The selectins are adhesion molecules belonging to the C-type lectin family. They are expressed constitutively on the surface of blood leukocytes and on activated endothelial cells and platelets. The selectins prominently bind O-linked glycans that are bound to Ser/Thr residues on a variety of glycoprotein scaffolds. Such binding between selectins and O-glycans initiate a series of steps that eventually results in the adhesion of leukocytes to blood vessel walls at sites of inflammation. This biomolecular interaction also conditions normal immune response, and it plays an important role in certain types of cardiovascular disorders and cancer metastatic processes. It is widely believed that controlling the rate of leukocyte adhesion by antagonizing selectin-ligand interactions can lead to new therapies to combat a variety of vascular ailments. Thus, in this proposal, we develop metabolic strategies to control leukocyte-endothelial interactions by engineering the glycans that are expressed on natural selectin-ligands.
The specific aims are:
Aim 1 : To evaluate the feasibility of using monosaccharide analogs to alter glycan structures and leukocyte cell adhesion properties. Here, we test the possibility that analogs of the naturally occurring monosaccharide, GalNAc and also Fucose, can be fed to cells in order to modify either the core or terminal structures of glycans that function as the natural ligands for the selectins. We evaluate the ability and mechanism by which these chemical inhibitors permeate cells, engage and modify glycan biosynthetic pathways, and inhibit cell adhesion.
Aim 2 : To define the precise 1(2,3) sialyltransferase(s) regulating leukocyte adhesion to L-, E- and P-selectin in humans. Here, we either over- express specific sialyltransferases belonging to the ST3Gal family in leukocytes, or stably silence one or more ST3Gals at the same time in these cells. The effect of this "system perturbation" on selectin-ligand glycan structure and leukocyte cell adhesion function under fluid shear is evaluated.
Aim 3 : To determine the role for reversible sialylation in regulating leukocyte selectin-ligand structure. Here, we quantify the extent to which human ST3Gals catalyze readily reversible reactions, a property called 'reversible sialylation'. We then evaluate if 'reversible sialylation'plays an important role during the biosynthesis of selectin-ligands. Success in this aim will demonstrate that besides enzymatic rate constants that drive glycoconjugate synthesis in living cells, equilibrium processes governed by reversible enzymatic activity may also control glycan biosynthesis. Diverse experimental methods are applied to accomplish the above aims. These include cell adhesion studies under controlled flow conditions, in vivo experiments in a mouse model of inflammation, and lentiviral strategies to silence or overexpress specific genes in human leukocytes and hematopoietic progenitor/stem cells. In the long run, we anticipate that novel strategies to antagonize selectin-ligand binding interactions will be identified from this work that may aid future drug design.
This proposal lies at the interface of bioengineering, biochemistry and medicine. It studies protein-carbohydrate binding interactions that regulate the adhesion of blood leukocytes (white blood cells) to endothelial cells that line blood vessel walls. The goal is to develop novel glycan/carbohydrate engineering approaches to modulate this cell adhesion event, since this can lead to new strategies to ameliorate inflammatory ailments.
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