Identification of novel β-mannan- and β-glucan-binding modules: evidence for a superfamily of carbohydrate-binding modules.
Sunna, A., Gibbs, M. D. & Bergquist, P. L. (2001). Biochem. J, 356(3), 791-798.
Many glycoside hydrolases, which degrade long-chain carbohydrate polymers, possess distinct catalytic modules and non-catalytic carbohydrate-binding modules (CBMs). On the basis of conserved protein secondary structure, we describe here the identification and experimental characterization of novel type of mannanase-associated mannan-binding module and also characterization of two CBM family 4 laminarinase-associated β-glucan-binding modules. These modules are predicted to belong to a superfamily of CBMs which include families 4, 16, 17, 22 and a proposed new family, family 27.
Lentinula edodes tlg1 encodes a thaumatin-like protein that is involved in lentinan degradation and fruiting body senescence.
Sakamoto, Y., Watanabe, H., Nagai, M., Nakade, K., Takahashi, M. & Sato, T. (2006). Plant Physiology, 141(2), 793-801.
Lentinan is an antitumor product that is purified from fresh Lentinula edodes fruiting bodies. It is a cell wall component, comprising β-1,3-glucan with β-1,6-linked branches, which becomes degraded during postharvest preservation as a result of increased glucanase activity. In this study, we used N-terminal amino acid sequence to isolate tlg1, a gene encoding a thaumatin-like (TL) protein in L. edodes. The cDNA clone was approximately 1.0 kb whereas the genomic sequence was 2.1 kb, and comparison of the two indicated that tlg1 contains 12 introns. The tlg1 gene product (TLG1) was predicted to comprise 240 amino acids, with a molecular mass of 25 kD and isoelectric point value of 3.5. The putative amino acid sequence exhibits approximately 40% identity with plant TL proteins, and a fungal genome database search revealed that these TL proteins are conserved in many fungi including the basidiomycota and ascomycota. Transcription of tlg1 was not detected in vegetative mycelium or young and fresh mushrooms. However, transcription increased following harvest. Western-blot analysis demonstrated a rise in TLG1 levels following harvest and spore diffusion. TLG1 expressed in Escherichia coli and Aspergillus oryzae exhibited β-1,3-glucanase activity and, when purified from the L. edodes fruiting body, demonstrated lentinan degrading activity. Thus, we suggest that TLG1 is involved in lentinan and cell wall degradation during senescence following harvest and spore diffusion.
Biochemical, molecular and structural analysis of multiple thaumatin-like proteins from the elderberry tree (Sambucus nigra L.).
Van Damme, E. J., Charels, D., Menu-Bouaouiche, L., Proost, P., Barre, A., Rougé, P. & Peumans, W. J. (2002). Planta, 214(6), 853-862.
Thaumatin-like proteins (TLPs) were isolated and characterized from fruits and leaves of elderberry (Sambucus nigra) and their corresponding genes cloned. In addition, the developmental regulation and induction of the different TLPs was followed in some detail. Ripening berries accumulated a fruit-specific TLP during the final stages of maturation. This fruit-specific TLP had no antifungal activity and was devoid of β-glucanase activity. Leaves constitutively expressed a TLP that closely resembled the fruit-specific homologue. Treatment with jasmonate methyl ester induced two additional TLPs in leaves but did not induce or enhance the expression of TLPs in immature berries. In contrast to jasmonate methyl ester, both ethephon and garlic extract induced the expression of aTLP in unripe berries that normally do not express any TLP. Sequence analysis and molecular modelling indicated that all elderberry thaumatin-like proteins share a high sequence similarity with group-5 pathogenesis-related proteins. However, the proteins encoded by the different sequences differed from each other in isoelectric point and the distribution of the charges on the surface of the molecule.
A molecular basis for the endo-β1, 3-glucanase activity of the thaumatin-like proteins from edible fruits.
Menu-Bouaouiche, L., Vriet, C., Peumans, W. J., Barre, A., Van Damme, E. J. M. & Rougé, P. (2003). Biochimie, 85(1), 123-131.
Fruit-specific thaumatin-like proteins were isolated from cherry, apple and banana, and their enzymatic and antifungal activities compared. Both the apple and cherry possess a moderate endo-β1,3-glucanase activity but are devoid of antifugal activity. In contrast, the banana thaumatin-like protein inhibits the in vitro hyphal growth of Verticillium albo-atrum but is virtually devoid of endo-β1,3-glucanase activity. Both structural and molecular modeling studies showed that all three thaumatin-like proteins possess an extended electronegatively charged cleft at their surface, which is believed to be a prerequisite for endo-β1,3-glucanase activity. Docking experiments showed that the positioning of linear (1,3)-β-D-glucans in the cleft of the apple and cherry proteins allows an interaction with the glutamic acid residues that are responsible for the hydrolytic cleavage of the glucan. Due to a different positioning in the cleft of the banana thaumatin-like protein, the linear β-glucans cannot properly interact with the catalytic glutamic acid residues and as a result the protein possesses no enzymatic activity. The possible function of the fruit-specific thaumatin-like proteins is discussed in view of the observed biological activities and structural features.
Some thaumatin‐like proteins hydrolyse polymeric β‐1,3‐glucans.
Grenier, J., Potvin, C., Trudel, J. & Asselin, A. (1999). The Plant Journal, 19(4), 473-480.
Thaumatin and 12 purified thaumatin-like (TL) proteins were surveyed for their capacity to hydrolyse β-1,3-glucans by using an in-gel glucanase assay. Six TL proteins identified by N-terminal amino acid microsequencing were found to be active on carboxymethyl(CM)-pachyman: a barley leaf stress-related permatin, two tomato fruit osmotins, a cherry fruit and two tobacco stigma proteins. TL enzymes ranged in specific activity from 0.07 to 89 nkat mg-1 with CM-pachyman as substrate. Hydrolytic activities were not restricted to TL proteins strongly binding to water-insoluble β-1,3-glucans since the two osmotins were active without tight binding to pachyman. Some TL proteins hydrolysed crude fungal walls and one barley TL enzyme even lysed fungal spores. No activity was observed on laminarin in the in-gel hydrolase assay. Thin-layer chromatography revealed that the six enzymes acted as endo-β-1,3-glucanases leading to the formation of various oligoglucosides. Thus far, the TL enzymes (EC 3.2.1.x) appeared different from the well-known β-1,3-glucanases (EC 220.127.116.11). No activity was found with thaumatin, zeamatin, tobacco leaf PR-R protein and four stress-related TL proteins from barley and pea. This is the first demonstration that diverse TL proteins are enzymatically active. The functions of some TL proteins must be reassessed because they display endo-β-1,3-glucanase activity on polymeric β-1,3-glucans.
Characterization of peach thaumatin‐like proteins and their identification as major peach allergens.
Palacin, A., Tordesillas, L., Gamboa, P., Sanchez‐Monge, R., Cuesta‐Herranz, J., Sanz, M. L., Barber, D., Salcedo G. & Díaz‐Perales, A. (2010). Clinical & Experimental Allergy, 40(9), 1422-1430.
Background Peach is the most important fruit related to food allergy in the Mediterranean area. Pru p 3, its lipid transfer protein, has been described as the principal allergen responsible for cross-reactivities with other foods and pollen and the severity of clinical symptoms. However, the involvement of other allergenic families cannot be ruled out. Thaumatin-like proteins (TLPs) have been described as food allergen in several fruits, such as apple, cherry, kiwi and banana, and pollen. Objective To identify members of the TLP family in peach fruit and to characterize putative allergens. Methods Through two-dimensional (2D) electrophoresis of peach extract and immunodetections with a pool of peach-allergic patients, IgE-binding spots were identified and the corresponding proteins purified and characterized as allergens by in vitro and in vivo assays. Three isoforms, belonging to the TLP family, were purified by different chromatographic systems and characterized by N-terminal amino acid sequences, molecular weight determination (MALDI) and enzymatic activity analysis (β-1,3-gluconase test and inhibition growth of fungi). In the same way, their IgE-binding capacity and allergenic activity were tested by ELISA assays, basophil activation tests and skin prick tests (SPT). Results Two peach-TLPs, Pru p 2.0101 and Pru p 2.0201, were identified as IgE-binding spots by 2D electrophoresis. Another peach-TLP, Pru p 2.0301, was cloned and produced as recombinant protein in a yeast system. The three isoforms were purified and characterized as TLPs by immunoblotting with anti-chestnut TLP antibodies and anti-plant N-asparagine complex glycan (anti-cross-reactive carbohydrate determinant). All of them showed β-1,3-glucanase activity and inhibition of fungal growth. The three TLPs were recognized by around 50% of the sera from 31 patients analysed in ELISA experiments. All three gave a positive response to an SPT and/or in basophil activation experiments. Conclusion Three isoforms, belonging to the TLP family, were identified in peach as principal allergens. Their prevalence, observed in in vitro, ex vivo and in vivo analyses, suggests that they are important allergens and should therefore be included in the routine diagnosis of peach allergy, at least in the Mediterranean area.
Endo-β-1,3-glucanase GLU1, from the fruiting body of Lentinula edodes, belongs to a new glycoside hydrolase family.
Sakamoto, Y., Nakade, K. & Konno, N. (2011). Applied and Environmental Microbiology, 77(23), 8350-8354.
The cell wall of the fruiting body of the mushroom Lentinula edodes is degraded after harvesting by enzymes such as β-1,3-glucanase. In this study, a novel endo-type β-1,3-glucanase, GLU1, was purified from L. edodes fruiting bodies after harvesting. The gene encoding it, glu1, was isolated by rapid amplification of cDNA ends (RACE)-PCR using primers designed from the N-terminal amino acid sequence of GLU1. The putative amino acid sequence of the mature protein contained 247 amino acid residues with a molecular mass of 26 kDa and a pI of 3.87, and recombinant GLU1 expressed in Pichia pastoris exhibited β-1,3-glucanase activity. GLU1 catalyzed depolymerization of glucans composed of β-1,3-linked main chains, and reaction product analysis by thin-layer chromatography (TLC) clearly indicated that the enzyme had an endolytic mode. However, the amino acid sequence of GLU1 showed no significant similarity to known glycoside hydrolases. GLU1 has similarity to several hypothetical proteins in fungi, and GLU1 and highly similar proteins should be classified as a novel glycoside hydrolase family (GH128).
Identification, cloning, and characterization of β-glucosidase from Ustilago esculenta.
Nakajima, M., Yamashita, T., Takahashi, M., Nakano, Y. & Takeda, T. (2012). Applied Microbiology and Biotechnology, 93(5), 1989-1998.
Hydrolytic enzymes responsible for laminarin degradation were found to be secreted during growth of Ustilago esculenta on laminarin. An enzyme involved in laminarin degradation was purified by assaying release of glucose from laminaribiose. Ion-exchange chromatography of the culture filtrate followed by size-exclusion chromatography yielded a 110-kDa protein associated with laminaribiose hydrolysis. LC/MS/MS analysis of the 110-kDa protein identified three peptide sequences that shared significant similarity with a putative glucoside hydrolase family (GH) 3 β-glucosidase in Ustilago maydis. Based on the DNA sequence of the U. maydis GH3 β-glucosidase, a gene encoding a putative GH3 β-glucosidase in U. esculenta (Uebgl3A) was cloned by PCR. Based on the deduced amino acid sequence, the protein encoded by Uebgl3A has a molecular mass of 91 kDa and shares 90% identity with U. maydis GH3 β-glucosidase. Recombinant UeBgl3A expressed in Aspergillus oryzae released glucose from β-1,3-, β-1,4-, and β-1,6-linked oligosaccharides, and from 1,3-1,4-β-glucan and laminarin polysaccharides, indicating that UeBgl3A is a β-glucosidase. Kinetic analysis showed that UeBgl3A preferentially hydrolyzed laminaritriose and laminaritetraose. These results suggest that UeBgl3A is a key enzyme that produces glucose from laminarioligosaccharides during growth of U. esculenta on laminarin.
Detection of enzymes active on various β‐1,3‐glucans after denaturing polyacrylamide gel electrophoresis.
Trudel, J., Grenier, J. & Asselin, A. (1998). Electrophoresis, 19(10), 1788-1792.
Enzymes were assayed for glucanase activity after denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in gels containing β-1,3-glucans embedded as substrate. Lentinan, curdlan, paramylon, baker's yeast alkali-insoluble glucan, baker's yeast alkali-soluble glucan and carboxymethyl (CM)-pachyman were compared to oligomeric laminarin, which is the usual substrate for assaying β-1,3-glucanase activities. Detecting enzyme activities by aniline blue fluorescent staining was also compared with the staining of released reducing sugars by 2,3,5-triphenyltetrazolium chloride (TTC). For the nonreduced proteins, the Driselase extract exhibited one major band at 32.5 kDa and one less intense band at 23 kDa for most substrates with the two detection procedures. No Lyticase enzyme was detected in either detection procedures for all tested substrates. For barley enzymes, no activity was revealed after aniline blue staining while one undescribed 19 kDa glucanase activity was best shown after TTC staining with curdlan, paramylon and CM-pachyman as substrates. In the case of reduced proteins, the Lyticase extract yielded three bands (33, 36 and 46 kDa) on several substrates with both detection procedures. This was the same for the barley leaf extract (32, 36 and 39 kDa). The Driselase extract showed one 42 kDa band. Many enzymes active on β-1,3-glucans are thus best revealed when proteins are denatured and reduced and when protein renaturation after SDS-PAGE involves a pH 8.0 treatment and the inclusion of 1 mM cysteine in buffers. However, some enzymes are only detected when proteins are denatured without reduction. Finally, the use of various polymeric β-1,3-glucan substrates different from oligomeric laminarin is necessary to detect new types of enzymes such as the 19 kDa barley glucanase.
Development of β‐1,3‐glucanase activity in germinated tomato seeds.
Morohashi, Y. & Matsushima, H. (2000). Journal of Experimental Botany, 51(349), 1381-1387.
Laminarin‐hydrolysing activity developed in the endosperm of tomato (Lycopersicon esculentum) seeds following germination. The enzyme was basic (pI>10) and the apparent molecular mass was estimated to be 35 kDa by SDS‐PAGE. It was specific for linear β‐1,3‐glucan substrates. Laminarin was hydrolysed by the enzyme to yield a mixture of oligoglucosides, indicating that the enzyme had an endo‐action pattern. Thus, the enzyme was identified as β‐1,3‐ endoglucanase (EC 18.104.22.168). The activity of the enzyme developed in the endosperm after radicle protrusion (germination) had occurred and the enzyme activity was localized exclusively in the micropylar region of the endosperm where the radicle had penetrated. When the lateral endosperm region, where no induction of the enzyme occurred, was wounded (cut or punctured), there was a marked enhancement of β‐1,3‐glucanase activity. Thus the post‐germinative β‐1,3‐glucanase activity in the micropylar endosperm portion might be brought about by wounding resulting from endosperm rupture by radicle penetration.