Subclade of flavin-monooxygenases involved in aliphatic glucosinolate biosynthesis

Glucosinolates (GSLs) are amino acid-derived secondary metabolites with diverse biological activities dependent on chemical modifications of the side chain. We previously identified the flavin-monooxygenase FMO(GS-OX1) as an enzyme in the biosynthesis of aliphatic GSLs in Arabidopsis (Arabidopsis thaliana) that catalyzes the S-oxygenation of methylthioalkyl to methylsulfinylalkyl GSLs. Here, we report the fine mapping of a quantitative trait locus for the S-oxygenating activity in Arabidopsis. In this region, there are three FMOs that, together with FMO(GS-OX1) and a fifth FMO, form what appears to be a crucifer-specific subclade. We report the identification of these four uncharacterized FMOs, designated FMO(GS-OX2) to FMO(GS-OX5). Biochemical characterization of the recombinant protein combined with the analysis of GSL content in knockout mutants and overexpression lines show that FMO(GS-OX2), FMO(GS-OX3), and FMO(GS-OX4) have broad substrate specificity and catalyze the conversion from methylthioalkyl GSL to the corresponding methylsulfinylalkyl GSL independent of chain length. In contrast, FMO(GS-OX5) shows substrate specificity toward the long-chain 8-methylthiooctyl GSL. Identification of the FMO(GS-OX) subclade will generate better understanding of the evolution of biosynthetic activities and specificities in secondary metabolism and provides an important tool for breeding plants with improved cancer prevention characteristics as provided by the methylsulfinylalkyl GSL.     

Cellular and subcellular localization of flavin-monooxygenases involved in glucosinolate biosynthesis

Glucosinolates are amino acid-derived secondary metabolites with diverse biological activities dependent on chemical modifications of the side chain. Five flavin-monooxygenases FMO(GS-OX1-5) have recently been identified as aliphatic glucosinolate side chain modification enzymes in Arabidopsis thaliana that catalyse the generation of methylsulphinylalkyl glucosinolates, which can be hydrolysed to products with distinctive benefits for human health and plant defence. Though the localization of most aliphatic glucosinolate biosynthetic enzymes has been determined, little is known about where the side chain modifications take place despite their importance. Hence, the spatial expression pattern of FMO(GS-OX1-5) genes in Arabidopsis was investigated by expressing green fluorescent protein (GFP) and β-glucuronidase (GUS) fusion genes controlled by FMO(GS-OX1-5) promoters. The cellular compartmentation of FMO(GS-OX1) was also detected by transiently expressing a FMO(GS-OX1)-yellow fluorescent protein (YFP) fusion protein in tobacco leaves. The results showed that FMO(GS-OX1-5) were expressed basically in vascular tissues, especially in phloem cells, like other glucosinolate biosynthetic genes. They were also found in endodermis-like cells in flower stalk and epidermal cells in leaf, which is a location that has not been reported for other glucosinolate biosynthetic genes. It is suggested that the spatial expression pattern of FMO(GS-OX1-5) determines the access of enzymes to their substrate and therefore affects the glucosinolate profile. FMO(GS-OX1)-YFP fusion protein analysis identified FMO(GS-OX1) as a cytosolic protein. Together with the subcellular locations of the other biosynthetic enzymes, an integrated map of the multicompartmentalized aliphatic glucosinolate biosynthetic pathway is discussed.     

拟南芥PMSR2对脂肪族芥子油苷侧链的修饰作用

芥子油苷是一类由氨基酸合成的次生代谢产物,脂肪族芥子油苷主要来源于甲硫氨酸,因侧链长度和结构的不同而拥有多样化的生物活性。根据拟南芥不同组织中芥子油苷组分和含量的特点及生物信息学分析,我们推断脂肪族芥子油苷的侧链修饰反应中可能存在由甲基亚磺酰基芥子油苷向甲硫基芥子油苷转化的还原反应,候选基因为甲硫氨酸硫还原酶2(Peptide Methionine Sulfoxide Reductase 2,PMSR2)。为了验证这一假设,我们构建了过量表达PMSR2基因的转基因拟南芥,对其芥子油苷组分及含量进行了测定,并与野生型和PMSR2基因缺失的突变体进行了对比分析,结果表明,PMSR2基因的过量表达并未使芥子油苷含量与组分发生明显变化,但PMSR2基因缺失的突变体与野生型相比,MS GSL/MT GSL的值显著提高,证明PMSR2参与了脂肪族芥子油苷侧链的修饰反应,可以将MS GSL中的硫还原生成MT GSL。该酶的鉴定进一步完善了对芥子油苷合成途径及其侧链修饰的认识,为深入研究脂肪族芥子油苷的生理功能奠定了理论基础。     

脂肪族芥子油苷侧链修饰酶基因FMOGS-OX4表达模式分析

芥子油苷是一类由氨基酸衍生而来的、在植物抗生物胁迫防御性反应中起重要作用的次生代谢产物,其生物活性与侧链结构密切相关。拟南芥中有5个黄素单氧化酶FMO_GS-OX1-5具有催化芥子油苷侧链上硫原子氧化的活性,使甲基硫烷芥子油苷转变为甲基亚磺酰烷芥子油苷。前期研究工作表明,在5个FMO_GS-OX基因缺失突变体中,除了fmo_gs-ox4外均表现出芥子油苷侧链结构变化的表型。为了深入揭示FMO_GS-OX4的表达特性和它对芥子油苷侧链的修饰作用,利用GFP和GUS为报告基因,系统地分析了FMO_GS-OX4在不同组织中的表达情况。结果表明FMO_GS-OX4主要在花梗、叶片及角果的维管组织中表达,在正常生长条件下,FMO_GS-OX4表达的空间位置与芥子油苷的分布不重叠,因而,酶与底物的分离可能是fmo_gs-ox4没有明显表型的主要原因。     

脂肪族芥子油苷侧链修饰酶基因FMO_（GS-OX4）表达模式分析

芥子油苷是一类由氨基酸衍生而来的、在植物抗生物胁迫防御性反应中起重要作用的次生代谢产物,其生物活性与侧链结构密切相关。拟南芥中有5个黄素单氧化酶FMOGS-OX1-5具有催化芥子油苷侧链上硫原子氧化的活性,使甲基硫烷芥子油苷转变为甲基亚磺酰烷芥子油苷。前期研究工作表明,在5个FMOGS-OX基因缺失突变体中,除了fmogs-ox4外均表现出芥子油苷侧链结构变化的表型。为了深入揭示FMOGS-OX4的表达特性和它对芥子油苷侧链的修饰作用,利用GFP和GUS为报告基因,系统地分析了FMOGS-OX4在不同组织中的表达情况。结果表明FMOGS-OX4主要在花梗、叶片及角果的维管组织中表达,在正常生长条件下,FMOGS-OX4表达的空间位置与芥子油苷的分布不重叠,因而,酶与底物的分离可能是fmogs-ox4没有明显表型的主要原因。     

Glucosinolate and amino acid biosynthesis in Arabidopsis

Enzymes that catalyze the condensation of acetyl coenzyme A and 2-oxo acids are likely to be important in two distinct metabolic pathways in Arabidopsis. These are the synthesis of isopropylmalate, an intermediate of Leu biosynthesis in primary metabolism, and the synthesis of methylthioalkylmalates, intermediates of Met elongation in the synthesis of aliphatic glucosinolates (GSLs), in secondary metabolism. Four Arabidopsis genes in the ecotype Columbia potentially encode proteins that could catalyze these reactions. MAM1 and MAML are adjacent genes on chromosome 5 at the Gsl-elong locus, while MAML-3 and MAML-4 are at opposite ends of chr 1. The isopropylmalate synthase activity of each member of the MAM-like gene family was investigated by heterologous expression in an isopropylmalate synthase-null Escherichia coli mutant. Only the expression of MAML-3 restored the ability of the mutant to grow in the absence of Leu. A MAML knockout line (KO) lacked long-chain aliphatic GSLs, which were restored when the KO was transformed with a functional MAML gene. Variation in expression of MAML did not alter the total levels of Met-derived GSLs, but just the ratio of chain lengths. MAML overexpression in Columbia led to an increase in long-chain GSLs, and an increase in 3C GSLs. Moreover, plants overexpressing MAML contained at least two novel amino acids. One of these was positively identified via MS/MS as homo-Leu, while the other, with identical mass and fragmentation patterns, was likely to be homo-Ile. A MAML-4 KO did not exhibit any changes in GSL profile, but had perturbed soluble amino acid content.     

Novel bioresources for studies of Brassica oleracea: identification of a kale MYB transcription factor responsible for glucosinolate production

Plants belonging to the Brassicaceae family exhibit species‐specific profiles of glucosinolates (GSLs), a class of defence compounds against pathogens and insects. GSLs also exhibit various human health–promoting properties. Among them, glucoraphanin (aliphatic 4‐methylsulphinylbutyl GSL) has attracted the most attention because it hydrolyses to form a potent anticancer compound. Increased interest in developing commercial varieties of Brassicaceae crops with desirable GSL profiles has led to attempts to identify genes that are potentially valuable for controlling GSL biosynthesis. However, little attention has been focused on genes of kale (Brassica oleracea var. acephala). In this study, we established full‐length kale cDNA libraries containing 59 904 clones, which were used to generate an expressed sequence tag (EST) data set with 119 204 entries. The EST data set clarified genes related to the GSL biosynthesis pathway in kale. We specifically focused on BoMYB29, a homolog of Arabidopsis MYB29/PMG2/HAG3, not only to characterize its function but also to demonstrate its usability as a biological resource. BoMYB29 overexpression in wild‐type Arabidopsis enhanced the expression of aliphatic GSL biosynthetic genes and the accumulation of aliphatic GSLs. When expressed in the myb28myb29 mutant, which exhibited no detectable aliphatic GSLs, BoMYB29 restored the expression of biosynthetic genes and aliphatic GSL accumulation. Interestingly, the ratio of methylsulphinyl GSL content, including glucoraphanin, to that of methylthio GSLs was greatly increased, indicating the suitability of BoMYB29 as a regulator for increasing methylsulphinyl GSL content. Our results indicate that these biological resources can facilitate further identification of genes useful for modifications of GSL profiles and accumulation in kale.     

VIP21/caveolin, glycosphingolipid clusters and the sorting of glycosylphosphatidylinositol-anchored proteins in epithelial cells.

We studied the role of the association between glycosylphosphatidylinositol (GPI)-anchored proteins and glycosphingolipid (GSL) clusters in apical targeting using gD1-DAF, a GPI-anchored protein that is differentially sorted by three epithelial cell lines. Differently from MDCK cells, where both gD1-DAF and glucosylceramide (GlcCer) are sorted to the apical membrane, in MDCK Concanavalin A-resistant cells (MDCK-ConAr) gD1-DAF was mis-sorted to both surfaces, but GlcCer was still targeted to the apical surface. In both MDCK and MDCK-ConAr cells, gD1-DAF became associated with TX-100-insoluble GSL clusters during transport to the cell surface. In dramatic contrast with MDCK cells, the Fischer rat thyroid (FRT) cell line targeted both gD1-DAF and GlcCer basolaterally. The targeting differences for GSLs in FRT and MDCK cells cannot be accounted for by a differential ability to form clusters because, in spite of major differences in the GSL composition, both cell lines assembled GSLs into TX-100-insoluble complexes with identical isopycnic densities. Surprisingly, in FRT cells, gD1-DAF did not form clusters with GSLs and, therefore, remained completely soluble. This clustering defect in FRT cells correlated with the lack of expression of VIP21/caveolin, a protein localized to both the plasma membrane caveolae and the trans Golgi network. This suggests that VIP21/caveolin may have an important role in recruiting GPI-anchored proteins into GSL complexes necessary for their apical sorting. However, since MDCK-ConAr cells expressed caveolin and clustered GPI-anchored proteins normally, yet mis-sorted them, our results also indicate that clustering and caveolin are not sufficient for apical targeting, and that additional factors are required for the accurate apical sorting of GPI-anchored proteins.     

Arabidopsis glucan synthase-like 10 functions in male gametogenesis

Callose or beta-1,3-glucan performs multiple functions during male and female gametophyte development. Callose is synthesized by 12 members of the glucan synthase-like (GSL) gene family in Arabidopsis thaliana. To elucidate the biological roles of Arabidopsis GSL family members during sexual development, we initiated a reverse genetic approach with T-DNA insertional mutagenesis lines. We screened T-DNA insertion lines for all members of the GSL gene family and detected homozygous mutant seedlings for all members except GSL10. Three independent alleles in GSL10, gsl10-1, gsl10-3 and gsl10-4 showed distorted segregation (1:1:0) of T-DNA inserts rather than Mendelian segregation (1:2:1). By genetic analysis through reciprocal cross, we determined that gsl10 pollen could not be transmitted to descendent. The mutant pollen of GSL10/gsl10 plants at tetrad and microspore stages were not different from that of wild type, suggesting that GSL10 is not essential for normal microspore growth. Analysis of GSL10/gsl10 hemizygous pollen during development revealed abnormal function in asymmetric microspore division. gsl10 mutant microspores failed to enter into mitosis. Unlike the previously described functions of GSL1, GSL2 and GSL5, GSL10 involves an independent process of pollen development at the mitotic division stage.     

Glucosinolate structures in evolution

By 2000, around 106 natural glucosinolates (GSLs) were probably documented. In the past decade, 26 additional natural GSL structures have been elucidated and documented. Hence, the total number of documented GSLs from nature by 2011 can be estimated to around 132. A considerable number of additional suggested structures are concluded not to be sufficiently documented. In many cases, NMR spectroscopy would have provided the missing structural information. Of the GSLs documented in the past decade, several are of previously unexpected structures and occur at considerable levels. Most originate from just four species: Barbarea vulgaris, Arabidopsis thaliana, Eruca sativa and Isatis tinctoria. Acyl derivatives of known GSLs comprised 15 of the 26 newly documented structures, while the remaining exhibited new substitution patterns or chain length, or contained a mercapto group or related thio-functionality.GSL identification methods are reviewed, and the importance of using authentic references and structure-sensitive detection methods such as MS and NMR is stressed, especially when species with relatively unknown chemistry are analyzed. An example of qualitative GSL analysis is presented with experimental details (group separation and HPLC of both intact and desulfated GSLs, detection and structure determination by UV, MS, NMR and susceptibility to myrosinase) with emphasis on the use of NMR for structure elucidation of even minor GSLs and GSL hydrolysis products. The example includes identification of a novel GSL, (R)-2-hydroxy-2-(3-hydroxyphenyl)ethylglucosinolate.Recent investigations of GSL evolution, based on investigations of species with well established phylogeny, are reviewed. From the relatively few such investigations, it is already clear that GSL profiles are regularly subject to evolution. This result is compatible with natural selection for specific GSL side chains. The probable existence of structure-specific GSL catabolism in intact plants suggests that biochemical evolution of GSLs has more complex implications than the mere liberation of a different hydrolysis product upon tissue disruption.     

Alterations in Glycosphingolipid Patterns in a Line of African Green Monkey Kidney Cells Infected with Herpesvirus

The major glycosphingolipids (GSLs) of a line of African green monkey kidney cells (BGM) were characterized as glucosylceramide, lactosylceramide, galactosyl-galactosyl-glucosylceramide, and N-acetylgalactosaminyl-galactosyl-galactosyl-glucosylceramide. Neutral GSLs accounted for approximately 80% of the total GSLs isolated. The predominant gangliosides were N-acetylneuraminyl-galactosyl-glucosylceramide, N-acetylgalactosaminyl-N-acetylneuraminyl-galactosyl- glucosylceramide, and galactosyl-N-acetylgalactosaminyl-N-acetylneuraminyl -galactosyl-glucosylceramide. The incorporation of labeled galactose into GSLs was compared in mock-infected and herpes simplex virus type 1-infected BGM cells. Herpes simplex virus type 1 infection resulted in a three- to four-fold increase in galactose incorporation into glucosylceramide and a decrease in galactose incorporation into galactosyl-galactosyl-glucosylceramide and N-acetyl-galactosaminyl-galactosyl-galactosyl-glucosylceramide. The virus-induced alteration in the GSL labeling pattern occurred early in infection, before the release of infectious virus, and was not prevented by the presence of cytosine arabinoside. Treatment of uninfected BGM cells with cycloheximide resulted in alterations in the GSL pattern which were similar to those observed in herpes simplex virus type 1-infected cells. These observations suggest that an early virus function such as inhibition of host cell protein synthesis is responsible for the observed alterations of GSL metabolism. Experiments with a syncytium-producing strain of herpes simplex virus type 1, herpes simplex virus type 2, and pseudorabies virus indicated that other herpes viruses altered GSL metabolism in a manner similar to herpes simplex virus type 1.     

Reduction of plasma glycosphingolipid levels has no impact on atherosclerosis in apolipoprotein E-null mice

Glycosphingolipids (GSLs) have been implicated as potential atherogenic lipids. Studies in apolipoprotein E-null (apoE(-/-)) mice indicate that exacerbated tissue GSL accumulation resulting from alpha-galactosidase deficiency promotes atherosclerosis, whereas the serine palmitoyl transferase inhibitor myriocin (which reduces plasma and tissue levels of several sphingolipids, including sphingomyelin, ceramide, sphingosine-1-phosphate, and GSLs) inhibits atherosclerosis. It is not clear whether GSL synthesis inhibition per se has an impact on atherosclerosis. To address this issue, apoE(-/-) mice maintained on a high-fat diet were treated with a potent glucosylceramide synthesis inhibitor, d-threo-1-ethylendioxyphenyl-2-palmitoylamino-3-pyrrolidino-propanol (EtDO-P4), 10 mg/kg/day for 94 days, and lesion development was compared in mice that were treated with vehicle only. EtDO-P4 reduced plasma GSL concentration by approximately 50% but did not affect cholesterol or triglyceride levels. Assessment of atherosclerotic lesions at four different sites indicated that EtDO-P4 had no significant impact on lesion area. Thus, despite the previously observed positive correlations between plasma and aortic GSL concentrations and the development of atherosclerosis, and the in vitro evidence implying that GSLs may be pro-atherogenic, our current data indicate that inhibition of GSL synthesis does not inhibit atherosclerosis in vivo.     

Selective stimulation of caveolar endocytosis by glycosphingolipids and cholesterol

Internalization of some plasma membrane constituents, bacterial toxins, and viruses occurs via caveolae; however, the factors that regulate caveolar internalization are still unclear. Here, we demonstrate that a brief treatment of cultured cells with natural or synthetic glycosphingolipids (GSLs) or elevation of cholesterol (either by acute treatment with mbeta-cyclodextrin/cholesterol or by alteration of growth conditions) dramatically stimulates caveolar endocytosis with little or no effect on other endocytic mechanisms. These treatments also stimulated the movement of GFP-labeled vesicles in cells transfected with caveolin-1-GFP and reduced the number of surface-connected caveolae seen by electron microscopy. In contrast, overexpression of caveolin-1 decreased caveolar uptake, but treatment with GSLs reversed this effect and stimulated caveolar endocytosis. Stimulation of caveolar endocytosis did not occur using ceramide or phosphatidylcholine and was not due to GSL degradation because similar results were obtained using a nonhydrolyzable GSL analog. Stimulated caveolar endocytosis required src kinase and PKC-alpha activity as shown by i) use of pharmacological inhibitors, ii) expression of kinase inactive src or dominant negative PKCalpha, and iii) stimulation of src kinase activity upon addition of GSLs or cholesterol. These results suggest that caveolar endocytosis is regulated by a balance of caveolin-1, cholesterol, and GSLs at the plasma membrane.     

Characterization of methylsulfinylalkyl glucosinolate specific polyclonal antibodies

Antibodies towards small molecules, like plant specialized metabolites, are valuable tools for developing quantitative and qualitative analytical techniques. Glucosinolates are the specialized metabolites characteristic of the Brassicales order. Here we describe the characterization of polyclonal rabbit antibodies raised against the 4-methylsulfinylbutyl glucosinolate, glucoraphanin that is one of the major glucosinolates in the model plant Arabidopsis thaliana (hereafter Arabidopsis). Analysis of the cross-reactivity of the antibodies against a number of glucosinolates demonstrated that it was highly selective for methionine-derived aliphatic glucosinolates with a methyl-sulfinyl group in the side chain. Use of crude plant extracts from Arabidopsis mutants with different glucosinolate profiles showed that the antibodies recognized aliphatic glucosinolates in a plant extract and did not cross-react with other metabolites. These methylsulfinylalkyl glucosinolate specific antibodies have prospective use in multiple applications such as ELISA, co-immunoprecipitation and immunolocalization of glucosinolates.     

Characterization of neutral glycosphingolipids from porcine erythrocyte membranes

1. Six neutral GSL fractions were purified from porcine erythrocyte membranes. 2. They were identified to be LacCer (14% of total neutral GSLs), 2-hydroxy acid-rich and -poor Gb3Cer (3 and 7%, respectively) and Gb4Cer (71%) by means of NMR spectrometry. 3. Monohexosylceramides (5%) were composed of GlcCer and GalCer with near amount. 4. All these GSL classes contained a high concentration (more than 20% of total acids in each class) of 2-hydroxy fatty acids. 5. GalCer and GlcCer contained considerable amounts of C16- and C18-acids, and of C18-phytosphingosine, whereas C24-acids and C18-sphingosine were predominant in the other GSLs. 6. A minor GSL fraction (less than 1% of total neutral GSLs) which migrated more slowly than Gb5Cer on a thin layer plate and composed of several GSL components contained L-fucose.     

Oxidation and base-catalyzed elimination of the saccharide portion of GSLs having very different polarities

Glycosphingolipids (GSLs), present in cell membranes, participate in a variety of biological functions. Although their exact role(s) may not be understood, it has been shown that 1) embryos lacking glucosylceramide synthase activity do not develop normally, 2) GSLs can affect neuritogenesis, and 3) they can function as receptors for some pathogens. To study the role of the saccharide portion of a GSL in any of these functions, it is necessary to either isolate it from the intact GSL or synthesize it. Because syntheses are more complex, modifications were made to the oxidation/elimination procedure previously described for the isolation of the saccharide portion of GM1 and GD1a to enable it to be used with GSLs of varying polarity. The key is to use a mixture of GSLs that differ in polarity. This appears to eliminate problems encountered when purified GSLs such as sulfatide or GT1b are used.     

Sphingolipids, cholesterol, and HIV-1: A paradigm in viral fusion

Our previous studies show that the depletion of cholesterol or sphingolipids (raft-associated lipids) from receptor-bearing adherent cell lines blocks HIV-1 entry and HIV-1 Env-mediated membrane fusion. Here we have evaluated the mechanism(s) by which these lipids contribute to the HIV-1 Env-mediated membrane fusion. We report the following: (1) GSL depletion from a suspension T lymphocyte cell line (Sup-T1) reduced subsequent fusion with HIV-1IIIB-expressing cells by 70%. (2) Cholesterol depletion from NIH3T3 cells bearing HIV-1 receptors (NIH3T3CD4R5/NIH3T3CD4X4) did not impair subsequent fusion with HeLa cells expressing the corresponding HIV-1 Envs. In contrast GSL depletion from these targets reduced fusion by 50% suggesting that GSL facilitate fusion in different ways. (3) GSL-deficient GM95 cells bearing high receptors fused with HIV-1 Env-expressing cells at 37°C with kinetics similar to that of GSL + NIH3T3 targets. Based on these observations, we propose that the plasma membrane cholesterol is required to maintain the integrity of receptor pools whereas GSLs are involved in stabilizing the coupling of inter-receptor pools.     

Glycosphingolipid dynamics in human embryonic stem cell and cancer: their characterization and biomedical implications

Glycosphingolipids (GSLs) are composed of complex glycans linked to sphingosines and various fatty acid chains. Antibodies against several GSLs designated as stage-specific embryonic antigens (SSEAs), have been widely used to characterize differentiation of embryonic stem (ES) cells. In view of the cross-reactivities of these antibodies with multiple glycans, a few laboratories have employed advanced mass spectrometry (MS) technologies to define the dynamic changes of surface GSLs upon ES differentiation. However, the amphiphilic nature and heterogeneity of GSLs make them difficult to decipher. In our studies, systematic survey of GSL expression profiles in human ES cells and differentiated derivatives was conducted, primarily with matrix-assisted laser desorption/ionization MS (MALDI-MS) and MS/MS analyses. In addition to the well-known ES-specific markers, SSEA-3 and SSEA-4, several previously undisclosed globo- and lacto-series GSLs, including Gb4Cer, Lc4Cer, fucosyl Lc4Cer, Globo H, and disialyl Gb5Cer were identified in the undifferentiated human ES and induced pluripotent stem cells. Furthermore, during differentiation to embryoid body outgrowth, the core structures of GSLs switched from globo- and lacto- to ganglio-series. Lineage-specific differentiation was also marked by alterations of specific GSLs. During differentiation into neural progenitors, core structures shifted to primarily ganglio-series dominated by GD3. GSL patterns shifted to prominent expression of Gb4Cer with little SSEA-3 and-?4 or GD3 during endodermal differentiation. Several issues relevant to MS analysis and novel GSLs in ES cells were discussed. Finally, unique GSL signatures in ES and cancer cells are exploited in glycan-targeted anti-cancer immunotherapy and their mechanistic investigations were discussed using anti-GD2 mAb and Globo H as examples.     

Influence of in vitro culture conditions on glucosinolate composition of microspore-derived embryos of Brassica napus

Microspore-derived embryos (MDEs) of Brassica napus were used to study the influence of sucrose, jasmonic acid (JA), and abscisic acid (ABA) on dry weight and total glucosinolate (GSL) content as well as on specific GSLs. An improved procedure was developed to enable the detection of alkenyl and indole GSLs in single MDEs although they were cultured in medium containing 13% sucrose, where the accumulation of GSL is very low. A sucrose content of 2% and below in the culture medium of the embryos was necessary to significantly increase the total GSL content in embryos of three different rapeseed cultivars. The increase in total GSL content was caused predominantly by higher contents of the indole GSL glucobrassicin (GBC). Contents of 4-hydroxy-3-indolyl glucosinolate (4OH), neoglucobrassicin (NEO), and 4-methoxyglucobrassicin (4ME) were also increased. Alkenyl GSL content remained largely unaffected and increased significantly only in embryos cultured at the lowest tested sucrose concentration of 1%. Growing the embryos in the presence of JA did not change the alkenyl GSL content but led to a 7-fold increase in the indole GSL content. Significant increases were found for GBC, 4OH and NEO, whereas 4ME content was not affected. The JA treatment did not affect the morphology or dry weight of the MDEs. In contrast, a treatment with ABA significantly reduced the dry weight and the indole GSL content of the embryos. In the combined JA and ABA treatment, the stimulative effect of JA on indole GSL biosynthesis could not override the inhibitory effect of ABA on growth and indole GSL biosynthesis of the embryos.