Fig 1.
Saccharification of E. bicyclis by the digestive fluid of sea hare (A. kurodai) and presence of BGLs inhibitors in the extract of E. bicyclis.
(A) Dried seaweeds (10 mg), E. bicyclis, U. pertusa, Saccharina sp. and U. pinnatifida were suspended in 50 mM acetate buffer (pH 5.5) containing 0.1 M NaCl and 10 mM CaCl2 (Buffer A), and incubated with several amounts of the digestive fluid (DF) of sea hare at 37°C for 20 h. The glucose content liberated from seaweeds was determined in three independent replicate. (B) Laminaran (2.5 mg) and E. bicyclis (10 mg) were digested with purified sea hare 110 kDa BGL, 210 kDa BGL, or A. kurodai digestive fluid at 37°C for 20 h. (C) The activities of 110 and 210 kDa BGLs were assayed in the presence of E. bicyclis extract. (D) Inhibition mechanism of 110 and 210 kDa BGLs by E. bicyclis extract. Open circles, extract 0 μl; closed circles, 64-fold diluted extract, 2 μl; open squares, 128-fold diluted extract, 2 μl.
Fig 2.
Inhibition of 110 and 210 kDa BGL by extracts of various seaweeds.
(A) Ten milligrams of E. bicyclis, U. pinnatifida, Saccharina sp., A. nodosum, and U. pertusa were extracted with 1.0 mL of Buffer A at 4°C for 20 h. After centrifugation, the supernatant (2 or 5 μL) was added into the assay mixture and the activity of each BGL was determined. (B) Inhibitory activity of E. bicyclis and A. nodosum extracts against 110 and 210 kDa BGLs. Effect of sequentially diluted extracts on the activity of each BGL was determined. All data (mean ± S.D.) were determined in three independent replicates.
Fig 3.
Purification of EHEP from the digestive fluid.
The EHEP present in the digestive fluid of the sea hare was purified by ammonium sulfate fractionation (0–30%), phenyl-Sepharose (A), and Sephacryl S-100 gel filtration (B) as described in Materials and Methods. (A) EHEA was eluted in the phenyl-Sepharose unbound fraction. (B) The fraction indicated by the horizontal bar was subjected to Sephacryl S-100 gel filtration. The EHEA fractions indicated by the horizontal bar was concentrated and analyzed by SDS-PAGE (C) and 2D-PAGE (D). Purified EHEP (5 μg) was resolved in both PAGE procedures and detected using Coomassie Brilliant Blue.
Fig 4.
Nucleotide sequence of the EHEP cDNA and the alignment of its deduced amino acid sequence with chondroitin proteoglycan-2-like protein sequences of A. californica predicted by automated computational analysis.
(I) The deduced amino acid sequence is displayed below the nucleotide sequence using a single-letter code. The predicted signal-peptide sequence is double underlined. The amino acid sequences identified by Edman degradation are underlined. The poly-(A) adenylation signal is boxed. (II) A, B, and C sequences are NCBI reference sequences XP_005103174.2, XP_005103176.2 and XP_005103175.1, respectively. Identical amino acid residues among aligned sequences are displayed in bold. The conserved cysteine residues are boxed. The accession number of the nucleotide sequence of EHEP cDNA in the DDBJ/EMBL/GenBank databases is LC122548.
Fig 5.
EHEP protects BGL activity from E. bicyclis extract inhibition.
(A) The 110 kDa BGL activity was assayed in a reaction mixture containing 1 mM 4MU-β-glucoside, E. bicyclis extract (0.5, 2.0, or 4.0 μL), and EHEP. The enzyme activity (mean ± S.D.) was determined in three independent replicates. (B) E. bicyclis (10 mg) was suspended in 1.0 mL Buffer A, and incubated with 210 or 110 kDa BGL in the absence or presence of EHEP at 37°C for 6 h. Glucose released was determined. (C) Saccharification of E. bicyclis by the synergistic action of 110 and 210 kDa BGLs in the presence of EHEP. E. bicyclis (10 mg) was incubated with a mixture of 110 and 210 kDa BGL in the absence or presence of EHEP in 1.0 mL Buffer A at 37°C for 24 h. Glucose (mean ± S.D.) was determined in at least three independent replicates. (D) U. pertusa (10 mg) was incubated with 45 kDa cellulase (0.5 U), 59 kDa α-amylase (0.5 U), 74 kDa α-glucosidase (AGL, 0.2 U), 210kDa BGL (0.4 U) and 110 kDa BGL (0.2 U) in the absence or presence of EHEP in 1.0 mL of Buffer A at 37°C for 24 h. Glucose (mean ± S.D.) was determined in three independent replicates.
Fig 6.
Interaction of EHEP with phlorotannin, tannic acid, phloroglucinol, and gallic acid.
(A) Inhibition of 110 kDa BGL by phlorotannin, tannic acid, phloroglucinol and gallic acid. The activity of the 110 kDa BGL was assayed in a reaction mixture containing phlorotannin A (diethyl ether fraction), phlorotannin B (aqueous fraction), tannic acid or phloroglucinol at the indicated concentrations. (B) Effect of EHEP on the inhibition of BGLs by phloroglucinol. The activities of the 110 and 210 kDa BGLs were assayed in the absence and presence of 2 mM phloroglucinol (PG) and EHEP (0.28 mg/mL). (C) Protection excerted by EHEP upon BGL’s inhibition by phlorotannin. The activities of 210 and 110 kDa BGLs were assayed in a reaction mixture containing 0.5 μg phlorotannin A in the absence and presence of the indicated EHEP concentration. (D) Reverse-correlation of EHEP precipitation and BGL inhibition by E. bicyclis extract. E. bicyclis extract (10 μL) was incubated with EHEP in 0.1 mL Buffer A at 25°C for 1 h. The precipitate formed was washed with the same buffer, and protein contents and composition were analyzed. The inhibitory activity of the supernatant was assayed. SDS-PAGE of precipitated proteins is shown in the inset. (E) Precipitation of E. bicyclis phlorotannins by EHEP. E. bicyclis extract (0.1 mL; 100 mg E. bicyclis /1.0 mL of Buffer A) was incubated with the indicated EHEP concentrations at 4°C for 16 h. After centrifugation at 12, 000 x g for 10 min, the supernatant and the precipitate were separated. The precipitate was washed twice with Buffer A and suspended in the same buffer. The content of phlorotannin in the supernatant and precipitate were determined as described in Materials and Methods.
Fig 7.
Specific precipitation of EHEP with phlorotannins.
Phlorotannin fractions A and B (10 μg of phloroglucinol equivalent) isolated from E. bicyclis, phloroglucinol (50 and 100 μg) or gallic acid (50 and 100 μg) were incubated with EHEP or BSA in 0.1 ml Buffer A at 25°C for 90 min. The reaction mixture was centrifuged at 12, 000 x g for 10 min. The resulting precipitate was dissolved in SDS-PAGE loading buffer containing 1% β-mercaptoethanol, treated at 95°C for 5 min and applied to 12% SDS-PAGE. Protein was detected by Coomassie Brilliant Blue staining. Precipitation of BSA with polyphenols was used as control, given that BSA is a polyphenol binding protein.