): The majority of human cancer deaths are due to metastasis, a process that is poorly understood at the molecular and cellular levels. In the multistep process of metastasis one expects significant roles for cell adhesion molecules. CD44, a cell surface receptor for hyaluronan (HA) that functions in cell adhesion, cell migration, and cell activation, has been implicated in the process of metastasis. CD44 has the property of regulated affinity for its ligand. The role of glycosylation in regulating CD44-mediated hyaluronan binding will therefore be examined. This study is consistent with the candidate's long-term objective of elucidating enough detail of the mechanism by which glycosylation regulates cell adhesion molecule function to enable modulation of this mechanism as an intervention f o r metastasis. Preliminary studies have identified oligosaccharide structures which impact the intrinsic HA-binding properties of CD44. The present proposal aims to determine which structures are biologically regulated and to elucidate the enzymatic basis for regulation of the functionally i m portant glycan structure. After identification of the key glycosyltransferase, the mechanism employed by the cell to regulate CD44 glycosylation as part of an integrated cell behavior will be defined. Mechanisms to be examined include synthetic glycosyltransferase expression, glycosyltransferase phosphorylation, CD44 alternative RNA splicing, and interacting glycosyltransferases. CD44 oligosaccharide structure will be c h a n ged by glycosidase digestion and glycosyltransferase modulation. I n t rinsic CD44 HA-affinity will be determined by affinity capillary e l e ctrophoresis. Structure/function correlations will be drawn. A physiologic change in CD44 glycosylation and hyaluronan-affinity will be tested upon cellular activation. The effects on metastasis of manipulating CD44 HA-affinity by glycosyltransferase modulation will be determined using an in vivo model. This proposal will serve as a basis to develop a general approach to defining the regulatory role of glycosylation in cell-cell and cell-matrix adhesion.
