Extracellular Glycolipids Research Articles

Glycolipids of Pseudomonas and Rhodococcus oil-degrading bacteria used in bioremediation preparations: Formation and structure

We studied formation and structural features of biosurfactants produced by five oil-degrading Pseudomonas and Rhodococcus strains. These bacteria were found to be capable of intense formation of extracellular glycolipid biosurfactants when grown on mineral salts medium with 2% hexadecane. Under these conditions, the surface tension of the cultures decreased from 77mN/m to 31–34mN/m. The strain Rhodococcus sp. S26 forming up to 780mg glycolipids/l of culture medium proved the most efficient biosurfactant producer. Extracellular glycolipids were purified from the crude extracts by column chromatography. Their structural features were determined by thin layer chromatography and electrospray ionization mass spectrometry. Strains Pseudomonas putida BS3701 and Pseudomonas fluorescens 142NF synthesized a number of glycolipids identified as rhamnolipid B and its homologues. Glycolipids produced by Rhodococcus sp. X5 and Rhodococcus sp. S26 were assigned to trehalose tetraesters.

Genome Sequence of the Basidiomycetous Yeast Pseudozyma antarctica T-34, a Producer of the Glycolipid Biosurfactants Mannosylerythritol Lipids

The basidiomycetous yeast Pseudozyma antarctica T-34 is an excellent producer of mannosylerythritol lipids (MELs), members of the multifunctional extracellular glycolipids, from various feedstocks. Here, the genome sequence of P. antarctica T-34 was determined and annotated. Analysis of the sequence might provide insights into the properties of this yeast that make it superior for use in the production of functional glycolipids, leading to the further development of P. antarctica for industrial applications.

The role of PaAAC1 encoding a mitochondrial ADP/ATP carrier in the biosynthesis of extracellular glycolipids, mannosylerythritol lipids, in the basidiomycetous yeast Pseudozyma antarctica

Pseudozyma antarctica produces large amounts of the glycolipid biosurfactants known as mannosylerythritol lipids (MEL), which show not only excellent surface-active properties but also versatile biochemical actions. A gene homologous with a mitochondrial ADP/ATP carrier was dominantly expressed in P. antarctica under MEL-producing conditions on the basis of previous gene expression analysis. The gene encoding the mitochondrial ADP/ATP carrier of P. antarctica (PaAAC1) contained a putative open reading frame of 954 bp and encodes a polypeptide of 317 amino acids. The deduced translation product shared high identity of 66%, 70%, 69%, 74%, 75% and 52% with the mitochondrial ADP/ATP carrier of Saccharomyces cerevisiae (AAC1), S. cerevisiae (AAC2), S. cerevisiae (AAC3), Kluyveromyces lactis (KlAAC), Neurospora crassa (NcAAC) and human (ANT1), respectively, and conserved the consensus sequences of all ADP/ATP carrier proteins. The gene expression by introducing a plasmid pUXV1-PaAAC1 into the yeast cells increased the MEL production. In addition, the expression of PaAAC1 in which the conserved arginine and leucine required for ATP transport activity were replaced with isoleucine and serine, respectively, failed to increase MEL production. Accordingly, these results suggest that PaAAC1 encoding a mitochondrial ADP/ATP carrier should be involved in MEL biosynthesis in the yeast.

Activation of Fibroblast and Papilla Cells by Glycolipid Biosurfactants, Mannosylerythritol Lipids.

Mannosylerythritol lipids (MELs), the extracellular glycolipids produced from feedstock by yeasts belonging to the genus Pseudozyma, are the most promising biosurfactants known due to its versatile interfacial and biochemical actions. In order to broaden the application in cosmetics, the cell activating property of MELs was investigated using cultured fibroblast and papilla cells, and a three-dimensional cultured human skin model. The di-acetylated MEL (MEL-A) produced from soybean oil significantly increased the viability of the fibroblast cells over 150% compared with that of control cells. On the other hand, no cell activation was observed by the treatment with MEL-A produced from olive oil. The mono-acetylated MEL (MEL-B) hardly increased the cell viability. The viability of the fibroblast cells decreased with the addition of more than 1 microg/L of MELs, whereas the cultured human skin cells showed high viability with 5 microg/L of MELs. Interestingly, the papilla cells were dramatically activated with 0.001 microg/L of MEL-A produced from soybean oil: the cell viability reached at 150% compared with that of control cells. Consequently, the present MEL-A produced from soybean oil should have a potential as a new hair growth agent stimulating the papilla cells.

Characterization of an extracellular glycolipid from Lentinus edodes (Berk.) Sing [Lentinula edodes (Berk.) Pegler

Submerged mycelium of a xylotrophic basidiomycete Lentinus edodes produces an extracellular glycolipid, S3, associated with a lectin. Galactose glycan residue, as well as the lipid pool composition, which includes nonhydroxylated short-chain fatty acids, is uncommon for basidiomycetes. The glycolipid consists of D-galactopyranose (15% of S3 contains galactose sulfate) acylated by octadecanoic and nonadecanoic fatty acid residues (28 and 72%, respectively). The glycolipid structure and composition are confirmed by physicochemical analysis. The glycolipid is assumed to be a regulator of lectin activity.

Production of glycolipid biosurfactants, mannosylerythritol lipids, by Pseudozyma siamensis CBS 9960 and their interfacial properties

The search for a novel producer of glycolipid biosurfactants, mannosylerythritol lipids (MELs), was undertaken on the basis of the analysis of ribosomal DNA sequences of yeast strains of the genus Pseudozyma. In the course of the investigation, Pseudozyma siamensis CBS 9960, which is closely related to Pseudozyma shanxiensis, a known MEL-C producer but with a different morphology, was found to accumulate a large amount of glycolipids. On thin layer chromatography, the extracellular glycolipids showed nearly the same spots as those of the MELs produced by P. shanxiensis. However, the result of high-performance liquid chromatography analysis revealed that the present strain has a much higher glycolipid production yield than P. shanxiensis. From the structural characterization by (1)H and (13)C NMR, the major glycolipid (more than 84% of the total) was identified as a mixture of 4-O-[(2',4'-di-O-acetyl-3'-O-alka(e)noyl)-beta-D-mannopyranosyl]-D-erythritol and 4-O-[(4'-O-acetyl-3'-O-alka(e)noyl-2'-O-butanoyl)-beta-D-mannopyranosyl]-D-erythritol, both of which are types of MEL-C. The present MEL-C possessed a short-chain acid (C(2) or C(4)) at the C-2' position and a long-chain acid (C(16)) at the C-3' position of the mannose moiety, and thus, the hydrophobic part was considerably different from that of conventional MELs, which mainly possess two medium-chain acids (C(10)) at the C-2' and C-3' positions. Under optimal growth conditions with safflower oil in a shake culture, the total amount of MELs reached approximately 19 g/l after 9 d at 25 degrees C. We further investigated the interfacial properties of the present MEL-C, considering its unique hydrophobic structure. The observed critical micelle concentration (CMC) and the surface tension at the CMC of the MEL were 4.5 x 10(-6) M and 30.7 mN/m, respectively. In addition, on a water penetration scan, the MEL efficiently formed the liquid crystal phases such as hexagonal (H) and lamella (L(a)) at a wide range of concentrations. These results demonstrated that the newly identified MEL-C produced by P. siamensis exhibits not only high surface activity but also excellent self-assembling properties, and should facilitate the development of promising yeast biosurfactants.

糖脂質バイオ活性剤のマンノシルエリトリトール脂質の産生者としてのＰｓｅｕｄｏｚｙｍａ ｇｒａｍｉｎｉｃｏｌａＣＢＳ１００９２の同定

A basidiomycetous yeast, Pseudozyma graminicola CBS 10092, was found to accumulate a large amount of glycolipids in the cultured medium when grown on soybean oil as the sole carbon source. Based on thin layer chromatography, the extracellular glycolipids gave spots corresponding to those of mannosylerythritol lipids (MELs), which are highly functional and promising biosurfactants. From the structural characterization by 1H and 13C NMR, the main product was identified as 4-O-[(4'-mono-O-acetyl-2', 3'-di-O-alka(e)noyl)-beta-D-mannopyranosyl]-D-erythritol, which is a highly hydrophilic derivative of MELs known as MEL-C. According to high-performance liquid chromatography analysis, the main product, MEL-C, comprised approximately 85% of all the MELs, and the total amount reached approximately 10 g/L for 7 days. The fatty acids of the present MEL-C consisted of mainly C6, C8 and C14 acids, considerably different from those of MEL-C produced by other Pseudozyma strains such as P. antarctica and P. shanxiensis. The observed critical micelle concentration (CMC) and the surface-tension at CMC of the MEL-C were 4.0 x 10(-6) M and 24.2 mN/m, respectively, while those of MEL-A, the most intensively studied MEL, were 2.7 x 10(-6) M and 28.4 mN/m, respectively. This implied that the MEL-C has higher hydrophilicity than conventional MELs hitherto reported. In addition, on a water-penetration scan, the MEL-C efficiently formed the lamella phase (Lalpha) at a wide range of concentrations, indicating its excellent self-assembling properties. From these results, the newly identified MELs produced by P. graminicola are likely to have great potential for use in oil-in-water type emulsifiers and/or washing detergents, and would thus facilitate a broad range of applications for the promising yeast biosurfactants.

Characterization of new glycolipid biosurfactants, tri-acylated mannosylerythritol lipids, produced by Pseudozyma yeasts

Mannosylerythritol lipids (MELs) are glycolipid biosurfactants produced by Pseudozyma yeasts. They show not only the excellent interfacial properties but also versatile biochemical actions. In the course of MEL production from soybean oil by P. antarctica and P. rugulosa, some new extracellular glycolipids (more hydrophobic than the previously reported di-acylated MELs) were found in the culture medium. The most hydrophobic one was identified as 1-O-alka(e)noyl-4-O-[(4',6'-di-O-acetyl-2',3'-di-O-alka(e)noyl)-beta-D-mannopyranosyl]-D-erythritol, namely tri-acylated MEL. Others were tri-acylated MELs bearing only one acetyl group. The tri-acylated MEL could be prepared by the lipase-catalyzed esterification of a di-acylated MEL with oleic acid implying that the new glycolipids are synthesized from di-acylated MELs in the culture medium containing the residual fatty acids.

Characterization of New Types of Mannosylerythritol Lipids as Biosurfactants Produced from Soybean Oil by a Basidiomycetous Yeast, Pseudozyma shanxiensis

Mannosylerythritol lipids (MELs) are glycolipid biosurfactants produced by the yeast strains of the genus Pseudozyma. These show not only the excellent surface-active properties but also versatile biochemical actions. In course of MEL production from soybean oil by P. shanxiensis, new extracellular glycolipids (more hydrophilic than the previously reported MELs) were found in the culture medium. As a result of the structural characterization, the glycolipids were identified as a mixture of 4-O-[(2', 4'-di-O-acetyl-3'-O-alka(e)noyl)-beta-D-mannopyranosyl]-D-erythritol and 4-O-[(4'-O-acetyl-3'-O-alka(e)noyl-2'-O-butanoyl)-beta-D-mannopyranosyl]-D-erythritol. Interestingly, the new MELs possessed a much shorter chain (C(2) or C(4)) at the C-2' position of the mannose moiety compared to the MELs hitherto reported, which mainly possess a medium-chain acid (C(10)) at the position. They would thus show higher hydrophilicity and/or water-solubility, and expand the development of the environmentally advanced yeast biosurfactants.

Amino Acid Conjugated Sophorolipids: A New Family of Biologically Active Functionalized Glycolipids

Sophorolipids (SLs) are extra cellular glycolipids produced by Candida bombicola ATCC 22214 when grown in the presence of glucose and fatty acids. These compounds have a disaccharide head group connected to a long-chain hydroxyl-fatty acid by a glycosidic bond. To explore structure-activity of modified SLs, a new family of amino acid-SL derivatives was prepared. Synthesized analogs consist of amino acids linked by amide bonds formed between their alpha-amino moiety and the carboxyl group of ring-opened SL fatty acids. Their preparation involved the following: (i) hydrolysis of a natural SL mixture with aqueous alkali to give SL free acids, (ii) coupling of free acids to protected amino acids using dicarbodiimide, and (iii) removing amino acid carboxyl protecting groups. These conjugates were evaluated for their antibacterial, anti-HIV, and spermicidal activity. All tested analogs showed antibacterial activity against both gram +ve and gram -ve organisms. Leucine-conjugated SL was most efficient. For example, the minimum inhibitory concentrations (MIC) for Moraxella sp. and E. coli were 0.83 and 1.67 mg/mL, respectively. Among the alkyl esters of amino acid conjugated SLs, the ethyl ester of leucine-SLs was most active. Against Moraxella sp., S. sanguinis, and M. imperiale, MIC values are 7.62 x 10(-4), 2.28 x 10-(3) and 1.67 mg/mL, respectively. All compounds displayed virus-inactivating activity with 50% effective concentrations (EC50) below 200 microg/mL. The EC50 of leucine-SL ethyl ester was 24.1 microg/mL, showing that it is more potent than commercial spermicide nonoxynol-9 (EC50 approximately 65 microg/mL).

Suppression of Cell Growth and Induction of Apoptosis of HTLV-I-Infected T-Cell Lines and Primary ATL Cells by Galectin-9.

Abstract Adult T-cell leukemia (ATL) is a fatal malignancy of T lymphocytes caused by infection with human T-cell leukemia virus type I (HTLV-I), and remains incurable. Therefore, novel treatments are urgently needed. Galectins are a family of animal lectins with diverse biological activities. They function both extracellularly, by interacting with cell-surface and extracellular matrix glycoproteins and glycolipids, and intracellularly, by interacting with cytoplasmic and nuclear proteins to modulate signaling pathways. The distribution of galectins is quite diverse, and their expression in various leukocytes has been observed. To determine whether expression of galectins in T cells correlates with HTLV-I infection, we surveyed a number of uninfected and HTLV-I-infected T-cell lines for galectin-3, -8, and -9 expression by RT-PCR. Expression of galectin-8 did not correlate with HTLV-I infection. Galectin-3 was abundantly expressed in HTLV-I-infected T-cell lines and primary ATL cells, but not in uninfected T-cell lines. In contrast, galectin-9 was abundantly expressed in uninfected T-cell lines and normal PBMCs, but not in HTLV-I-infected T-cell lines and primary ATL cells. HTLV-I transformed protein, Tax, did not affect the expression of galectin-3 and -9. It was previously shown that galectin-8 and -9 are proapoptotic proteins. We found that galectin-9 prevented cell growth of HTLV-I-infected T-cell lines and primary ATL cells compared with galectin-8. Galectin-9 induced cell cycle arrest by reducing the expression of cyclin D1, cyclin D2, cyclin B1, Cdk1, Cdk4, Cdk6, and Cdc25C, and apoptosis by reducing the expression of XIAP and c-IAP2. Most of these genes are known to be regulated by NF-κB, which plays a critical role in oncogenesis by HTLV-I. Galectin-9 suppressed phosphorylation of IκBα. Most importantly, treatment with galectin-9 reduced tumor formation from an HTLV-I-infected T-cell line, HUT-102, when these cells were inoculated s.c. into severe combined immunodeficient mice. Our results suggest that galectin-9 may be a new approach for management of ATL.

Genetic analysis of biosurfactant production in Ustilago maydis.

The dimorphic basidiomycete Ustilago maydis produces large amounts of surface-active compounds under conditions of nitrogen starvation. These biosurfactants consist of derivatives of two classes of amphipathic glycolipids. Ustilagic acids are cellobiose lipids in which the disaccharide is O-glycosidically linked to 15,16-dihydroxyhexadecanoic acid. Ustilipids are mannosylerythritol lipids derived from acylated beta-d-mannopyranosyl-d-erythritol. Whereas the chemical structure of these biosurfactants has been determined, the genetic basis for their biosynthesis and regulation is largely unknown. Here we report the first identification of two genes, emt1 and cyp1, that are essential for the production of fungal extracellular glycolipids. emt1 is required for mannosylerythritol lipid production and codes for a protein with similarity to prokaryotic glycosyltransferases involved in the biosynthesis of macrolide antibiotics. We suggest that Emt1 catalyzes the synthesis of mannosyl-d-erythritol by transfer of GDP-mannose. Deletion of the gene cyp1 resulted in complete loss of ustilagic acid production. Cyp1 encodes a cytochrome P450 monooxygenase which is highly related to a family of plant fatty acid hydroxylases. Therefore we assume that Cyp1 is directly involved in the biosynthesis of the unusual 15,16-dihydroxyhexadecanoic acid. We could show that mannosylerythritol lipid production is responsible for hemolytic activity on blood agar, whereas ustilagic acid secretion is required for long-range pheromone recognition. The mutants described here allow for the first time a genetic analysis of glycolipid production in fungi.

The fungicidal activity of an extracellular glycolipid from Sympodiomycopsis paphiopedili sugiyama et al.

The yeast Sympodiomycopsis paphiopedili (Ustilaginomycetes) produces an extracellular glycolipid, which possesses the maximum antifungal activity at the pH of the medium equal to 4.0-4.5. Among the approximately 300 tested species of yeastlike and mycelial fungi, more than 80% (including species pathogenic for plants, animals, and humans) were found to be susceptible to this glycolipid.

LC/MS analysis and lipase modification of the sophorolipids produced by Rhodotorula bogoriensis.

The extracellular glycolipids produced by the yeast, Rhodotorula bogoriensis (formerly Candida bogoriensis), were analyzed using an LC/API-MS method. The analysis confirmed that the predominant form the sophorolipid structure contained a C22 hydroxy carboxylic acid. A minor amount (<10%) of a C24 hydroxy carboxylic acid in the sophorolipid was also found, which had not been reported previously. The sophorolipid product, which contained varying degrees of acetylation at the primary hydroxy groups of the sophorose sugar, was deacetylated with sodium methoxide. The des-acetylated sophorolipid was esterified using an immobilized lipase as catalyst in tetrahydrofuran and the product analyzed by mass spectrometric techniques. The product was screened for dimer or polymer formation but only a monomeric lactonized sophorolipid structure was detected.

RhlA is required for the production of a novel biosurfactant promoting swarming motility in Pseudomonas aeruginosa: 3-(3-hydroxyalkanoyloxy)alkanoic acids (HAAs), the precursors of rhamnolipids.

Pseudomonas aeruginosa produces extracellular glycolipids composed of L-rhamnose and 3-hydroxyalkanoic acid called rhamnolipids. Although these compounds are usually regarded as biosurfactants or haemolysins, their exact physiological function is not well understood. Rhamnolipids are synthesized by a rhamnosyltransferase, encoded by the rhlAB operon, which catalyses the transfer of TDP-L-rhamnose to 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA) moieties of various lengths. RhlB is the catalytic protein of the rhamnosyltransferase. rhlA is indispensable for rhamnolipid synthesis, but its function is unknown. Using a liquid chromatography/mass spectrometry method, the production of extracellular HAAs by P. aeruginosa was detected previously and it was demonstrated that they are the actual precursors of rhamnolipid biosynthesis. In this report, evidence is presented indicating that rhlA is required for production of HAAs and that these HAAs display potent surface-active properties. P. aeruginosa can colonize surfaces by swarming motility, a form of organized translocation requiring the production of wetting agents. Using rhlA and rhlB mutants it was observed that swarming requires the expression of the rhlA gene but does not necessitate rhamnolipid production, as HAAs act as surfactants. Finally, it was shown that the use of ammonium instead of nitrate as source of nitrogen and an excess of available iron both decrease rhlA expression and swarming motility.

ATP leakage from yeast cells treated by extracellular glycolipids of Pseudozyma fusiformata.

The ustilaginaceous yeast Pseudozyma fusiformata secreted glycolipids which were lethal to many yeasts and fungi more active at pH of about 4.0, and in the temperature range of 20-30 degrees C. Purified glycolipids enhanced non-specific permeability of the cytoplasmic membrane in sensitive cells, which resulted in ATP leakage and susceptibility of the cells to staining with bromocresol purple. Cells of Saccharomyces cerevisiae lost the ability to acidify the medium. Basidiomycetous yeasts were more sensitive to the glycolipids than ascomycetous ones. The minimal effective glycolipid concentration was 0.13 and 0.26 mg ml(-1) for Cryptococcus terreus and Filobasidiella neoformans, while for Candida albicans and Saccharomyces cerevisiae it was 1.0 and 1.6 mg ml(-1).

Protein Kinase Cα Plays a Critical Role in Mannosylerythritol Lipid-induced Differentiation of Melanoma B16 Cells

Mannosylerythritol lipid (MEL), a novel extracellular glycolipid from yeast, was found to inhibit the proliferation of mouse melanoma B16 cells in a dose-dependent manner and to induce the apoptosis of B16 cells at concentrations higher than 10 microm (Zhao, X., Wakamatsu, Y., Shibahara, M., Nomura, N., Geltinger, C., Nakahara, T., Murata, T., and Yokoyama, K. K. (1999) Cancer Res. 59, 482-486). We show here that exposure of B16 cells to MEL (5 microm) for 2 days resulted in an increase of the levels of differentiation-associated markers of melanoma cells such as melanogenesis and tyrosinase activity, which were accompanied by morphological changes. The MEL-induced differentiation of B16 cells at this concentration was closely associated with arrest of the cell cycle at G(1) phase, but no significant population of apoptotic cells was identified. Expression of protein kinase Calpha (PKCalpha) was enhanced after exposure of B16 cells to MEL for 48 h. Antisense oligodeoxynucleotides against the mouse gene for PKCalpha prevented MEL-induced melanogenesis in B16 cells. Conversely, the effects of the expression of a constitutively active form of PKCalpha mimicked the effects of MEL on B16 cells. These data suggest that MEL, a yeast-derived glycolipid, triggers the differentiation of B16 melanoma cells through a signaling pathway that involves PKCalpha.

The Glycolipid Defense

Host cells can recognize the protein flagellin, which is a common component of bacterial flagella, and this contributes to host defense against infection. Asialoganglioside M1 (ASGM1) is a glycolipid present on the surface of epithelial cells that binds flagellin and initiates host-cell defense responses, such as cytokine and mucin production. McNamara et al. show that agents that bind ASGM1 (flagellin or an antibody against ASGM1) stimulate an increase in extracellular adenosine triphosphate (ATP), which activates purinergic PY2 receptors, leading to increased mucin expression. Increases in mucin expression were measured by a reporter gene assay, and treatment of the cells with specific inhibitors of PY2 receptors that blocked mucin expression confirmed PY2 receptor involvement. Increased mucin expression in response to ASGM1 binding also required activation of phospholipase C and an increase in intracellular [Ca 2+ ], which is consistent with the activation of G protein-coupled PY2 receptors. ASGM1 binding led to increased phosphorylation of extracellular signal-regulated kinases 1 and 2, but increased mucin expression was only partially blocked by inhibition of the upstream kinases MEK1 and MEK2, suggesting that at least two pathways downstream of the calcium transient for increased mucin expression. Whereas the intracellular signaling pathways are becoming well delineated for host responses to flagellin exposure, the mechanism by which binding of an extracellular glycolipid can trigger ATP release remains unknown. N. McNamara, A. Khong, D. McKemy, M. Caterina, J. Boyer, D. Julius, C. Basbaum, ATP transduces signals from ASGM1, a glycolipid that functions as a bacterial receptor. Proc. Natl. Acad. Sci. U.S.A. 98 , 9086-9091 (2001). [Abstract] [Full Text]

Mannosylerythritol lipid induces characteristics of neuronal differentiation in PC12 cells through an ERK-related signal cascade.

Rat pheochromocytoma PC12 cells undergo neuronal differentiation in response to nerve growth factor (NGF). The differentiation involves protein kinase cascades that include the kinases MEK and ERK, as well as activation of the transcription factors c-Jun and c-Fos. We show here, that exposure of PC12 cells to mannosylerythritol lipid (MEL), a yeast extracellular glycolipid, enhances the activity of acetylcholinesterase and interrupts the cell cycle at the G1 phase, with resulting outgrowth of neurites and partial cellular differentiation. Treatment with MEL stimulates the phosphorylation of ERK to a similar extent as treatment with NGF, although, the appearance of phosphorylated ERK is somewhat delayed. Both the MEL-induced outgrowth of neurites and the increase in the activity of acetylcholinesterase are prevented by PD98059, a specific inhibitor of MEK. Northern blotting analysis of c-jun transcripts and analysis of transcription in PC12 cells of a c-jun/CAT reporter construct demonstrated a significant increase in the rate of transcription of the c-jun gene upon treatment with MEL. The sequence elements required for the MEL-mediated activation of transcription of the c-jun gene are located between nucleotides -126 and -79 in the 5' flanking region. Our results suggest that MEL induces characteristics of neuronal differentiation in PC12 cells, with transactivation of the c-jun gene, via an ERK-related signal cascade that is partially overlapping the pathways activated in response to NGF. These results might provide the groundwork for the use of microbial extracellular glycolipids as novel reagents for the treatment of cancer cells.

Mannosylerythritol lipid, a yeast extracellular glycolipid, shows high binding affinity towards human immunoglobulin G

BackgroundThere have been many attempts to develop new materials with stability and high affinity towards immunoglobulins. Some of glycolipids such as gangliosides exhibit a high affinity toward immunoglobulins. However, it is considerably difficult to develop these glycolipids into the practical separation ligand due to their limited amounts. We thus focused our attention on the feasible use of "mannosylerythritol lipid A", a yeast glycolipid biosurfactant, as an alternative ligand for immunoglobulins, and undertook the investigation on the binding between mannosylerythritol lipid A (MEL-A) and human immunoglobulin G (HIgG).ResultsIn ELISA assay, MEL-A showed nearly the same binding affinity towards HIgG as that of bovine ganglioside GM1. Fab of human IgG was considered to play a more important role than Fc in the binding of HIgG by MEL-A. The bound amount of HIgG increased depending on the attached amount of MEL-A onto poly (2-hydroxyethyl methacrylate) (polyHEMA) beads, whereas the amount of human serum albumin slightly decreased. Binding-amount and -selectivity of HIgG towards MEL-A were influenced by salt species, salt concentration and pH in the buffer solution. The composite of MEL-A and polyHEMA, exhibited a significant binding constant of 1.43 × 106 (M-1) for HIgG, which is approximately 4-fold greater than that of protein A reported.ConclusionsMEL-A shows high binding-affinity towards HIgG, and this is considered to be due to "multivalent effect" based on the binding molar ratio. This is the first report on the binding of a natural human antibody towards a yeast glycolipid.