Structural characterization of the oligosaccharides of a human monoclonal anti-lipopolysaccharide immunoglobulin M

The oligosaccharide side chains of a human anti-lipopolysaccharide IgM produced by a human-human-mouse heterohybridoma were analyzed at each of its five conserved N-glycosylation sites. This antibody also has a potential sixth N-glycosylation site in the variable region of its heavy chain which is not glycosylated. The oligosaccharides were released by digestion with various endo- and exoglycosidases and analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and fluorophore-assisted carbohydrate electrophoresis. The antibody has various complex- and hybrid-type oligosaccharide structures at Asn 171, various sialylated complex-type oligosaccharides at Asn 332 and 395, and high-mannose-type oligosaccharides at Asn 402 and 563. Of note is the presence in this human IgM of oligosaccharides containing N-glycolylneuraminic acid and N-acetylneuraminic acid in the ratio of 98:2 as determined using anion- exchange chromatography. Furthermore, we observed oligosaccharide structures containing Gal alpha (1,3)Gal that have not been reported as components of human glycoproteins.      

Can Nep1-like proteins form oligomers?

Nep1-like proteins (NLPs) are a novel family of microbial elicitors of plant necrosis that induce a hypersensitive-like response in dicot plants. The spatial structure and role of these proteins are yet unknown. In a paper published in BMC Plant Biology (2008; 8:50) we have proposed that the core region of Nep1-like proteins (NLPs) belong to the Cupin superfamily. Based on what is known about the Cupin superfamily, in this addendum to the paper we discuss how NLPs could form oligomers.Key words: quaternary structure, necrosis and ethylene inducing proteins, NLPs, MpNEP1, MpNEP2, NPP1, Moniliophthora perniciosa, Phytophthora parasiticaCupins may be organized as monomers, dimers, hexamers and octamers of β-barrel domains.¹ To the best of our knowledge trimers have not been detected yet. The interaction of two monomers building up a dimeric structure is basically performed by three types of interactions: hydrophobic interactions between β-strands in different subunits, salt bridges and hydrogen bonds between β-strands. In cupin dimers, the hydrophobic interactions occur between two βI strands in different subunits (Fig. 1A and B). This strand represents the central axis of rotation of the dimer as one residue in βI interacts with the corresponding residue in the other subunit (Fig. 1B). Therefore, all residues in βI must be hydrophobic, as one residue interacts with the other subunit and the next one in the sequence interacts with the interior of the protein. Charged residues in βI would disrupt such interactions. Most cupin dimers have strong hydrophobic residues such as tryptophan (W), phenylalanine (F) and methionine (M) pointing towards the own subunit (↓), while small hydrophobic residues such as leucine (L), isoleucine (I), and valine (V) point to the other subunit (↑). A particular case is leucine that interacts with other subunits, for instance, βI = liaW (positions 217–220 in Fig. 1B) and βI = LVsw of type I and II NLP consensuses, respectively. Therefore, the pattern of hydropathicity suggests that the side chain orientation is βI = l217 ↑ i218 ↓ a219 ↑ W220 ↓ d221 ↑. However we observe that just after βI there is a charged residue (aspartate D221) which would point outwards disrupting the dimer or at least making it less stable. It is interesting to observe that the requirement for a negatively charged residue at this last position is very high: 96% of all type I NLPs contains an aspartate (D) or glutamate (E) indicating an important role for it, maybe in avoiding dimerization of the NLPs. A second interesting hypothesis is as follows: several cupins are oxygenases, decarboxylases, etc. and use a negatively charged residue, such as aspartate or glutamate as proton donor.¹ Now, if the alternate pattern of side chains of the residues is βI = l217 ↓ i218 ↑ a219 ↓ W220 ↑ d221 ↓, instead of the previous one, then the aspartate or glutamate residue would point to the hydrophobic pocket and would be positioned to interact with the metal ion, as in cupins with enzymatic activity. However, there are no experimental evidences that the NLPs have enzymatic activity.Open in a separate windowFigure 1(A) Three-dimensional structure prediction for type I NLP consensus, (B) Interface between two βI strands in type I NLP consensus. From the left to the right: EF-coil with the conserved residue H162, βC and βH strands (superposed) with the conserved histidines H133 and H135 in βC, H193 and leucine L195 in βH, W220 in βI and W118 in βB. The strands in the right subunit follow the same pattern but rotated.The second type of interaction is salt bridges between charged residues in different subunits. Analyzing all interacting side chains in the 1VJ2 protein (dimer), we verify that the charged side chains of N35 and E57 (numbers in original structure) are only 2.72 Å apart. In the NLPs, this corresponds to N108^36% (Q108^60%) at the border of βB and E138^98%. The negatively charged residue D125 helps to correct the orientation of the subunits in relation to each other avoiding any disorientation. The high conservation level of these residues suggests that NLPs are dimeric structures. However, as we will see next, only hydrophobic and charged interactions are not enough to build a dimer.Garcia et al. (2007)² have used small angle X-ray scattering (SAXS) to show that, in solution, at low concentrations (<2 mg/ml) the two copies of the NLPs of Moniliophthora perniciosa, MpNEP1 and MpNEP2, exist as dimers and monomers, respectively. The same technique showed that at higher concentrations, >5 mg/ml, both proteins exist as dimers, as is the case for PpNPP1.² They also reported, based on electrophoresis analysis, that PpNPP1 and MpNEP1 exist as oligomers and MpNEP2 as monomers.² However, experiments with the PpNPP1 in size exclusion chromatography using myoglobin as size standard suggest that PpNPP1 is a monomer.³ Figure 2 compares MpNEP1, MpNEP2 and PpNPP1, where the most relevant differences in sequence are marked with asterisks (*) and are possibly related to the differences in oligomeric properties between MpNEP1 and PpNPP1 with MpNEP2. These positions are methionine M27 and leucine L35, which occur only in MpNEP2, glycine G250, which occurs only in MpNEP2 and NEP1 (Fusarium oxysporum) and lysine K31, which occurs only MpNEP2, {"type":"entrez-protein","attrs":{"text":"BAB04114","term_id":"10173008"}}BAB04114 (Bacillus halodurans) and {"type":"entrez-protein","attrs":{"text":"AAU23136","term_id":"52003194"}}AAU23136 (Bacillus licheniformis). The other residues are aspartate D28, which occurs 9 times and alanine A37 which occurs 7 times of all investigated NLPs. Thus, the sequence mdHDkiakl at the start of the NLPs seems to explain the monomeric state of MpNEP2, although at higher concentrations they form dimers. Besides the weak hydrophobic interactions, dimeric cupins and bicupins (two β barrels in the same sequence building up a dimeric-like 4d-structure) are stable structures (see Fig. 1 in ref. ⁴). By aggregating the first β-strand in the start domain of one β-barrel to the ABIDG β-sheet of the other β-barrel, composing a big ABIDGY β-sheet (Y is the first β-strand). For instance, using the bicupin 1L3J (oxalate decarboxylase) as template, the low confidence level β-strand at position 26–33 (v in H29D30 avv) in type I NLPs corresponds to the first β-strand. Since this proceeds from both barrels they can build a stable structure (see Fig. 1 in ref. ⁴). The quaternary structure is related to the presence of interaction residues in the BID β-sheet of the cupin structure. These are present in the NLPs and would enable them to form dimers.Open in a separate windowFigure 2Alignment of type I NLP consensus, PpNPP1, MpNEP1 and MpNEP2. Solid line boxes are β-strands, double line boxes are α-helices. The sequence positions marked with asterisks (*) are possibly related to the differences in oligomeric properties between MpNEP1 and PpNPP1 with MpNEP2.     

Higher‐order assemblies of oligomeric cargo receptor complexes form the membrane scaffold of the Cvt vesicle

Selective autophagy is the mechanism by which large cargos are specifically sequestered for degradation. The structural details of cargo and receptor assembly giving rise to autophagic vesicles remain to be elucidated. We utilize the yeast cytoplasm‐to‐vacuole targeting (Cvt) pathway, a prototype of selective autophagy, together with a multi‐scale analysis approach to study the molecular structure of Cvt vesicles. We report the oligomeric nature of the major Cvt cargo Ape1 with a combined 2.8 Å X‐ray and negative stain EM structure, as well as the secondary cargo Ams1 with a 6.3 Å cryo‐EM structure. We show that the major dodecameric cargo prApe1 exhibits a tendency to form higher‐order chain structures that are broken upon interaction with the receptor Atg19 in vitro. The stoichiometry of these cargo–receptor complexes is key to maintaining the size of the Cvt aggregate in vivo. Using correlative light and electron microscopy, we further visualize key stages of Cvt vesicle biogenesis. Our findings suggest that Atg19 interaction limits Ape1 aggregate size while serving as a vehicle for vacuolar delivery of tetrameric Ams1.     

Interleukin-7-aggravated joint inflammation and tissue destruction in collagen-induced arthritis is associated with T-cell and B-cell activation

Introduction

We sought to investigate the capacity of interleukin (IL)-7 to enhance collagen-induced arthritis and to study by what mechanisms this is achieved.

Methods

Mice received multiple injections with IL-7 or phosphate-buffered saline (PBS) as a control. Arthritis severity and incidence were determined by visual examination of the paws. Joint destruction was determined by assessing radiographs and immunohistochemistry of the ankle joints. Total cellularity and numbers of T-cell and B-cell subsets were assessed, as well as ex vivo production of interferon-γ (IFN-γ), IL-17, and IL-4. Proinflammatory mediators were measured in serum with multianalyte profiling.

Results

IL-7 increased arthritis severity and radiology-assessed joint destruction. This was consistent with IL-7-increased intensity of cell infiltrates, bone erosions, and cartilage damage. Splenic CD19⁺ B cells and CD19⁺/GL7⁺ germinal center B cells, as well as CD4 and CD8 numbers, were increased by IL-7. IL-7 expanded memory T cells, associated with increased percentages of IFN-γ-, IL-4-, and IL-17-producing CD4⁺ T cells. On antigen restimulation of draining lymph node cells in vitro IL-7 treatment was found to increase IFN-γ and IL-17 production, whereas IL-4 was reduced. IL-7 also increased concentrations of proinflammatory mediators, indicative of T-cell activation (sCD40L), vascular activation (VCAM-1, VEGF), tissue destruction (fibroblast growth factor-basic (FGF-b), LIF), and chemotaxis (MIP-1γ, MIP-3β, lymphotactin, MDC, and MCP-5).

Conclusions

In arthritic mice, IL-7 causes expansion of T and B cells, associated with increased levels of proinflammatory mediators. IL-7 intensifies arthritis severity and joint destruction, accompanied by increased Th1 and Th17 activity. These data indicate that IL-7 could be an important mediator in arthritic conditions and that targeting IL-7 or its receptor represent novel therapeutic strategies.     

A comparison of the polysaccharides extracted from dried and non-dried walls of suspension-cultured sycamore cells

Suspension-cultured sycamore cells (Acer pseudoplatanus) were disrupted in aqueous K-Pi buffer and the insoluble residue (the cell wall) purified by extraction with organic solvents and air-dried (dry cell walls) or by washing with aqueous sodium dodecyl sulphate and stored frozen (wet cell walls). Polysaccharides solubilized from the purified wet and dry cell walls by enzymatic digestion and chemical extraction were isolated and their glycosyl-residue compositions compared. No significant differences were found in the types or yields of the polysaccharides solubilized by enzymatic digestion and chemical extraction of the wet and dry cell wall preparations. Moreover, the glycosyl-residue compositions of the so-called ‘-cellulose’ fraction that remains after extraction of the wet and dry cell wall preparations with alkali was indistinguishable from the glycosyl-residue compositions of the walls prior to extraction.     

Genomic organization and chromosomal localization of three members of the UDP-N-acetylgalactosamine: polypeptide N- acetylgalactosaminyltransferase family

A homologous family of UDP- N -acetylgalactosamine: polypeptide N - acetylgalactosaminyltransferases (GalNAc-transferases) initiate O- glycosylation. These transferases share overall amino acid sequence similarities of approximately 45-50%, but segments with higher similarities of approximately 80% are found in the putative catalytic domain. Here we have characterized the genomic organization of the coding regions of three GalNAc-transferase genes and determined their chromosomal localization. The coding regions of GALNT1 , -T2 , and -T3 were found to span 11, 16, and 10 exons, respectively. Several intron/exon boundaries were conserved within the three genes. One conserved boundary was shared in a homologous C. elegans GalNAc- transferase gene. Fluorescence in situ hybridization showed that GALNT1 , -T2 , and -T3 are localized at chromosomes 18q12-q21, 1q41-q42, and 2q24-q31, respectively. These results suggest that the members of the polypeptide GalNAc-transferase family diverged early in evolution from a common ancestral gene through gene duplication.      

Oxidative stress in skin fibroblasts cultures from patients with Parkinson's disease

Background  

In the substantia nigra of Parkinson's disease (PD) patients, increased lipid peroxidation, decreased activities of the mitochondrial complex I of the respiratory chain, catalase and glutathione-peroxidase, and decreased levels of reduced glutathione have been reported. These observations suggest that oxidative stress and mitochondrial dysfunction play a role in the neurodegeneration in PD. We assessed enzymatic activities of respiratory chain and other enzymes involved in oxidative processes in skin fibroblasts cultures of patients with PD.     

Structure of the xyloglucan produced by suspension-cultured tomato cells

The xyloglucan secreted by suspension-cultured tomato (Lycopersicon esculentum) cells was structurally characterized by analysis of the oligosaccharides generated by treating the polysaccharide with a xyloglucan-specific endoglucanase (XEG). These oligosaccharide subunits were chemically reduced to form the corresponding oligoglycosyl alditols, which were isolated by high-performance liquid chromatography (HPLC). Thirteen of the oligoglycosyl alditols were structurally characterized by a combination of matrix-assisted laser-desorption ionization mass spectrometry and two-dimensional nuclear magnetic resonance (NMR) spectroscopy. Nine of the oligoglycosyl alditols (GXGGol, XXGGol, GSGGol, XSGGol, LXGGol, XTGGol, LSGGol, LLGGol, and LTGGol, [see, Fry, S.C.; York, W.S., et al., Physiologia Plantarum 1993, 89, 1-3, for this nomenclature]) are derived from oligosaccharide subunits that have a cellotetraose backbone. Very small amounts of oligoglycosyl alditols (XGGol, XGGXXGGol, XXGGXGGol, and XGGXSGGol) derived from oligosaccharide subunits that have a cellotriose or celloheptaose backbone were also purified and characterized. The results demonstrate that the xyloglucan secreted by suspension-cultured tomato cells is very complex and is composed predominantly of 'XXGG-type' subunits with a cellotetraose backbone. The rigorous characterization of the oligoglycosyl alditols and assignment of their 1H and 13C NMR spectra constitute a robust data set that can be used as the basis for rapid and accurate structural profiling of xyloglucans produced by Solanaceous plant species and the characterization of enzymes involved in the synthesis, modification, and breakdown of these polysaccharides.     

l-Galactose replaces l-fucose in the pectic polysaccharide rhamnogalacturonan II synthesized by the l-fucose-deficient mur1 Arabidopsis mutant

Arabidopsis thaliana mur1 is a dwarf mutant with altered cell-wall properties, in which l-fucose is partially replaced by l-galactose in the xyloglucan and glycoproteins. We found that the mur1 mutation also affects the primary structure of the pectic polysaccharide rhamnogalacturonan II (RG-II). In mur1 RG-II a non-reducing terminal 2-O-methyl l-galactosyl residue and a 3,4-linked l-galactosyl residue replace the non-reducing terminal 2-O-methyl l-fucosyl residue and the 3,4-linked l-fucosyl residue, respectively, that are present in wild-type RG-II. Furthermore, we found that a terminal non-reducing l-galactosyl residue, rather than the previously reported d-galactosyl residue, is present on the 2-O-methyl xylose-containing side chain of RG-II in both wild type and mur1 plants. Approximately 95% of the RG-II from wild type and mur1 plants is solubilized as a high-molecular-weight (>100 kDa) complex, by treating walls with aqueous potassium phosphate. The molecular mass of RG-II in this complex was reduced to 5–10 kDa by treatment with endopolygalacturonase, providing additional evidence that RG-II is covalently linked to homogalacturonan. The results of this study provide additional information on the structure of RG-II and the role of this pectic polysaccharide in the plant cell wall.Abbreviations AIR Alcohol-insoluble residue - d-Gal d-Galactosyl - EPG Endopolygalacturonase - ESI–MS Electrospray ionization mass spectrometry - GC–MS Gas chromatography–mass spectrometry - ¹H-NMR Proton nuclear magnetic resonance spectroscopy - l-Fuc l-Fucosyl - l-Gal l-Galactosyl - 2-O-MeFuc 2-O-Methyl l-fucosyl - 2-O-MeGal 2-O-Methyl l-galactosyl - 2-O-MeXyl 2-O-Methyl d-xylosyl - MWCO Molecular weight cut-off - RG-II Rhamnogalacturonan II - ppm Parts per million - RI Refractive index - SEC Size-exclusion chromatography - TFA Trifluoroacetic acid - WT Wild type