Primary structures of the N-glycans of two major pollen allergens (Lol p 11 and Ole e 1) and a major peanut allergen (Ara h 1) were determined. Ole e 1 and Ara h 1 carried high mannose and complex N-glycans, whereas Lol p 11 carried only the complex. The complex structures all had a ␤(1,2)-xylose linked to the core mannose. Substitution of the proximal N-acetylglucosamine with an ␣(1,3)-fucose was observed on Lol p 11 and a minor fraction of Ole e 1 but not on Ara h 1. To elucidate the structural basis for IgE recognition of plant N-glycans, radioallergosorbent test analysis with protease digests of the three allergens and a panel of glycoproteins with known N-glycan structures was performed. It was demonstrated that both ␣(1,3)-fucose and ␤(1,2)-xylose are involved in IgE binding. Surprisingly, xylose-specific IgE antibodies that bound to Lol p 11 and bromelain did not recognize closely related xylose-containing structures on horseradish peroxidase, phytohemeagglutinin, Ole e 1, and Ara h 1. On Lol p 11 and bromelain, the core ␤-mannose is substituted with just an ␣(1,6)-mannose. On the other xylose-containing N-glycans, an additional ␣(1,3)-mannose is present. These observations indicate that IgE binding to xylose is sterically hampered by the presence of an ␣(1,3)-antenna.In the early 1980s, it was reported for the first time that IgE antibodies in sera of pollen allergic patients can be directed to carbohydrate determinants on glycoproteins (1-3). The carbohydrate nature of these epitopes was supported by several characteristic properties, such as their periodate sensitivity and their resistance to heating and protease digestion. IgE antibodies directed to these carbohydrate structures were shown to be extremely cross-reactive not only between different plant-derived glycoproteins but also to glycoproteins from invertebrate animals (e.g. seafood and insect venoms) (2, 4 -7).This high degree of cross-reactivity was explained by the conserved structure of N-glycans from plants and invertebrate animals, sharing several features that are not found in mammalian N-glycans (8). More recently, several research groups have confirmed the role of carbohydrate epitopes in IgE reactivity (9 -22).In plants, the N-glycosylation of proteins starts by the transfer of the oligosaccharide precursor Glc 3 Man 9 GlcNAc 2 in the endoplasmic reticulum (reviewed in Ref. 23). This structure can subsequently be modified by glycosidases and glycosyltransferases during transport of the glycoprotein through the endoplasmic reticulum, the Golgi apparatus, and the vacuole. Depending on the accessibility of the glycan side chain, these enzymes can convert the precursor to high mannose-type Nglycans ranging from Man 9 GlcNAc 2 to Man 5 GlcNAc 2 and then to complex-type N-glycans having an ␣(1,3)-fucose attached to the proximal glucosamine residue and/or a ␤(1,2)-xylose residue attached to the ␤-mannose. These linkages of fucose and xylose are typical for complex N-glycans from plants and invertebrate animals and are not found in mammals. Mo...

A liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method using reversed-phase chromatography was developed for the analysis of phospholipids from bacterial extracts of a wild-type strain of Escherichia coli. Product ion mass spectra from [M--H](-) precursor ions allowed an identification of individual phospholipid species that includes both fatty acid composition and fatty acyl location on the glycerol backbone using diagnostic product ions. Thus, complete assignment, including sn-1/sn-2 fatty acyl position, was achieved for this strain of E. coli. In addition, the phospholipids were quantified relative to one another using an internal standard method.

Chlamydomonas reinhardtii is a green unicellular eukaryotic model organism for studying relevant biological and biotechnological questions. The availability of genomic resources and the growing interest in C. reinhardtii as an emerging cell factory for the industrial production of biopharmaceuticals require an in-depth analysis of protein N-glycosylation in this organism. Accordingly, we used a comprehensive approach including genomic, glycomic, and glycoproteomic techniques to unravel the N-glycosylation pathway of C. reinhardtii. Using mass-spectrometry-based approaches, we found that both endogenous soluble and membrane-bound proteins carry predominantly oligomannosides ranging from Man-2 to Man-5. In addition, minor complex N-linked glycans were identified as being composed of partially 6-Omethylated Man-3 to Man-5 carrying one or two xylose residues. These findings were supported by results from a glycoproteomic approach that led to the identification of 86 glycoproteins. Here, a combination of in-source collision-induced dissodiation (CID) for glycan fragmentation followed by mass tag-triggered CID for peptide sequencing and PNGase F treatment of glycopeptides in the presence of 18 O-labeled water in conjunction with CID mass spectrometric analyses were employed. In conclusion, our data support the notion that the biosynthesis and maturation of N-linked glycans in the endoplasmic reticulum and Golgi apparatus occur via a GnT I-independent pathway yielding novel complex N-linked glycans that maturate differently from their counterparts in land plants. Molecular & Cellular