applied sciences Review Wheat Germ Agglutinin—From Toxicity to Biomedical Applications Gabriele˙ Balˇciunait¯ e-Murzien˙ e˙ 1,2,* and Mindaugas Dzikaras 2 1 Institute of Pharmaceutical Technologies, Faculty of Pharmacy, Academy of Medicine, Lithuanian University of Health Sciences, 44307 Kaunas, Lithuania 2 Panevežys˙ Institute of Technologies and Business, Kaunas University of Technology, 44249 Kaunas, Lithuania; [email protected] * Correspondence: [email protected]; Tel.: +370-638-85187 Featured Application: Wheat germ agglutinin has the potential for enabling and improving tar- geted drug delivery systems, anticancer drugs, and antibacterial and antifungal therapeutics due to its cytotoxic mechanisms and specific carbohydrate binding. Abstract: Wheat germ agglutinin is a hevein class N-Acetylglucosamine–binding protein with specific toxicity and biomedical potential. It is extractable from wheat germ—a low-value byproduct of the wheat industry—using well–established extraction methods based on salt precipitation and affinity chromatography. Due to its N-Acetylglucosamine affinity, wheat germ agglutinin exhibits antifungal properties as well as cytotoxic properties. Its anticancer properties have been demonstrated for various cancer cells, and toxicity mechanisms are well described. Wheat germ agglutinin has been demonstrated as a viable solution for various biomedical and therapeutic applications, such as chemotherapy, targeted drug delivery, antibiotic-resistant bacteria monitoring and elimination. This is performed mostly in conjunction with nanoparticles, liposomes, and other carrier mechanisms via surface functionalization. Combined with abundant wheat byproduct sources, wheat germ agglutinin has the potential to improve the biomedical field considerably. Citation: Balˇciunait¯ e-Murzien˙ e,˙ G.; Dzikaras, M. Wheat Germ Keywords: wheat; germ; wheat byproducts; agglutinin; WGA; toxicity; glycosylation; N-Acetylglucosamine; Agglutinin—From Toxicity to GlcNAc; carbohydrate Biomedical Applications. Appl. Sci. 2021, 11, 884. https://doi.org/ 10.3390/app11020884 1. Introduction Received: 2 December 2020 Accepted: 15 January 2021 Wheat (Triticum aestivum L.) is one of the most essential agricultural staple foods used Published: 19 January 2021 for human consumption and animal feed. Approximately 21% of the world’s food supplies depend on annual wheat crop harvest [1], which causes the production of byproducts that Publisher’s Note: MDPI stays neutral are not always used efficiently or, in some cases, entirely discarded as waste [2]. Increasing with regard to jurisdictional claims in trends of consumption-based economy restructuring to more ecologically-minded circular published maps and institutional affil- economy models encourage the scientific development of new technologies for byproduct iations. valorization to create products with high nutritional value. One of the wheat processing byproducts which takes 2–3% of whole wheat grain [3] is wheat germ. They are a source of oils [4], tocopherols [5], various polyphenols [6], and specific proteins, such as agglutinins (lectins) [7]. Even though agglutinins are considered to be antinutrients [8], they might have Copyright: © 2021 by the authors. various prospective applications in biomedicine, biotechnology, and agriculture itself. Con- Licensee MDPI, Basel, Switzerland. sidering the high quantities of wheat germ available as low-value byproducts throughout This article is an open access article the world, any valorization attempts by extracting specific proteins may lead to high impact distributed under the terms and worldwide. This review is focused on wheat germ agglutinin, its structure and specificity, conditions of the Creative Commons extraction and purification methods, biological activity, and possible applications. Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Appl. Sci. 2021, 11, 884. https://doi.org/10.3390/app11020884 https://www.mdpi.com/journal/applsci Appl. Sci. 2021, 11, x FOR PEER REVIEW 2 of 11 Appl. Sci. 2021, 11, 884 2 of 10 2. Wheat Germ Agglutinin Structure 2. Wheat Germ Agglutinin Structure Wheat germ agglutinin (WGA) is one of the first purified lectins extracted at the very Wheat germ agglutinin (WGA) is one of the first purified lectins extracted at the beginning of the lectinomics field. WGA structure was described during the 1970s. WGA very beginning of the lectinomics field. WGA structure was described during the 1970s. is a mixture of three closely related major isoforms, named WGA1, WGA2, and WGA3 WGA is a mixture of three closely related major isoforms, named WGA1, WGA2, and [9], which are a 36.0 kDa size stable 18.0 kDa polypeptide chain homodimer [10] with WGA3 [9], which are a 36.0 kDa size stable 18.0 kDa polypeptide chain homodimer [10] twofold axis symmetry [11]. Variability of these three isolectins is observed at 10 sequence with twofold axis symmetry [11]. Variability of these three isolectins is observed at 10 positions, with WGA3 being the most distinct form, differing from WGA1 by 8 positions sequence positions, with WGA3 being the most distinct form, differing from WGA1 by and from WGA2 by 7 positions [12]. The polypeptide chain is stable under high-tempera- 8 positions and from WGA2 by 7 positions [12]. The polypeptide chain is stable under ture exposure [13]. Moreover, WGA monomers are highly resistant to acidity, and confor- high-temperature exposure [13]. Moreover, WGA monomers are highly resistant to acidity, mationaland conformational changes can changes be reversed can be by reversed increasing by increasingpH [14]. pH [14]. Each polypeptidepolypeptide chain chain is is composed composed of fourof four hevein hevein domains, domains, named named A, B, C,A,and B, C D, [and15,16 D] [1(Figure5,16] 1(Figure). WGA3 1). exhibits WGA3 higherexhibits interdomain higher interdomain similarity thansimilarity WGA1 than or WGA2,WGA1 suggestingor WGA2, suggestingcloser relatedness closer relatedness to the common to the ancestral common molecule ancestral [12 molecule]. Amino [12] acid. Amino analysis acid shows analysis that showsWGA containsthat WGA high contains amounts high of glycineamounts and of half-cystine,glycine and featureshalf-cystine, not typical features to mostnot typical of the tolectins most [ 17of ].the Additionally, lectins [17]. theAdditionally, protein is rich the inprotein disulfide is rich bridges, in disulfide with each bridges hevein, with domain each heveincontaining domain eight containing disulfide-forming eight disulfide cysteines-forming [18]. Thesecysteines disulfides [18]. These have disulfides been shown have to beenplay shown an important to play an role important in hevein role stability in hevein since stability heveins since lack heveins a hydrophobic lack a hydrophobic core [19]. coreAdditionally, [19]. Additionally, the disulfides the disulfides seem to explain seem to low explain pH value low pH stability value [ 14stability]. WGA [14] has. WGA been hasshown been to shown undergo to cotranslationalundergo cotranslational processing processing of glycan addition of glycan to addition the C-terminus, to the C which-termi- is nuslater, which post-translationally is later post-translationally removed before removed WGA reaches before mature WGA reaches form [20 mature]. Affinity form studies [20]. Affinityshow that studies subunit show specificity that subunit to oligosaccharides specificity to isoligosaccharides much better than is much to monosaccharides. better than to monosaccharides.A, B, and C subunits A, bind B, andN-Acetylglucosamine C subunits bind N (GlcNAc)-Acetylglucosamine residues, such (GlcN as chitin,Ac) residues whereas, suchsubunit as chitin, D accommodates whereas subunit the glycoside D accommodates aglycones. the Monosaccharides glycoside aglycones. are only Monosaccha- bound to ridessubunit are C only [21]. bound However, to subunit low affinity C [21]. to However, other carbohydrates, low affinity to such other as N-Acetylneuraminiccarbohydrates, such as(sialic) N-Acetylneuraminic acid, plays an important (sialic) acid role, plays in WGA an important activity [role22]. in Based WGA on activity the structure [22]. Based and oncarbohydrate the structure specificity, and carbohydrate WGA is classified specificity as, WGA chitin-binding is classified lectin as composedchitin-binding of hevein lectin composeddomains [ 23of]. hevein Lectin domains structure [2 and3]. Lectin carbohydrate structure specificity and carbohydrate enable its specificity broad spectrum enable its of broadbiological spectrum activities. of biological activities. Figure 1. Dimeric wheat germ agglutinin 3 (WGA3) structure by [ 24]. Red, Red, orange, orange, yellow, yellow, and brown brown colors represent the subunits A, B, C, and D of the first first protein, respectively. Blue, light blue, pink, and purple represent the subunits A, A, B, B, C, C, and D of the second protein, respectively. TheThe sugar-bindingsugar-binding sitesite isis locatedlocated atat thethe interfaceinterface ofof bothboth WGAWGA monomers.monomers. Appl. Sci. 2021, 11, 884 3 of 10 3. WGA Extraction and Purification The most widely used natural WGA purification from wheat germ strategies include a series of protein precipitation and chromatographic purification steps. Untreated wheat germ material goes through a defatting step followed by material disruption with a labora- tory mill. The material preparation is followed by extraction in water or aqueous buffers. Crude wheat extracts are used for