This application describes a proposal to investigate the specificity and biological roles of the siglecs (sialic acid binding lectins) expressed on leukocytes. In the first aim the applicant will use the surface plasmon resonance system to determine siglec Kd values against soluble oligosaccharides, both commercially available as well as synthesized de novo (in collaboration with another postdoctoral fellow). This will allow for the direct quantitative comparison of all marine and human siglecs, important for the future use of murine systems as models for human siglec function. Additionally, based on the monovalent Kd values, polyvalent probes will be developed and screened for their ability to bind to siglec expressing cells, the goal of this being the development of the polyvalent fluorescent probe specific for each immune siglec. Emphasis will be placed on developing a probe for Siglec 2 (CD22) which is an important regulator in B-cell stimulation, including a potential role in the control of autoimmunity. Additionally, recent data has illustrated that the siglecs may be functionally regulated by glycosylation, making them unavailable for ligand recognition. As the proposed function of many of the siglecs is based on their expression profile, understanding the timing of this regulation will allow a better understanding of siglec function. These unique probes will allow for the evaluation of CD22 cell surface expression as compared to ligand accessibility (aim III) which may be controlled by the expression of a specific sialyltransferase or neuraminidase. As current data is contradictory concerning the role of CD22 and its ligand in B- cell regulation (aim IV) these probes will be used in the analysis of CD22 localization and function in B-cells from ST6GalI -/- mice that lack the endogenous (cis or traps) ligands for CD22. As time allows the applicant will use the probes to subsequently investigate the expression of other immune siglecs.
