Biomedical subjects

C R Bertozzi

Publications and source records attributed to C R Bertozzi.

45 records · Page 3Linked to original sources

PubMed1997

An ELISA for selectins based on binding to a physiological ligand.

Members of the selectin family of adhesion receptors, consisting of L-, P- and E-selectin, mediate the initial interaction between leukocytes and endothelium during leukocyte trafficking from the blood into tissue sites. These receptors have attracted great attention in recent years due to their participation in a number of acute and chronic inflammatory diseases. We describe here a new ELISA that measures the binding between selectin-IgG chimeras and a physiological ligand for L-selectin and can be used to screen selectin inhibitors. The ligand used is a mucin-like glycoprotein known as GlyCAM-1, which is derived from high endothelial venules in secondary lymphoid organs. We demonstrate binding of all three selectins to GlyCAM-1 and demonstrate that the binding interactions satisfy a number of important criteria. The advantage of this ELISA over previous assays is that a macromolecular physiological ligand is employed, rather than a fortuitous or simplified carbohydrate ligand. Thus, the protein-carbohydrate interactions, as well as other interactions contributing to ligand recognition, can be investigated. The assay is suitable for high-throughout screening of compounds and may find use in the identification of selectin antagonists with anti-inflammatory potential.

Animals↗

PubMed1997

Engineered cell surfaces: fertile ground for molecular landscaping.

The cell surface contains a wealth of information that determines how cells interact with their environment. Methods for directing the cell surface expression of novel protein-based and oligosaccharide-based epitopes are stimulating new directions in biotechnology and biomedical research.

Biotechnology↗

PubMed1996

L-selectin-carbohydrate interactions: relevant modifications of the Lewis x trisaccharide.

Protein-carbohydrate interactions are known to mediate cell-cell recognition and adhesion events. Specifically, three carbohydrate binding proteins termed selectins (E-, P-, and L-selectin) have been shown to be essential for leukocyte rolling along the vascular endothelium, the first step in the recruitment of leukocytes from the blood into inflammatory sites or into secondary lymphoid organs. Although this phenomenon is well-established, little is known about the molecular-level interactions on which it depends. All three selectins recognize sulfated and sialylated derivatives of the Lewis x [Le(x):Gal beta 1-->4(Fuc alpha 1-->3)GlcNAc] and Lewis a [Le(a): Gal beta 1-->3(Fuc alpha 1-->4)GlcNAc] trisaccharide cores with affinities in the millimolar range, and it is believed that variants of these structures are the carbohydrate determinants of selectin recognition. Recently it was shown that the mucin GlyCAM-1, a secreted physiological ligand for L-selectin, is capped with sulfated derivatives of sialyl Lewis x [sLe(x): Sia alpha 2-->3Gal beta 1-->4(Fuc alpha 1-->3)GlcNAc] and that sulfation is required for the high-affinity interaction between GlyCAM-1 and L-selectin. To elucidate the important sites of sulfation on Le(x) with respect to L-selectin recognition, we have synthesized six sulfated Le(x) analogs and determined their abilities to block binding of a recombinant L-selectin-Ig chimera to immobilized GlyCAM-1. Our results suggest that 6-sulfo sLe(x) binds to L-selectin with higher affinity than does sLe(x) or 6'-sulfo sLe(x) and that sulfation of sLe(x) capping groups on GlyCAM-1 at the 6-position is important for L-selectin recognition.

Carbohydrate Metabolism↗

PubMed1996

Two selectins converge on sulphate. Leukocyte adhesion.

Selectins mediate the attachment of leukocytes to endothelial cells and to platelets under conditions of blood flow. Sulphation of the ligands for two of the selectins is required for optimal cell-cell interactions.

Animals↗

PubMed1995

Sulfated disaccharide inhibitors of L-selectin: deriving structural leads from a physiological selectin ligand.

The selectins are a family of three adhesion molecules (L-, P-, and E-) that direct the interaction of circulating leukocytes with endothelial cells during the first step in recruitment to tissue sites. Their involvement in inflammatory disease makes the selectins attractive targets for anti-inflammatory therapy. The sialyl Lewis x tetrasaccharide binds weakly to all three selectins and has demonstrated anti-inflammatory activity in vivo. However, the synthetic difficulties inherent to sialylated and fucosylated oligosaccharides motivate the search for alternative antagonists. Here we demonstrate that information gained from the biochemical analysis of a physiological selectin ligand can provide new leads for small molecule design. Previous structural analysis of the oligosaccharide chains on GlyCAM-1, an endothelial-derived ligand for L-selectin, revealed two novel structures: 6'-sulfo sialyl Lewis x and 6-sulfo sialyl Lewis x. The sulfate esters on these structures are thought to be essential for high-affinity binding to L-selectin. By incorporating sulfate esters on the analogous positions of the disaccharide lactose, we generated a simple small molecule (lactose 6',6-disulfate) with greater inhibitory potency for L-selectin than sialyl Lewis x.

Carbohydrate Sequence↗

PubMed1995

Cracking the carbohydrate code for selectin recognition.

Selectins are central in the inflammatory response; the discovery that they bind to carbohydrate ligands has galvanized carbohydrate chemists to search for inhibitors of the process. Recent progress in identifying and analyzing physiological selectin counter-receptors suggests new approaches to the design of ligands that bind to specific selectins.

Animals↗

PubMed1994

Identification of the sulfated monosaccharides of GlyCAM-1, an endothelial-derived ligand for L-selectin.

L-Selectin, a receptor bearing a C-type lectin domain, mediates the initial attachment of lymphocytes to high endothelial venules of lymph nodes. One of the endothelial-derived ligands for L-selectin is GlyCAM-1 (previously known as Sgp50), a mucin-like glycoprotein with sulfated, sialylated, and fucosylated O-linked oligosaccharide chains. Sialylation, sulfation, and fucosylation appear to be required for the avid interaction of this ligand with L-selectin, but the exact carbohydrate structures involved in recognition remain undefined. In this study, we examine the nature of the sulfate-modified carbohydrates of GlyCAM-1. GlyCAM-1 was metabolically labeled in lymph node organ culture with 35SO4 and a panel of tritiated carbohydrate precursors. Mild hydrolysis conditions were established that released sulfated oligosaccharides without cleavage of sulfate esters. Low molecular weight and singly charged fragments, obtained by a combination of gel filtration and anion-exchange chromatography, were analyzed. The structural identification of the fragments relied on the use of a variety of radiolabeled sugar precursors, further chemical and enzymatic hydrolysis, and high-pH anion-exchange chromatography analysis. Sulfated constituents of GlyCAM-1 were identified as Gal-6-SO4, GlcNAc-6-SO4, (SO4-6)Gal beta 1-->4GlcNAc, and Gal beta 1-->4(SO4-6)GlcNAc. In the accompanying paper [Hemmerich, S., & Rosen, S.D. (1994) Biochemistry 33, 4830-4835] evidence is presented that (SO4-6)Gal beta 1-->4GlcNAc forms the core of a sulfated sialyl Lewis x structure that may comprise a recognition determinant on GlyCAM-1.

Acetylglucosamine↗

PubMed1994

The selectins and their ligands.

The selectins are a family of carbohydrate-binding proteins, or lectins, that have stimulated tremendous interest because of their involvement in a wide array of interactions between leukocytes and endothelial cells. Highlights of recent progress include an extension of the list of instances of selectin participation in inflammatory diseases, further definition of selectin carbohydrate specificities, and identification of their carbohydrate-based ligands.

Animals↗

PubMed1992

Fluorescence probes in biochemistry: an examination of the non-fluorescent behavior of dansylamide by photoacoustic calorimetry.

Photoacoustic calorimetry is shown to be a simple, precise, and accurate method for the quantification of the photophysics of a fluorescence probe, e.g., dansylamide, in a variety of solvents. This technique, which is described in detail, provides a direct measurement of the energy that is released nonradiatively following photostimulation, and can therefore be used to indirectly determine the amount of energy released via luminescent pathways. Photoacoustic calorimetry combined with established absorption and fluorescence methodologies provides a complete arsenal for characterizing the photophysical properties of many systems. Comparison of the photoacoustic signal for dansylamide versus standard compounds (ferrocene, tetraphenylethylene, 8-anilinonaphthalene-1-sulfonate, and/or 5,5'-dithiobis(2-nitrobenzoic acid) in 12 different solvents gave fh values (fraction of each absorbed 337.1-nm photon returned as heat) from a low of 0.530 in 1,4-dioxane to a high of 0.973 in water. The trend noted with solvent polarity is different and more revealing than that determined by the more classical approach of examining either the wavelength of the emission maximum or the fluorescence quantum yield.

Calorimetry↗