algae (average 81% of total cover) and followed by sponges (13%), bryozoans (5%), cnidarians (0.5%), polychaetes (0.5%) and tunicates (0.5%). The most common species were the algae Caulerpella ambigua and Caulerpa racemosa var. peltata; the bryozoan Margaretta buski; and the sponges Scopalina ruetzleri, Chondrosia collectrix and Clathria calla. The Manly resource selection function showed that H. ciliaris preferred the sponges Geodia neptuni, Erylus latens, Clathria calla and Asteropus niger, among others, and avoided common species such as the sponges Scopalina ruetzleri, Dysidea etheria and Hemimycale insularis and the algae Caulerpella ambigua, Bryopsis plumosa and Neomeris annulata. Kendall's rank correlation index showed no significant correlation between prey abundance in the field and in the diet of H. ciliaris, which seems to actively choose relatively rare and less defended prey.