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Microalgae of the genus Chlorella vulgaris are candidates for the production of lipids for biofuel production. Besides that, Chlorella vulgaris is marketed as protein and vitamin rich food additive. Its potential as a novel expression system for recombinant proteins inspired us to study its asparagine-linked oligosaccharides (N-glycans) by mass spectrometry, chromatography and gas chromatography. Oligomannosidic N-glycans with up to nine mannoses were the structures found in culture collection strains as well as several commercial products. These glycans co-eluted with plant N-glycans in the highly shape selective porous graphitic carbon chromatography. Thus, Chlorella vulgaris generates oligomannosidic N-glycans of the structural type known from land plants and animals. In fact, Man5 (Man5GlcNAc2) served as substrate for GlcNAc-transferase I and a trace of an endogenous structure with terminal GlcNAc was seen. The unusual more linear Man5 structure recently found on glycoproteins of Chlamydomonas reinhardtii occurred - if at all - in traces only. Notably, a majority of the oligomannosidic glycans was multiply O-methylated with 3-O-methyl and 3,6-di-O-methyl mannoses at the non-reducing termini. This modification has so far been neither found on plant nor vertebrate N-glycans. It’s possible immunogenicity raises concerns as to the use of C. vulgaris for production of pharmaceutical glycoproteins.

The many emerging applications of microalgae such as Chlorella also instigate interest in their ability to conduct protein modifications such as N-glycosylation. Chlorella vulgaris has recently been shown to equip its proteins with highly O-methylated oligomannosidic N-glycans. Two other frequently occurring species names are Chlorella sorokiniana and Chlorella pyrenoidosa—even though the latter is taxonomically ill defined. We analyzed by mass spectrometry and nuclear magnetic resonance spectroscopy the N-glycans of type culture collection strains of C. sorokiniana and of a commercial product labeled C. pyrenoidosa. Both samples contained arabinose, which has hitherto not been found in N-glycans. Apart from this only commonality, the structures differed fundamentally from each other and from that of N-glycans of land plants. Despite these differences, the two algae lines exhibited considerable homology in their ITS1-5.8S-ITS2 rDNA sequences. These drastic differences of N-glycan structures between species belonging to the very same genus provoke questions as to the biological function on a unicellular organism.

The purpose of determining specific IgE (sIgE) in serum-based allergy diagnosis is assigning the allergen(s) that cause(s) notable allergic symptoms. This attractively simple principle is, however, perturbed by the occurrence of clinically insignificant IgEs 1. A number of candidates for these interfering IgEs have been identified and classified into IgEs that bind to peptide epitopes and IgEs that bind to carbohydrate epitopes 1. Weighting of these different reasons has never been made. The extraordinarily wide distribution of a particular carbohydrate epitope was addressed three decades ago, when the now well-known term ‘cross-reactive carbohydrate determinant’ (CCD) was coined 2. The structural basis of CCDs is complex-type Asn-linked oligosaccharides on glycoproteins 3,4. In particular, it is the presence of core-α1,3-linked fucose that implants the same epitope onto glycoproteins from insect venoms, plant pollens, vegetable foodstuffs and even latex 5–9. Consequently, a patient who develops IgE against CCDs on whichever allergen reacts with other allergens that contain typical plant or insect glycosylation. Up to a quarter of all patients undergoing sIgE testing show these multiple reactions 1,8,10–12. The problem is that anti-CCD IgE, from what we know today, has no clinical significance 1,11,13–18. The picture was further complicated around 2001 as multivalent CCD-containing proteins displayed relevant biological potency in in vitro histamine release tests 12,19,20. Since then, no patient has been presented who reacted against CCDs in a way clearly addressable as an allergic reaction. Thus, it appears prudent to adhere to the notion that anti-CCD IgE has no clinical significance. While we can only speculate about the reasons for this remarkable circumstance 4, the serious consequence is that for a large cohort of patients, any sIgE test will return a positive result, which will, however, be false positive for most or all of the allergens. The severity of the problem may have been underestimated in single allergen testing, where only small numbers of allergens carefully selected on the basis of anamnesis are tested, for example, with the ImmunoCAP system. Positive results are expected, and false positives escape notice as they do not raise suspicion. By contrast, array tests return a multitude of positive results for CCD-positive patients. The problem has been known for several years, and more or less promising solutions have been suggested. Some laboratories determine anti-CCD IgE with a MUXF-CAP (Thermo Scientific/Phadia; ‘MUXF’ is explained in Fig. ​Fig.2).2). This identifies problematic results, but cannot help to discriminate false from truly positive results. Removal of anti-CCD IgE with immobilized CCDs has also been suggested 15, but dismissed as too laborious for routine application 21. The German guideline on allergy diagnosis 14 as well as newer literature 22 mentions inhibition of anti-CCD IgE but does not state how the inhibition should be achieved. A mixture of natural plant glycoproteins to be used for CCD inhibition is available from Mediwiss Analytics (Moers, Germany). Natural glycoproteins could contain peptide epitopes that cause unwanted inhibitions. For many years, our group has used a semisynthetic CCD blocker consisting of bromelain glycopeptides coupled to bovine serum albumin (BSA) 20,23,24. The proteolytic digestion of the starting material ensures the destruction of peptide epitopes. However, only rudimentary glycopeptide purification has been performed and BSA may itself bind IgE in patients who are allergic to meat or milk. Figure 2 Preparation of the CCD blocker. Highly purified glycopeptides containing core α1,3-fucose and xylose are chemically coupled to human serum albumin (HSA). The glycopeptides contain 2-4 amino acids at maximum, which is verified by MALDI-TOF MS (panel ... In the present work, we used a new, highly pure and specific version of our CCD blocker to determine sIgEs in single allergen tests as well as on multi-allergen strips and component arrays. For several patients, laboratory diagnosis was augmented by skin prick tests.

Analysis of the monosaccharides of complex carbohydrates is often performed by liquid chromatography with fluorescence detection. Unfortunately, methylated sugars, unusual amino- or deoxysugars and incomplete hydrolysis can lead to erroneous assignments of peaks. Here, we demonstrate that a volatile buffer system is suitable for the separation of anthranilic acid labeled sugars. It allows off-line examination of peaks by electrospray mass spectrometry. Approaches towards on-line mass spectrometric detection using reversed-phase or porous graphitic carbon columns fell short of achieving sufficient separation of the relevant isobaric sugars. Adequate chromatographic performance for isomeric sugars was achieved with reversed-phase chromatography of “hyper”-methylated anthranilic acid-labeled monosaccharides. Deuteromethyl iodide facilitates the discovery of naturally methylated sugars and identification of their parent monosaccharide as demonstrated with N-glycans of the snail Achatina fulica, where two thirds of the galactoses and a quarter of the mannoses were methylated.

The Bifibobacterium longum subsp. longum 35624™ strain (formerly named Bifidobacterium longum subsp. infantis) is a well described probiotic with clinical efficacy in Irritable Bowel Syndrome clinical trials and induces immunoregulatory effects in mice and in humans. This paper presents (a) the genome sequence of the organism allowing the assignment to its correct subspeciation longum; (b) a comparative genome assessment with other B. longum strains and (c) the molecular structure of the 35624 exopolysaccharide (EPS624). Comparative genome analysis of the 35624 strain with other B. longum strains determined that the sub-speciation of the strain is longum and revealed the presence of a 35624-specific gene cluster, predicted to encode the biosynthetic machinery for EPS624. Following isolation and acid treatment of the EPS, its chemical structure was determined using gas and liquid chromatography for sugar constituent and linkage analysis, electrospray and matrix assisted laser desorption ionization mass spectrometry for sequencing and NMR. The EPS consists of a branched hexasaccharide repeating unit containing two galactose and two glucose moieties, galacturonic acid and the unusual sugar 6-deoxy-L-talose. These data demonstrate that the B. longum 35624 strain has specific genetic features, one of which leads to the generation of a characteristic exopolysaccharide.