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.
