Selectins on activated vascular endothelium mediate inflammation by binding to complementary carbohydrates on circulating neutrophils. The human neutrophil receptor for E-selectin has not been established. We report here that sialylated glycosphingolipids with 5 Nacetyllactosamine (LacNAc, Gal␤1-4Glc-NAc␤1-3) repeats and 2 to 3 fucose residues are major functional E-selectin receptors on human neutrophils. Glycolipids were extracted from 10 10 normal peripheral blood human neutrophils. Individual glycolipid species were resolved by chromatography, adsorbed as model membrane monolayers and selectinmediated cell tethering and rolling under fluid shear was quantified as a function of glycolipid density. E-selectin-expressing cells tethered and rolled on selected glycolipids, whereas P-selectin-expressing cells failed to interact. Quantitatively minor terminally sialylated glycosphingolipids with 5 to 6 LacNAc repeats and 2 to 3 fucose residues were highly potent E-selectin receptors, constituting more than 60% of the E-selectin-binding activity in the extract. These glycolipids are expressed on human blood neutrophils at densities exceeding those required to support E-selectin-mediated tethering and rolling. Blocking glycosphingolipid biosynthesis in cultured human neutrophils diminished E-selectin, but not Pselectin, adhesion. The data support the conclusion that on human neutrophils the glycosphingolipid NeuAc␣2-3Gal IntroductionMultiple mechanisms ensure that granulocytes efficiently move into tissues when and where needed. 1 Activation of the blood vessel endothelium results in circulating granulocytes tethering and rolling, stopping, flattening, and squeezing into the surrounding tissue. A 3-step model describes leukocyte extravasation 2 : (1) Eand P-selectin on activated endothelia bind to glycans on leukocytes, initiating tethering and rolling under the shear stress of blood flow (L-selectin on some leukocytes contributes to this process).(2) Upon tethering, leukocytes are exposed to chemoattractants (eg, chemokines) generated by activated endothelium and tissueresident cells, resulting in integrin mobilization. (3) Strong cell adhesion is mediated by leukocyte integrin binding to immunoglobulin superfamily proteins on the endothelium. Each step is required for recruitment of leukocytes; blocking any one diminishes inflammation. Despite exceptions, 3 this scheme is broadly applicable.Each selectin (E-, L-, and P-) 4 has a carbohydrate recognition domain that mediates binding to specific glycans on apposing cells. They have remarkably similar protein folds and carbohydrate binding residues, 5 leading to overlap in the glycans to which they bind. Nevertheless, each selectin has distinct receptors to which it binds with high affinity.Selectins bind to the sialyl Lewis x (SLe x ) determinant "NeuAc␣2-3Gal␤1-4(Fuc␣1-3)GlcNAc." [6][7][8][9] However, SLe x , per se, does not constitute an effective selectin receptor. [10][11][12] Instead, SLe x and related sialylated, fucosylated glycans are components of more ext...

Infrared atmospheric pressure matrix-assisted laser desorption/ionization on an ion trap mass spectrometer is used to study sialylated oligosaccharides desorbed from the liquid phase. Glycerol doped with various cations provides the opportunity to produce cation-adducted intact molecular ions of sugars. Distinct combinations of cations allow for sialic acid stabilization, as well as differential cleavage, resulting in more complete fragmentation coverage of the oligosaccharide. Alkali and transition metal cations are utilized to create three distinct molecular ion species, involving the adduction of a singly charged cation, two singly charged cations, or a doubly charged cation. From these different molecular ion types, complementary sequence, branching, and linkage information for sialylated oligosaccharides can be deduced.

A 2.94-microm Er:YAG laser for IR atmospheric pressure matrix-assisted laser desorption/ionization on an ion trap mass spectrometer is used for the analysis of sialylated oligosaccharides. This approach provided the opportunity to utilize liquid matrixes and is effective in determining structural features--sequence, branching, and linkage--of intact, fully sialylated molecular species.

An investigation of sugar-sugar noncovalent complex fragmentation was conducted using a 2.94 m Er:YAG laser for infrared (IR) atmospheric pressure matrix-assisted laser desorption/ ionization (AP MALDI) on an ion trap mass spectrometer (ITMS). This approach allowed the analysis of weak noncovalent complexes between a variety of biologically relevant oligosaccharides. The strength of interaction varied with different sugar structures, potentially due to varying strength of hydrogen bonding networks. In some cases, fragmentation of intramolecular sugar bonds preceded breakdown of the noncovalent complex. This result appeared primarily when complexes contained sugars with at least one sialic acid. Globotrios dimers also showed intramolecular fragmentation in preference to breakdown of the noncovalent dimer. This technique will allow further study of sugar-sugar interactions known to play a role

Infrared atmospheric pressure matrix-assisted laser desorption/ionization quadrupole ion trap mass spectrometry was applied to the study of siglec binding to oligosaccharide ligands. Peptides were designed to mimic the binding sites of three members of the siglec family: sialoadhesin, MAG and CD22. These peptides were tested for their ability to complex with their carbohydrate ligands 3'-sialyllactose (3'SL) and 6'-sialyllactose (6'SL). All peptides demonstrated the ability to bind to the carbohydrates, with the peptide representing sialoadhesin maintaining its binding specificity for 3'SL in preference to 6'SL. This technique can be used to study other protein-sugar interactions and can be expanded to create high-throughput screening techniques.

Infrared atmospheric pressure matrix-assisted laser desorption/ionization on an ion trap mass spectrometer is used to analyze frozen samples generated using a Peltier-cooled sample stage. This allows for the analysis of samples in water without the addition of matrix, in near-native conditions, and with minimal loss of water due to evaporation. Analysis of frozen samples is extended to study peptides, carbohydrates, and glycolipids.