Bioscience, Biotechnology, and Biochemistry, 2021, Vol. 85, No. 3, 630-633

doi: 10.1093/bbb/zbaa058 Advance access publication date: 31 December 2020 NOTE

NOTE An efficient heterologous Escherichia coli-based Downloaded from https://academic.oup.com/bbb/article/85/3/630/6056114 by guest on 24 September 2021 expression system for lectin production from porrigens Tomohiro Suzuki ,1,∗,† Luna Nakamura,1,† Satomi Inayoshi,2 Yuki Tezuka,1 Akiko Ono ,1 Jae-Hoon Choi ,2,3 Hideo Dohra ,3 Tomohiro Sasanami ,2 Hirofumi Hirai,2,3 and Hirokazu Kawagishi 2,3,4

1Center for Bioscience Research and Education, Utsunomiya University, Mine-machi, Utsunomiya, Tochigi, Japan; 2Graduate School of Integrated Science and Technology, Shizuoka University, Ohya, Suruga-ku, Shizuoka, Japan; 3Research Institute of Green Science and Technology, Shizuoka University, Ohya, Suruga-ku, Shizuoka, Japan; and 4Graduate School of Science and Technology, Shizuoka University, Ohya, Suruga-ku, Shizuoka, Japan

∗Correspondence: Tomohiro Suzuki, [email protected] †Contributed equally to this work and share first-author status

ABSTRACT In this study, we report a more efficient heterologous expression of lectin from (PPL) using an Escherichia coli-based expression system. The yield (9.3 mg/L culture broth) of recombinant PPL (rPPL) using this expression system was increased approximately 9-fold compared to our previous study. The rPPL obtained in this study exhibited the same biochemical properties as the native PPL.

Keywords: Heterologous expression, lectin, Pleurocybella porrigens, Escherichia coli

The term “lectin” defines a protein (or glycoprotein) that is detection of characteristic glycans in tumor cells using lectins neither an enzyme nor an antibody. Lectins show specific can accelerate the detection of tumor development. For exam- carbohydrate-binding activity and are ubiquitous in plants, an- ple, α-1-6 fucosylation (core fucosylation) is a biomarker for imals, and microorganisms (Goldstein et al. 1980). Many lectins hepatocellular carcinoma. The mushroom lectin Aleuria aurantia contribute to diverse biological functions, such as host defense lectin (AAL) is used as a biomarker for liver cancer because it has mechanisms, inflammation, and cancer metastasis (Imberty an affinity for fucosyl residues (Wimmerova et al. 2003). Recently, et al. 2000; Carrizo et al. 2005). Lectins have a unique sugar- our research group discovered that a lectin from Pholiota squar- binding specificity and can be used as biomarkers for sugar rosa (PhoSL) specifically binds to core α-1-6 fucose and conse- chain detection. Abnormal glycosylation patterns, such as fu- quently has potential use as a novel diagnostic agent (Kobayashi cosylation, sialylation, or the aberrant expression of β-1,6- et al. 2012). Therefore, investigation of the detailed sugar-binding branched N-glycans or Tn antigen (truncated O-glycan) occur specificity of lectins is essential for enhancing their use andap- in many developmental stages in cancer (Syed et al. 2016). The plication in tumor diagnostic studies.

Received: 28 September 2020; Accepted: 27 October 2020 © The Author(s) 2020. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry. All rights reserved. For permissions, please e-mail: [email protected]

630 An efficient heterologous Escherichia coli-based expression system for lectin production 631 Downloaded from https://academic.oup.com/bbb/article/85/3/630/6056114 by guest on 24 September 2021

Figure 1. Molecular properties of rPPL. (a) SDS-PAGE of rPPL expressed in E. coli. Lane M: marker proteins, Lane 1: soluble fraction of BL21-Codon Plus containing pCold I-ppl, Lane 2: inclusion body fraction of BL21-Codon Plus containing pCold I-ppl, Lane 3: purified rPPL. (b) MALDI-TOF mass spectrometry analysis of rPPL.

The Pleurocybella porrigens (Sugihiratake in Japan) be- then prepared using BugBuster Protein Extraction Reagent longs to the family Tricholomataceae (order , class according to manufacturer’s protocols. The results of SDS-PAGE ). It has been consumed as a foodstuff for a long analysis of these two fractions indicated that the target PPL time in Japan, particularly in the Hokuriku and Tohoku regions, band (around 15 kDa) was present in both fractions (Figure 1a). until 2004. In that year, acute encephalopathy was found to be The soluble fraction was then subjected to His GraviTrap affinity caused by eating this mushroom. To date, we have succeeded in chromatography (GE Healthcare, Uppsala, Sweden). the isolation, purification, characterization, and cloning of the After purification using the His-tag affinity column, thepu- full-length cDNA sequence of P.porrigens lectin (PPL) (Suzuki et al. rified protein was subjected to SDS-PAGE analysis. As a result, 2009). As reported previously, we constructed a heterologous ex- a single band around 15 kDa in size was observed (Figure 1a). pression system using the basidiomycete Phanerochaete sordida To confirm the detailed molecular weight of the purified lectin, YK-624, which resulted in the extracellular secretion of recom- MALDI-TOF-MS analysis was performed. For MALDI-TOF-MS binant PPL (rPPL) in its active form using a signal peptide of lignin analysis, super-DHB and Protein Calibration Standard I (Bruker, peroxidase (Suzuki et al. 2014). However, the expression of rPPL Billerica, MA, USA) were used as the matrix and mass cali- was quite low, with a yield of only 1 mg/L culture broth after bration standards, respectively. As a result, single (M + H]+) purification by acid-treated Sepharose 4B affinity chromatogra- and double ([M + 2H]2+) charged ions were observed as domi- phy, and its hemagglutination activity was 128 titer/mg protein. nant peaks at m/z 17,176 and 8,592, respectively (Figure 1b). The Thus, in the present study, we attempted to construct a more ef- molecular weight estimated from the amino acid sequence of ficient heterologous expression system of PPL in Escherichia coli rPPL was 17,217; therefore, the result of MALDI-TOF-MS analysis using the pCold I DNA vector and characterized the properties was consistent with that of the estimated molecular weight in of rPPL produced. SDS-PAGE. Furthermore, the purified protein was digested with Two oligonucleotide primers, pcold-ppl-F1 (5-CTCGAGGGAT trypsin in-gel and the total digest was subjected to LC-MS/MS. CCGCGTCCATCCCTGCCGGAACCTAC-3) and pcold-ppl-R1 (5- LC-MS/MS analyses using a Triple TOF 5600 mass spectrome- TATCTAGACTGCAGGTCAAACGGCTTCGAAGACCCAG-3), were ter (AB Sciex, Framingham, MA, USA) were performed using the designed to amplify the full-length sequence of the coding methods described in our previous study (Kruger et al. 2002). sequence of ppl for introduction into the pCold I DNA vector The results of protein identification using PEAKS Studio version (Takara, Shiga, Japan). To construct an expression vector (pCold 8.5 (Bioinformatics Solutions Inc., Waterloo, Canada) against I-ppl), we performed restriction enzyme treatment of pCold I the Swiss-Prot database identified PPL with a high sequence DNA (1000 μg/mL, 4407 bp) with EcoRI and SalI. The restric- coverage (76%), which indicated that the recombinant protein tion enzyme-treated pCold I DNA was ligated to the ppl gene was successfully expressed and purified. Purification using this sequence using the Gibson Assembly system (New England method was carried out in triplicate, and the average yield of BioLabs, Beverly, MA, USA), yielding the plasmid pCold I-ppl.The rPPL was determined to be 9.3 mg/L culture broth (Table S1). constructed pCold I-ppl was transformed into E. coli BL21-Codon After the purification procedure, the hemagglutination activity Plus (Takara), a competent cell system for protein expression. of rPPL was found to be 4.2 × 103 titer/mg protein. The rPPL The obtained transformed colony was then inoculated into LB obtained in this study showed higher hemagglutination activ- liquid medium supplemented with 50 μg/mL ampicillin and cul- ity than that reported previously (128 titer/mg protein) (Suzuki tured with shaking at 37 °C to an optical density (OD) at 600 nm et al. 2014). Since the purification method was different from our in the range of 0.4 to 0.8. The culture was chilled by cooling on previous study, there was possibility that the purity and hemag- ice for 30 min and then isopropyl β-D-1-thiogalactopyranoside glutination activity of rPPL obtained in this expression system (IPTG) (to a final concentration of 0.1 mM) was added to the was high. medium and cultured at 15 °C for 24 h. The cells were collected The rPPL obtained in this study was characterized using the by centrifugation (16,000 × g, 20 min, 4 °C) and lysed using method described previously (Suzuki et al. 2009). All tests were the BugBuster Protein Extraction Reagent (Merck Millipore, performed in duplicate. Furthermore, native PPL (nPPL) was pu- Billerica, MA, USA). Soluble and inclusion body fractions were rified in the same method as our previous reports in orderto 632 Bioscience, Biotechnology, and Biochemistry, 2021, Vol. 85, No. 3 Downloaded from https://academic.oup.com/bbb/article/85/3/630/6056114 by guest on 24 September 2021 Figure 2. Thermostability and pH stability testing of rPPL. (a) Thermostability of rPPL. (b) pH stability of rPPL. use nPPL as a control (Suzuki et al. 2009). The hemagglutination Table 1. Inhibition of native and recombinant PPL-mediated hemag- activity test was performed to investigate whether the lectin ob- glutination by monosaccharides, oligosaccharides, and glycoproteins tained in this study has lectin activity. Rabbit erythrocytes alone MICb and rabbit erythrocytes treated with actinase E (Pronase) were used in the assay. rPPL showed stronger hemagglutination ac- Inhibitora rPPL nPPL tivity in rabbit erythrocytes treated with actinase E compared to untreated rabbit erythrocytes, similar to nPPL (Table S2). The (mM) (mM) thermostability and pH stability of rPPL were tested to further N-acetylgalactosamine 0.39 0.1 determine its biochemical properties. For temperature stability, Lactose 25 1.56 an rPPL solution adjusted to a titer of 8 was incubated at tem- Lactulose 25 1.56 peratures ranging from 40 °C to 100 °C for 30 min, cooled on ice, Lactitol 50 6.25 α and then tested for hemagglutination activity. To examine the Methyl -galactoside 100 6.25 Fucose 50 100 pH stability of the lectin, it was dissolved in buffer at different Rhamnose n.d. 100 pH levels from 2.0 to 10.0, incubated at 4 °C for 16 h, and then subjected to the hemagglutination activity test. The lectin activ- (μg/mL) (μg/mL) ity of rPPL was stable below 60 °C, but it was gradually inacti- Asialo-BSM 0.12 0.12 vated at temperatures above 60 °C (Figure 2a). rPPL was stable BSM 0.12 0.98 over a wide pH range but exhibited highest activity between pH PSM 0.12 0.98 6.0 and 7.0 (Figure 2b). These results suggest that the properties Asialo-PSM 0.12 1.95 of rPPL obtained in this study are almost identical to those of Asialo-fetuin 62.5 n.d. nPPL (Suzuki et al. 2009). Fetuin 125 n.d. We evaluated the sugar-binding specificity of rPPL by Transferrin 125 n.d. the hemagglutination inhibition test using various monosac- a charides, oligosaccharides, polysaccharides, and glycoproteins. Glucose, mannose, l-fucose, saccharose, xylose, melibiose, arabinose, 2- deoxyglucose, 2-deoxyribose, cellobiose, l-sorbose, sorbitol, levulose, fructose, Table 1 shows a comparison of the sugar-binding specificities of α-gluconoic acid, N-acetylglucosamine, and methyl α-glucopyranoside exhibited rPPL and nPPL. rPPL showed the strongest sugar-binding speci- no inhibition at concentrations up to 400 mM (data not shown). ficity to GalNAc (0.39 mM) among the monosaccharides and Mannan, albumin (chicken egg), and α-1-acid glycoprotein did not inhibit at con- oligosaccharides tested, followed by lactose (25 mM) and lac- centrations up to 1 mg/mL (data not shown). b turase (25 mM). These data demonstrated that rPPL had al- Minimum inhibitory concentrations required for inhibition of hemagglutina- tion of titer 4 of lectin. most similar sugar-binding specificity to nPPL. Furthermore, n.d.: Activity was not detected. rPPL showed the highest affinity to asialo-porcine submaxillary mucin (PSM), asialo-bovine submaxillary mucin (BSM), PSM, and BSM among the tested glycoproteins. To date, several studies antiviral signaling through recognition of galactosyl groups on have reported that the sugar-binding specificity of lectins from the cell surface of macrophages, which can eventually suppress some mushrooms is slightly changed as a result of heteroge- HIV infection (Zhou et al. 2018). As described above, Gal/GalNAc- neous expression (Kruger et al. 2002; Tateno and Goldstein 2003; specific lectins containing PPL have tremendous potential for Tateno et al. 2004). Taken together, these data show that rPPL is use in various medical applications and in studies investigating likely to recognize O-linked oligosaccharides in the same man- glycan structures. This efficient heterologous expression system ner as nPPL. constructed in this study facilitates the analysis of the crystal Mammalian cell surfaces contain a variety of N-andO-linked structure and the more detailed sugar-binding specificity of PPL glycans, including galactosyl groups associated with membrane- in the future and may be useful for further applications and uses bound proteins and glycolipids, which are involved in essen- as described above. tial biological functions (Varki 1993). For example, macrophage galactose-type C-type lectins have been reported to interfere Supplementary material with regulatory T cell function by binding to cell surface GalNAc (Zizzari et al. 2015). Recently, soybean lectin (SBL), which is Supplementary material is available at Bioscience, Biotechnology, a Gal/GalNAc-specific lectin, has been reported to activate and Biochemistry online. An efficient heterologous Escherichia coli-based expression system for lectin production 633

Acknowledgments sis lectins complexed with sialic acid-containing oligosaccha- rides. J Biol Chem 2000;275:17541-8. L.N. thanks the Saito Yutaka Scholarship of Utsunomiya Univer- Kobayashi Y, Tateno H, Dohra H et al. A novel core fucose- sity Foundation for Graduate Fellowship. specific lectin from the mushroom Pholiota squarrosa. J Biol Chem 2012;287:33973-82. Author contribution Kruger RP, Winter HC, Simonson-Leff N et al. Cloning, expres- sion, and characterization of the Galα 1,3Gal high affinity T.Su. conceived the project and designed most of the experimen- lectin from the mushroom Marasmius oreades. J Biol Chem tal outlines. T.Su., L.N., S.I., Y.K., A.O., J.C., H.D., and T.Sa. per- 2002;277:15002-5. formed the experiments. T.Su., H.D., H.H., and H.K. contributed Suzuki T, Amano Y, Fujita M et al. Purification, characterization, to discussions of the project. T.Su., L.N., and H.K. wrote the and cDNA cloning of a lectin from the mushroom Pleurocybella manuscript. porrigens. Biosci Biotechnol Biochem 2009;73:702-9. Suzuki T, Dohra H, Omae S et al. Heterologous expression of a Funding lectin from Pleurocybella porrigens (PPL) in Phanerochaete sordida Downloaded from https://academic.oup.com/bbb/article/85/3/630/6056114 by guest on 24 September 2021 YK-624. J Microbiol Methods 2014;100:70-6. This work was partially supported by JSPS KAKENHI under Syed P, Gidwani K, Kekki H et al. Role of lectin microarrays in Grants JP19K05808 and 26850110; the Novartis Foundation for cancer diagnosis. Proteomics 2016;16:1257-65. Gerontological Research under a research grant; Sugiyama Tateno H, Goldstein IJ. Molecular cloning, expression, and char- Chemical & Industrial Laboratory under a grant; and the UU-COE acterization of novel hemolytic lectins from the mushroom research project at Utsunomiya University to T. Su. This work Laetiporus sulphureus, which show homology to bacterial tox- was also partially supported by Scientific Research on Innovative ins. J Biol Chem 2003;278:40455-63. Areas “Frontier Research on Chemical Communications” Grant Tateno H, Winter HC, Goldstein IJ. Cloning, expression in JP17H06402 and Scientific Research (A) Grant 16H02546 to H.K. Escherichia coli and characterization of the recombinant Neu5Acα2,6Galβ1,4GlcNAc-specific high-affinity lectin and Disclosure statement its mutants from the mushroom Polyporus squamosus. Biochem J 2004;382:667-75. No potential conflict of interest was reported by the authors. Varki A. Biological roles of oligosaccharides: All of the theories are correct. Glycobiology 1993;3:97-130. References Wimmerova M, Mitchell E, Sanchez JF et al. Crystal structure of fungal lectin: Six-bladed beta-propeller fold and novel fu- Carrizo ME, Capaldi S, Perduca M et al. The antineoplastic lectin cose recognition mode for Aleuria aurantia lectin. J Biol Chem of the common edible mushroom (Agaricus bisporus)hastwo 2003;278:27059-67. binding sites, each specific for a different configuration ata Zhou R, Wang X, Liu H et al. GalNAc-specific soybean lectin in- single epimeric hydroxyl. J Biol Chem 2005;280:10614-23. hibits HIV infection of macrophages through induction of an- Goldstein IJ, Hughes RC, Monsigny M et al. What should be called tiviral factors. J Virol 2018;92:e01720-17. alectin?Nature 1980;285:66. Zizzari IG, Martufi P, Battisti F et al. The macrophage galactose- Imberty A, Gautier C, Lescar J et al. An unusual carbohydrate type C-type lectin (MGL) modulates regulatory T cell func- binding site revealed by the structures of two Maackia amuren- tions. PLoS One 2015;10:e0132617.