Sialic acids are a family of 9-carbon carboxylated sugars, where different substitutions of the backbone define over 30 members. Biological roles of these substitutions have been missed until recently because of their low abundance and lability to conventional isolation/purification methods. This new approach characterizes sialic acids using electrospray ionization-mass spectrometry (ESI-MS) to monitor the HPLC separation of their DMB (1,2-diamino-4,5-methylenedioxy-benzene) derivatives (quinoxalinones). A combination of retention times and spectra characteristics allows definition of the type and position of the various substituents. This approach requires no previous purification, involving a simple derivatization reaction followed by direct injection on the microbore HPLC column. A complete spectrum, including molecular ions and CAD fragments of a sialic acid quinoxalinone, is obtained by injecting 10-20 pmol of the compound. Individual quinoxalinones can be purified by regular RP-HPLC and analyzed by direct-injection ESI-MS or LSIMS. Using this approach, we identified 28 different sialic acids, including the following new species: Neu5Gc9Lt (BSM), anhydro derivatives of Neu5Ac other than the 4,8-anhydro (horse serum hydrolyzates), KDN5(7)Ac and KDN5(7),9Ac2 (amphibian Pleurodeles waltl), four isomers of Neu5Gc8MexAc and three anhydro derivatives of Neu5Gc8Me (glycolipids of the starfish Pisaster brevispinus), and Neu5Ac8S (in addition to Neu5Gc8S, in the glycolipids of the sea urchin Lovenia cordiformis). Results show the usefulness of LC-ESI-MS to study sialic acid diversity, and identification of small amounts of unexpected sialic acids or new members of their family.

No abstract

We previously reported an unusual carboxylated modification on N-glycans isolated from whole bovine lung. We have now raised IgG mAbs against the modification by immunization with biotinylated aminopyridine-derivatized glycans enriched for the anionic species and screening for Abs whose reactivities were abrogated by carboxylate neutralization of bovine lung glycopeptides. One such Ab (mAb GB3.1) was inhibited by carboxylated bovine lung glycopeptides and other multicarboxylated molecules, but not by glycopeptides in which the carboxylate groups were modified. The Ab recognized an epitope constitutively expressed on bovine, human, and other mammalian endothelial cells. Stimulated, but not resting, neutrophils bound to immobilized bovine lung glycopeptides in a carboxylate-dependent manner. The binding of activated neutrophils to immobilized bovine lung glycopeptides was inhibited both by mAb GB3.1 and by soluble glycopeptides in a carboxylate-dependent manner. The Ab also inhibited extravasation of neutrophils and monocytes in a murine model of peritoneal inflammation. This inhibition of cell trafficking correlated with the increased sequestration but reduced transmigration of leukocytes that were found to be adherent to the endothelium of the mesenteric microvasculature. Taken together, these results indicate that these novel carboxylated N-glycans are constitutively expressed on vascular endothelium and participate in acute inflammatory responses by interaction with activated neutrophils.

Larvae of the specialist marine herbivore Alderia modesta (Opisthobranchia: Ascoglossa) metamorphose in response to a chemical settlement cue from the alga Vaucheria longicaulis, the obligate adult prey. Bioactivity coeluted with both high and low molecular weight carbohydrates in solution, and with insoluble high molecular weight carbohydrates associated with the algal cell wall. Larvae metamorphosed in response to water conditioned by V. longicaulis, as well as to frozen and homogenized algal tissue. The inducer was efficiently extracted from the algae with boiling water, but after all soluble activity was extracted, residual tissue still induced larval settlement. Ethanol precipitation of a boiled-water extract followed by gel filtration chromatography showed that the precipitate contained carbohydrates of >100,000 Da molecular weight, while the supernatant contained only low molecular weight carbohydrates (<2,000 Da); in both cases all activity was associated with the carbohydrate peak. An aqueous-insoluble 4% NaOH extract was chromatographed in 7 M urea to yield a bioactive high molecular weight carbohydrate peak. Activity was not affected by proteinase K or mild acid hydrolysis, but was significantly decreased by periodate treatment. The results indicate that larvae of A. modesta metamorphose in response to both water-soluble and surface-associated carbohydrates of V. longicaulis, and that the soluble cue exists as both high and low molecular weight isoforms.

Fluorescent tagging of free oligosaccharides by reductive amination permits sensitive detection and fractionation of these molecules. To expand the scope of this approach, we have synthesized a fluorescent reagent, 2-amino-(6-amidobiotinyl)pyridine. This reagent can tag oligosaccharides under nondegradative conditions with high efciency. The resulting adducts show excellent fractionation by reversephase HPLC with sensitive detection in the low picomole range. When combined with sequential exoglycosidase digestion, stepwise sequencing of the sugar chains is possible. The biotinyl group can also be used to recover the sugar chain from reaction mixtures. The high-affinity interaction of the biotinyl group with multivalent avidin or streptavidin can be used to create the functional equivalent of neoglycoproteins carrying multiple copies of oligosaccharides of dermed structure. These complexes allow the production of IgG antibodies directed against the oligosaccharide chain. They can also harness the power of (strept)avidin-biotin technology for the detection and isolation of oligosaccharide-specific receptors from native sources of recombinant libraries.Many important biological roles of oligosaccharides involve their interaction with specific receptors (1-4). Discovery and exploration of such interactions require comprehensive structural analyses of oligosaccharides, which necessitate their release and fractionation into structurally distinct species. Critical to the fractionation of oligosaccharides is their sensitive and specific detection. However, detection by UV absorption is neither sensitive nor specific, metabolic labeling (5) has limited applicability, and chemical labeling (6, 7) or pulsed-amperometric detection (8) exposes the sugar chains to strongly basic conditions. Also, the last method requires specialized equipment. Alternatively, free oligosaccharides can be tagged via reductive amination with neutral (9, 10) or acidic (11)(12)(13) (18). This can also harness the well-known power of avidin-biotin technology (19). However, as with neoglycoproteins, the underivatized oligosaccharide or glycopeptide must first be purified to homogeneity, prior to biotin coupling. Another approach to studying oligosaccharide biology is to obtain monospecific antibodies (20-23). However, immune responses to free sugar chains are T-cell-independent, and most antibodies are low-affinity IgMs (23), which are technically difficult to work with and likely to show nonspecific cross-reactivity (22). To obtain high-affinity IgG antibodies, the oligosaccharide must be covalently coupled to carrier proteins (20, 23), i.e., neoglycoproteins. Thus, each current approach to the tagging, fractionation, and biological study of oligosaccharides has its own advantages and limitations. Here, we report a reagent for making fluorescent oligosaccharide adducts that allows a more versatile approach to their subsequent study.