Differential regulation of T cell plasma membrane proteins by N-glycan branching and clathrin-mediated endocytosis - PROJECT SUMMARY/ABSTRACT Nearly every aspect of T cell biology is determined in part by the relative expression of a multitude of cell-surface proteins. For example, the relative expression of co-stimulatory and co-inhibitory receptors impact pro- versus anti-inflammatory outcomes. N-glycosylation is a critical but poorly understood regulator of cell- surface protein turnover. Virtually every cell-surface and secreted protein is modified with covalently attached complex carbohydrates at asparagine (N) residues. These N-glycans are variably edited by a large number of glycosylation enzymes along the secretory pathway, producing many possible glycan structures. The N-glycan branching pathway serves as a key gateway between relatively simple high-mannose structures and more complex N-glycans that carry glyco-epitopes recognized by numerous families of carbohydrate-binding proteins, known as lectins. The branching pathway controls N-acetyllactosamine (LacNAc) incorporation into N-glycans, which are recognized by the galectin family of soluble lectins. At the cell surface, the interaction of multivalent soluble galectins with glycoproteins carrying LacNAc units, leads to the formation of a molecular lattice-like structure. This galectin-glycoprotein lattice affects cell surface organization, receptor mobility in the plane of the membrane, and endocytosis rates. In part by regulating cell surface expression of CD4, CD8, IL2Rα, CTLA-4, and the T cell receptor (TCR), N-glycan branching regulates T cell development, TCR signaling, T cell activation, T cell proliferation and pro-inflammatory versus anti-inflammatory differentiation. Based on studies of these receptors, a model has emerged of the lattice as a unidirectional regulator of cell- surface retention which opposes glycoprotein loss and promotes cell surface retention. However counter- examples to this model have recently emerged demonstrating that some receptors are regulated in the opposite manner. Thus a comprehensive and unbiased analysis of branching regulated changes is needed. Furthermore, an understanding of the mechanisms involved that promote expression of some receptors while hindering expression of others is also lacking. Without such detailed information, therapeutic targeting of the complex glycosylation pathways that regulate T cell function will be limited. We have developed an approach to examine branching mediated effects on cell-surface expression at the proteome scale. We provide proof-of- concept that this approach is reliable and informative and propose to use it to tackle this outstanding aspect of T cell biology. In Aim1, we will extend this approach to more physiologically relevant primary T cells and examine a range of N-glycan branching states to derive a complete and informative picture. We will also adapt our approach to globally determine endocytosis and recycling rates of branching regulated proteins. In Aim 2, we will test the hypothesis that cargo adapter proteins in the clathrin-mediated endocytosis pathway cooperate with N-glycan branching to differentially regulate receptor turnover and expression. Together these studies will dramatically increase our knowledge of branching mediated regulation of T cell biology.
