This Project will examine the distribution and biosynthesis of ligands for the two lectins CD33 and Sialoadhesin (Sn), the distribution of the lectins themselves, and identify functional roles for these lectins in myelopoiesis and immune function. CD33 and Sn are two I-type lectins with considerable similarity including a measurable binding affinity for alpha2-3 sialylated oligosaccharides. Sn is found on subsets of mature macrophages and CD33 on both early myeloid precursors and mature macrophages. While both lectins bind to cells expressing alpha2-3 linked Sia residues, not all cells react to the same extent, indicating that these lectins recognize specific and distinct alpha2-3 sialylated ligands. This observation is of considerable interest, as alpha2-3 Sia residues are the product of a family of alpha2-3 sialyltransferases. Thus, these lectins are potentially capable of discriminating between the products of these different sialyltransferases. The objectives of this Project will be accomplished by pursuing four aims: (1) Determine the expression of ligands for Sn and CD33 in wild type mice and in mice with null mutations in four different alpha2-3 sialyltransferase genes (ST3Gal I, ST3Gal II, ST3Gal III, and ST3Gal IV; from Project 1). Similar studies will be done in mice with a null mutation in sialate:9-O-acetylesterase (from Project 4) which are anticipated to over express 9-O-acetylated Sia residues which can block lectin binding. These studies will establish which tissues express potential ligands, and the relative contributions of different enzymes to their synthesis. (2) Establish the cellular distribution of CD33 in mice. (3) Employ ES cell technology to establish CD33 null or Sn null mice. Tissue specific deletion, using the Cre-loxP approach, will be utilized to circumvent potential embryonic lethality. (4) Examine these mice for specific defects in myelomonocytic cell function. This will be approached by testing specific hypotheses for CD33 and Sn involvement in cell adhesion, by examining the following: hematopoiesis, monocyte trafficking and margination, monocyte-dependent lymphocyte responses, clearance of pathogenic organisms, and granuloma formation. Functional defects demonstrated will likewise be probed for in mice with null alleles of alpha2-3 sialyltransferases or sialate:9-O-acetylesterase, to corroborate a role of lectin-sialic acid recognition in the observed phenotype. These studies will establish the functional roles of specific Sia structures in normal myelopoiesis and relate their function to human disease.
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