Search

Studying the interaction between the hemibiotrophic bacterium Pseudomonas syringae pv. tomato DC3000 and Arabidopsis thaliana has shed light onto the various forms of mechanisms plants use to defend themselves against pathogen attack. While a lot of emphasis has been put on investigating changes in protein expression in infected plants, only little information is available on the effect infection plays on the plants N-glycan composition. To close this gap in knowledge, total N-glycans were enriched from P. syringae DC3000-infected and mock treated Arabidopsis seedlings and analyzed via MALDI-TOF–MS. Additionally, fluorescently labelled N-glycans were quantified via HPLC-FLD. N-glycans from infected plants were overall less processed and displayed increased amounts of oligomannosidic N-glycans. As multiple peaks for certain oligomannosidic glycoforms were detected upon separation via liquid chromatography, a porous graphitic carbon (PGC)-analysis was conducted to separate individual N-glycan isomers. Indeed, multiple different N-glycan isomers with masses of two N-acetylhexosamine residues plus 8, 9 or 10 hexoses were detected in the infected plants which were absent in the mock controls. Treatment with jack bean α-mannosidase resulted in incomplete removal of hexoses from these N-glycans, indicating the presence of glucose residues. This hints at the accumulation of misfolded glycoproteins in the infected plants, likely because of endoplasmic reticulum (ER) stress. In addition, poly-hexose structures susceptible to α-amylase treatment were found in the DC3000-infected plants, indicating alterations in starch metabolism due to the infection process.

The glycosylation abilities of snails deserve attention, because snail species serve as intermediate hosts in the developmental cycles of some human and cattle parasites. In analogy to many other host-pathogen relations, the glycosylation of snail proteins may likewise contribute to these host-parasite interactions. Here we present an overview on the O-glycan structures of 8 different snails (land and water snails, with or without shell): Arion lusitanicus, Achatina fulica, Biomphalaria glabrata, Cepaea hortensis, Clea helena, Helix pomatia, Limax maximus and Planorbarius corneus. The O-glycans were released from the purified snail proteins by β-elimination. Further analysis was carried out by liquid chromatography coupled to electrospray ionization mass spectrometry and – for the main structures – by gas chromatography/mass spectrometry. Snail O-glycans are built from the four monosaccharide constituents: N-acetylgalactosamine, galactose, mannose and fucose. An additional modification is a methylation of the hexoses. The common trisaccharide core structure was determined in Arion lusitanicus to be N-acetylgalactosamine linked to the protein elongated by two 4-O-methylated galactose residues. Further elongations by methylated and unmethylated galactose and mannose residues and/or fucose are present. The typical snail O-glycan structures are different to those so far described. Similar to snail N-glycan structures they display methylated hexose residues.

Neuropilins are involved in angiogenesis and neuronal development. The membrane proximal domain of neuropilin-1, called c or MAM domain based on its sequence conservation, has been implicated in neuropilin oligomerization required for its function. The c/MAM domain of human neuropilin-1 has been recombinantly expressed to allow for investigation of its propensity to engage in molecular interactions with other protein or carbohydrate components on a cell surface. We found that the c/MAM domain was heavily O-glycosylated with up to 24 monosaccharide units in the form of disialylated core 1 and core 2 O-glycans. Attachment sites were identified on the chymotryptic c/MAM peptide ETGATEKPTVIDSTIQSEFPTY by electron-transfer dissociation mass spectrometry (ETD-MS/MS). For highly glycosylated species consisting of carbohydrate to about 50 %, useful results could only be obtained upon partial desialylation. ETD-MS/MS revealed a hierarchical order of the initial O-GalNAc addition to the four different glycosylation sites. These findings enable future functional studies about the contribution of the described glycosylations in neuropilin-1 oligomerization and the binding to partner proteins as VEGF or galectin-1. As a spin-off result the sialidase from Clostridium perfringens turned out to discriminate between galactose- and N-acetylgalactosamine-linked sialic acid.

N-acetylglucosaminyltransferase II (GnTII, EC 2.4.1.143) is a Golgi enzyme involved in the biosynthesis of glycoprotein-bound N-linked oligosaccharides, catalysing an essential step in the conversion of oligomannose-type to complex N-glycans. GnTII activity has been detected in both animals and plants. However, while cDNAs encoding the enzyme have already been cloned from several mammalian sources no GnTII homologue has been cloned from plants so far. Here we report the molecular cloning of an Arabidopsis thalianaGnTII cDNA with striking homology to its animal counterparts. The predicted domain structure of A. thalianaGnTII indicates a type II transmembrane protein topology as it has been established for the mammalian variants of the enzyme. Upon expression of A. thalianaGnTII cDNA in the baculovirus/insect cell system, a recombinant protein was produced that exhibited GnTII activity.

Baculovirus-mediated expression in insect cells has become well-established for the production of recombinant glycoproteins. Its frequent use arises from the relative ease and speed with which a heterologous protein can be expressed on the laboratory scale and the high chance of obtaining a biologically active protein. In addition to Spodoptera frugiperda Sf9 cells, which are probably the most widely used insect cell line, other mainly lepidopteran cell lines are exploited for protein expression. Recombinant baculovirus is the usual vector for the expression of foreign genes but stable transfection of - especially dipteran - insect cells presents an interesting alternative. Insect cells can be grown on serum free media which is an advantage in terms of costs as well as of biosafety. For large scale culture, conditions have been developed which meet the special requirements of insect cells.

The N-glycans from soluble extracts of ten pollens were examined. The pyridylaminated oligosaccharides derived from these sources were subject to gel filtration and reverse-phase HPLC, in conjunction with exoglycosidase digests, and in some cases matrix-assisted laser desorption-ionisation mass spectrometry. In comparison to known structures, it was possible to determine the major structures of the N-glycans derived from Kentucky blue grass (Poa pratensis), rye (Secale cerale), ryegrass (Lolium perenne), short ragweed (Ambrosia elatior), giant ragweed (Ambrosia trifida), birch (Betula alba), hornbeam (Carpinus betulus), horse chestnut (Aesculus hippocastanum), olive (Olea europaea) and snake-skin pine (Pinus leucodermis) pollen extracts. For grass pollens the major glycans detected were identical in properties to: [structure in text] Grass pollens also contained some minor structures with one or two non-reducing terminal N-acetylglucosamine residues. In the ragweed pollens, the major structures carried core alpha1,3-linked fucose with or without the presence of xylose. In tree pollen extracts, the major structures were either xylosylated, with or without fucose and terminal N-acetylglucosamine residues, with also significant amounts of oligomannose structures. These results are compatible with the hypothesis that the carbohydrate structures are another potential source of immunological cross-reaction between different plant allergens.

The N-glycan moiety of ascorbic acid oxidase from zucchini (Cucurbita pepo) has been described to be a core-pentasaccharide with a xylose [D'Andrea et al. (1988) Glycoconjugate J 5:151-7]. Ascorbic acid oxidase is sometimes used to characterize antibodies directed against carbohydrate determinants on plant glycoproteins. To prevent misinterpretations of immunological data, the structure of the N-glycan of ascorbic acid oxidase has been reinvestigated. The oligosaccharides were released by almond N-glycosidase and analysed as their pyridylamino derivatives by 2D-HPLC and exoglycosidase digestions. The main structure resembled the typical complex plant N-glycan consisting of a core-pentasaccharide decorated with xylose and 3-linked fucose. The other abundant species lacked the fucose residue. Small amounts of these glycans carried a GlcNAc residue on the 6-arm. Therefore, ascorbic acid oxidase will not only react with antibodies directed against the xylosylated region but also with those binding to N-glycans with 3-linked fucose.

The binding to concanavalin A (Con A) by pyridylaminated oligosaccharides derived from bromelain (Man alpha 1,6(Xyl beta 1, 2) Man beta 1, 4GlcNAc beta 1, 4(Fuc alpha 1, 3)GlcNAc), horseradish peroxidase (Man alpha 1,6(Man alpha 1, 3) (Xyl beta 1, 2)Man beta 1, 4GlcNAc beta 1,4(Fuc alpha 1, 3) GlcNAc), bee venom phospholipase A2 (Man alpha 1,6Man beta 1,4GlcNAc beta 1,4GlcNAc and Man alpha 1,6(Man alpha 1, 3)Man beta 1,4GlcNAc beta 1, 4 (Fuc alpha 1, 3)GlcNAc) and zucchini ascorbate oxidase (Man alpha 1,6(Man alpha 1, 3) (Xyl beta 1, 2)Man beta 1, 4 GlcNAc beta 1, 4GlcNAc) was compared to the binding by Man3GlcNAc2, Man5GlcNAc2 and the asialo-triantennary complex oligosaccharide from bovine fetuin. While the fetuin oligosaccharide did not bind, bromelain, zucchini, Man2GlcNAc2 and horseradish peroxidase were retarded (in that order). The alpha 1, 3-fucosylated phospholipase, Man3GlcNAc2 and Man5GlcNAc2 structures were eluted with 15 M alpha -methylmannoside. It is concluded that core alpha 1,3-fucosylation has little or no effect on ConA binding while xylosylation decreases affinity for ConA.

Based on the previous demonstration of surface (S-) layer protein glycosylation in Lactobacillus buchneri 41021/251 and because of general advantages of lactic acid bacteria for applied research, protein glycosylation in this bacterial species was investigated in detail. The cell surface of L. buchneri CD034 is completely covered with an oblique 2D crystalline array (lattice parameters, a = 5.9 nm; b = 6.2 nm; γ ~ 77°) formed by self-assembly of the S-layer protein SlpB. Biochemical and mass spectrometric analyses revealed that SlpB is the most abundant protein and that it is O-glycosylated at four serine residues within the sequence S(152)-A-S(154)-S(155)-A-S(157) with, on average, seven Glc(α1-6) residues, each. Subcellular fractionation of strain CD034 indicated a sequential order of SlpB export and glucosylation as evidenced by lack of glucosylation of cytosolic SlpB. Protein glycosylation analysis was extended to strain L. buchneri NRRL B-30929 where an analogous glucosylation scenario could be detected, with the S-layer glycoprotein SlpN containing an O-glycosylation motif identical to that of SlpB. This corroborates previous data on S-layer protein glucosylation of strain 41021/251 and let us propose a species-wide S-layer protein O-glucosylation in L. buchneri targeted at the sequence motif S-A-S-S-A-S. Search of the L. buchneri genomes for the said glucosylation motif revealed one further ORF, encoding the putative glycosyl-hydrolase LbGH25B and LbGH25N in L. buchneri CD034 and NRRL B-30929, respectively, for which we have indications of a glycosylation comparable to that of the S-layer proteins. These findings demonstrate the presence of a distinct protein O-glucosylation system in Gram-positive and beneficial microbes.

Hyaluronidase from the venom of the honeybee (Apis mellifera) has been purified by gelpermeation and cation exchange chromatography. Its asparagine-linked carbohydrate chains were released from tryptic glycopeptides with N-glycosidase A and reductively aminated with 2-aminopyridine. Separation of the fluorescent derivatives by size-fractionation and reversed-phase HPLC afforded eighteen fractions which were analysed by two-dimensional HPLC mapping combined with exoglycosidase digestions. The bulk of the N-linked glycans of hyaluronidase consisted of small oligosaccharides (Man1-3GlcNAc2), most of which were either alpha 1,3-monofucosylated or alpha 1,3-(alpha 1,6-)difucosylated at the innermost GlcNAc residue. High-mannose type structures constituted the minor fractions, together making up about 5% of the oligosaccharide pool from hyaluronidase. Four fractions, making up 8% of the N-linked glycans, contained the terminal trisaccharide GalNAc beta 1-4[Fuc alpha 1-3]GlcNAc beta 1- in beta 1,2-linkage to the core alpha 1,3-mannosyl residue. No evidence for the presence of O-glycans or sialic acids could be found.

Chymotryptic glycopeptides were prepared from a honeybee (Apis mellifica) venom phospholipase A2 (E.C. 3.1.1.4) fraction, with high affinity towards lentil (Lens culinaris) lectin. Treatment of the glycopeptide mixture with peptide-N4-(N-acetyl-β-glucosaminyl)asparagine amidase A, followed by HPLC fractionation, yielded two oligosaccharides, which were analysed by 500 MHz1H-NMR spectroscopy to give the following structures $$\begin{array}{*{20}c} {Man\alpha 1} & \backslash & {Fuc\alpha 1} & | \\ {} & 6 & {Man\beta 1 - 4GlcNAc\beta 1 - 4GlcNAc} & 6 \\ {} & 3 & {} & {} \\ {Man\alpha 1} & / & {} & {} \\ \end{array} \begin{array}{*{20}c} {Man\alpha 1} & \backslash & {Fuc\alpha 1} & | \\ {} & 6 & {Man\beta 1 - 4GlcNAc\beta 1 - 4GlcNAc} & 6 \\ {} & 3 & 3 & | \\ {Man\alpha 1} & / & {Fuc\alpha 1} & {} \\ \end{array}$$ This is the first report on a naturally occurring glycoprotein N-glycan with two fucose residues linked to the asparagine-boundN-acetylglucosamine.

UDP-GlcNAc:α3-D-mannoside β1,2-N-acetylglucosaminyltransferase I (GnTI; EC 2.4.1.101) is a Golgi-resident glycosyltransferase that is essential for the processing of oligomannose to hybrid and complex N-glycans in higher eukaryotes. The cDNA of Nicotiana tabacum GnTI has been cloned and characterised previously. To assess the influence of GnTI expression levels on the formation of complex N-glycans we used posttranscriptional gene silencing to knock down the expression of GnTI in the tobacco related species Nicotiana benthamiana. 143 independent transgenic plants containing GnTI constructs in either sense or antisense orientation were generated. 23 lines were selected for measurement of GnTI activity and 10 lines thereof showed a reduction of more than 85% in in vitro assays as compared to wildtype plants. GnTI reduction was stably inherited and did not interfere with the viability of the transformants. Noteworthy one line, 34S/2, exhibited a residual GnTI activity below the detection limit. β1,2-N-acetylglucosaminyltransferase II (GnTII), an enzyme which acts further downstream in the N-glycosylation pathway, as well as other control enzymes (α-mannosidase, β-N-acetylglucosaminidase) were not affected indicating the specific downregulation of GnTI. Remarkably, immunoblots and mass spectrometric N-glycan profiling revealed no significant changes of the total N-glycan pattern. Thus, even the undetectable residual GnTI activity was sufficient for the synthesis of complex N-glycans comparable to wildtype plants. Published in 2003.

An alpha 1,3-fucosyltransferase was purified 3000-fold from mung bean seedlings by chromatography on DE 52 cellulose and Affigel Blue, by chromatofocusing, gelfiltration and affinity chromatography resulting in an apparently homogenous protein of about 65 kDa on SDS-PAGE. The enzyme transferred fucose from GDP-fucose to the Asn-linked N-acetylglucosaminyl residue of an N-glycan, forming an alpha 1,3-linkage. The enzyme acted upon N-glycopeptides and related oligosaccharides with the glycan structure GlcNAc2Man3 GlcNAc2. Fucose in alpha 1,6-linkage to the asparagine-linked GlcNAc had no effect on the activity. No transfer to N-glycans was observed when the terminal GlcNAc residues were either absent or substituted with galactose. N-acetyllactosamine, lacto-N-biose and N-acetylchito-oligosaccharides did not function as acceptors for the alpha 1,3-fucosyltransferase. The transferase exhibited maximal activity at pH 7.0 and a strict requirement for Mn2+ or Zn2+ ions. The enzyme's activity was moderately increased in the presence of Triton X-100. It was not affected by N-ethylmaleimide.

The occurrence of alpha-D-mannosidase II activity in insect cells was studied using pyridylaminated oligosaccharides as substrates and two-dimensional HPLC and glycosidase digestion for the analysis of products. GlcNAcMan5GlcNAc2 was converted to GlcNAcMan3GlcNAc2 by each of the three cell lines investigated (Bm-N, Sf-21, and Mb-0503). The respective activity was highest in Bm-N cells which were used for further experiments. Man5GlcNAc2 was not degraded by the Bm-N cell homogenate. Thus, this alpha-mannosidase essentially exhibits the same substrate specificity as mammalian and plant Golgi alpha-mannosidase II. The alpha-mannosidase II-like activity from Bm-N cells exhibits a pH optimum of 6.0-6.5, has no requirement for divalent metal ions, and is highly sensitive to swainsonine. The alpha 1,6-linked mannosyl residue is removed first as deduced from the elution time on reversed phase HPLC of the intermediate product. The same branch preference was found with alpha-mannosidase II from mung bean seedlings and Xenopus liver. Upon ultracentrifugation of Bm-N cell homogenate, 72% of the mannosidase acting on the GlcNAcMan5GlcNAc2 substrate was found in the microsomal pellet indicating the enzyme to be membrane-bound.

The initial velocities of hydrolysis of nineteen glycopeptides by peptide: N-glycosidase F and A were determined. Substrates were prepared from bovine fetuin, hen ovalbumin, pineapple stem bromelain, bovine fibrin and taka-amylase. From these glycopeptides, several variants with regard to peptide and carbohydrate structure were prepared and derivatized with dabsyl chloride, dansyl chloride or activated resorufin. Tyrosine containing glycopeptides were also used without an additional chromophore. Enzymatic hydrolysis of glycopeptides was quantified by narrow bore, reversed phase HPLC with turnaround cycle times of down to 6 min, but usually 15 min. KM values ranging from 30 to 64 microM and from 4 to 36 microM were found for N-glycosidase F and A, respectively. Relative velocities of hydrolysis of the different substrates by each enzyme varied considerably. Little, if any, similarity of the performance of N-glycosidase F and A with the different substrates was observed. The minimal carbohydrate structure released by peptide: N-glycosidase F was a di-N-acetylchitobiose. N-glycosidase A could release even a single N-acetylglucosamine, albeit 3000 times slower than a di-N-acetylchitobiose or larger glycans. In general the structure of the intact glycan had little effect on activity, and with both enzymes the rate of hydrolysis appeared to be primarily governed by peptide structure and length. However, N-glycosidase F did not release glycans alpha 1,3-fucosylated at the asparagine linked N-acetylglucosamine irrespective of the presence of xylose in the substrate.

O-glycosylation is a widely occurring posttranslational modification of proteins. The glycosylation status of a specific site may influence the location, activity and function of a protein. The initiating enzyme of mucin-type O-glycosylation is UDP-GalNAc:polypeptide GalNAc transferase (ppGalNAcT; EC 2.4.1.41). Using electron-transfer dissociation mass spectrometry, ppGalNAcT from the snail Biomphalaria glabrata was characterized regarding its ability to glycosylate threonine and serine residues in different peptide sequence environments. The preferences of the snail enzyme for flanking amino acids of the potential glycosylation site were very similar to vertebrate and insect members of the family. Acceptor sites with adjacent proline residues were highly preferred, while other residues caused less pronounced effects. No specific O-glycosylation consensus sequence was found. The results obtained from synthetic peptides were in good correlation with the observed glycosylation patterns of native peptides and with the order of attachment in a multi-glycosylated peptide. The snail enzyme clearly preferred threonine over serine in the in vitro assays. No significant differences of transfer speed or efficiency could be detected using a mutant of the enzyme lacking the lectin domain. This is the first characterisation of the substrate specificity of a member of the ppGalNAcT family from mollusc origin.