The biochemical mechanisms underlying phagocytosis are still largely unknown. Two groups have previously determined that the glucose analogue 2-deoxyglucose (2DG) inhibits opsonin-dependent phagocytosis in a manner unrelated to energy production. We have recently described a cytoplasmic phosphoglycoprotein of 63 kDa (pgp 63) possessing an oligosaccharide that appears to exist in two interconvertible states, one as an O-linked mannose disaccharide and the other containing, in addition, a phosphodiester-linked glucose residue. We have also described the two antagonistic enzymes responsible for the removal and addition of the alphaGlc-1-P. Our data in other systems suggest that pgp63 may be intimately involved in the CA++-dependent cascade leading to exocytic vesicle release and that the removal and addition of alphaGlc-1-P is an integral part of this cascade. We intend to critically test the related hypotheses that a) the inhibitory effect of 2DG seen in opsonin-dependent phagocytosis is due to its conversion to UDP-2DG and the nucleotide sugar's subsequent effect on alphaGlc-1-P turnover on pgp63 and b) that pgp63 plays an important role in macrophage and neutrophil phagocytic function.
The specific aims of the proposal are: 1. To partially characterize the Glc phosphotransferase and its pgp63 acceptor in human neutrophils and mouse macrophages and to determine if a variety of treatments that modulate various aspects of neutrophil or macrophage function affect the levels of alphaGlc-1-P transfer to pgp63. 2. To determine if the inhibition of phagocytosis caused by 2DG can be attributed to its conversion to the 2-deoxy analogue of UDP-Glc, and also to determine if the beta-phosphorothioate analogue of UDP-Glc has similar inhibitory effects. 3. To determine the topographic distribution of pgp63 in macrophages at rest and as phagocytosis proceeds. 4. To determine if decrease in functional pgp63, induced by the introduction into the cytoplasm of either a monospecific antibody, phosphorothioate-containing pgp63, or a specific antisense oligonucleotide, affects phagocytic function in macrophages.
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