Johannis P. Kamerling

Nonulosonic acids or non-2-ulosonic acids (NulOs) are an ancient family of 2-ketoaldonic acids (α-ketoaldonic acids) with a 9-carbon backbone. In nature, these monosaccharides occur either in a 3-deoxy form (referred to as “sialic acids”) or in a 3,9-dideoxy “sialic-acid-like” form. The former sialic acids are most common in the deuterostome lineage, including vertebrates, and mimicked by some of their pathogens. The latter sialic-acid-like molecules are found in bacteria and archaea. NulOs are often prominently positioned at the outermost tips of cell surface glycans, and have many key roles in evolution, biology and disease. The diversity of stereochemistry and structural modifications among the NulOs contributes to more than 90 sialic acid forms and 50 sialic-acid-like variants described thus far in nature. This paper reports the curation of these diverse naturally occurring NulOs at the NCBI sialic acid page (https://www.ncbi.nlm.nih.gov/glycans/sialic.html) as part of the NCBI-Glycans initiative. This includes external links to relevant Carbohydrate Structure Databases. As the amino and hydroxyl groups of these monosaccharides are extensively derivatized by various substituents in nature, the Symbol Nomenclature For Glycans (SNFG) rules have been expanded to represent this natural diversity. These developments help illustrate the natural diversity of sialic acids and related NulOs, and enable their systematic representation in publications and online resources.

Conotoxins are small peptides present in the venom of cone snails. The snail uses this venom to paralyze and capture prey. The constituent conopeptides display a high level of chemical diversity and are of particular interest for scientists as tools employed in neurological studies and for drug development, because they target with exquisite specificity membrane receptors, transporters, and various ion channels in the nervous system. However, these peptides are known to contain a high frequency and variability of post-translational modifications—including sometimes O-glycosylation—which are of importance for biological activity. The potential application of specific conotoxins as neuropharmalogical agents and chemical probes requires a full characterization of the relevant peptides, including the structure of the carbohydrate part. In this review, the currently existing knowledge of O-glycosylation of conotoxins is described.

The conformational properties of the neutral exopolysaccharide produced by Lactobacillus delbrueckii ssp. bulgaricus LBB.B26 have been studied by NMR measurements and molecular modelling. The exopolysaccharide, with an average molecular mass of 1.3 × 10⁶ Da, was previously determined to consist of pentasaccharide repeating units with the following structure: →3)-β-D-Galp-(1→4)-β-D-Glcp-(1→3)-β-D-Galf-(1→3)-[α-D-Glcp-(1→6)-]α-D-Galp-(1→. Adiabatic maps were generated for each of the disaccharide fragments in the repeating unit. In addition, free energy maps calculated from MD simulations were obtained for each of the glycosidic linkages in an extended repeating unit and the influence of the Galf residue on the conformational properties of the repeating unit was investigated. The values of the global energy minima provided by the free energy maps were used to build a polymer chain. The polysaccharide was shown to have a random coil structure, without stable extended helical motifs.

Cell aggregation in the marine sponge Microciona prolifera is mediated by a multimillion molecular-mass aggregation factor, termed MAF. Earlier investigations revealed that the cell aggregation activity of MAF depends on two functional domains: (i) a Ca²⁺-independent cell-binding domain and (ii) a Ca²⁺-dependent proteoglycan self-interaction domain. Structural analysis of involved carbohydrate fragments of the proteoglycan in the self-association established a sulfated disaccharide β-d-GlcpNAc3S-(1→3)-α-l-Fucp and a pyruvated trisaccharide β-d-Galp4,6(R)Pyr-(1→4)-β-d-GlcpNAc-(1→3)-α-l-Fucp. Recent UV, SPR, and TEM studies, using BSA conjugates and gold nanoparticles of the synthetic sulfated disaccharide, clearly demonstrated self-recognition on the disaccharide level in the presence of Ca²⁺-ions. To determine binding forces of the carbohydrate–carbohydrate interactions for both synthetic MAF oligosaccharides, atomic force microscopy (AFM) studies were carried out. It turned out that, in the presence of Ca²⁺-ions, the force required to separate the tip and sample coated with a self-assembling monolayer of thiol-spacer-containing β-d-GlcpNAc-(1→3)-α-l-Fucp-(1→O)(CH₂)₃S(CH₂)₆S- was found to be quantized in integer multiples of 30 ± 6 pN. No binding was observed between the two monolayers in the absence of Ca²⁺-ions. Cd²⁺-ions could partially induce the self-interaction. In contrast, similar AFM experiments with thiol-spacer-containing β-d-Galp4,6(R)Pyr-(1→4)-β-d-GlcpNAc-(1→3)-α-l-Fucp-(1→O)(CH₂)₃S(CH₂)₆S- did not show a binding in the presence of Ca²⁺-ions. Also TEM experiments of gold nanoparticles coated with the pyruvated trisaccharide could not make visible aggregation in the presence of Ca²⁺-ions. It is suggested that the self-interaction between the sulfated disaccharide fragments is stronger than that between the pyruvated trisaccharide.

The species-specific cell adhesion in the marine sponge Microciona prolifera involves the interaction of an extracellular proteoglycan-like macromolecular complex, otherwise known as aggregation factor. In the interaction, two highly polyvalent functional domains play a role: a cell-binding and a self-interaction domain. The self-recognition has been characterized as a Ca²⁺-dependent carbohydrate–carbohydrate interaction of repetitive low affinity carbohydrate epitopes. One of the involved epitopes is the pyruvated trisaccharide β-D-Galp4,6(R)Pyr-(1→4)-β-D-GlcpNAc-(1→3)-L-Fucp. To evaluate the role of this trisaccharide in the proteoglycan–proteoglycan self-recognition, β-D-Galp4,6(R)Pyr-(1→4)-β-D-GlcpNAc-(1→3)-α-L-Fucp-(1→O)(CH₂)₃S(CH₂)₆SH was synthesized, and partially converted into gold glyconanoparticles. These mimics are being used to explore the self-interaction phenomenon for the trisaccharide epitope, viaTEM aggregation experiments (gold glyconanoparticles) and atomic force microscopy (AFM) experiments (self assembled monolayers; binding forces).

The methylotrophic yeast Pichia pastoris is widely used for the production of recombinant glycoproteins. With the aim to generate biologically active ¹⁵N-labeled glycohormones for conformational studies focused on the unravelling of the NMR structures in solution, the P. pastoris strains GS115 and X-33 were explored for the expression of human chorionic gonadotropin (phCG) and human follicle-stimulating hormone (phFSH). In agreement with recent investigations on the N-glycosylation of phCG, produced in P. pastoris GS115, using ammonia/glycerol-methanol as nitrogen/carbon sources, the N-glycosylation pattern of phCG, synthesized using NH₄Cl/glucose–glycerol–methanol, comprised neutral and charged, phosphorylated high-mannose-type N-glycans (Man_8–15GlcNAc₂). However, the changed culturing protocol led to much higher amounts of glycoprotein material, which is of importance for an economical realistic approach of the aimed NMR research. In the context of these studies, attention was also paid to the site specific N-glycosylation in phCG produced in P. pastoris GS115. In contrast to the rather simple N-glycosylation pattern of phCG expressed in the GS115 strain, phCG and phFSH expressed in the X-33 strain revealed, besides neutral high-mannose-type N-glycans, also high concentrations of neutral hypermannose-type N-glycans (Man_up-to-30GlcNAc₂). The latter finding made the X-33 strain not very suitable for generating ¹⁵N-labeled material. Therefore, ¹⁵N-phCG was expressed in the GS115 strain using the new optimized protocol. The ¹⁵N-enrichment was evaluated by ¹⁵N-HSQC NMR spectroscopy and GLC-EI/MS. Circular dichroism studies indicated that ¹⁵N-phCG/GS115 had the same folding as urinary hCG. Furthermore, ¹⁵N-phCG/GS115 was found to be similar to the unlabeled protein in every respect as judged by radioimmunoassay, radioreceptor assays, and in vitro bioassays.