The objective of this proposal is to characterize the structural, functional and molecular biological properties of a neutrophil surface glycoprotein (GP110). These studies are based upon the observation that there is a decrease in the cell surface expression of GP110 in neutrophils from patients with Localized Juvenile Periodontitis (LJP) which exhibits a strong correlation with chemotactic dysfunction in these subjects. Neutrophils represent a highly mature stage inflammatory cell differentiation and they are not a rich source of mRNA that would code for GP110. It is, therefore, reasonable to look for a model system which can provide sufficient quantities for mRNA coding for GP110. Cells of the HL-60 line (acute leukemic cell line) have been shown to be capable of producing GP110 after chemical induction to differentiation. Therefore, differentiating HL-60 cells can be used as a substitute for neutrophils from healthy individuals. Non- differentiating HL-60 cells do not make GP110 and, therefore, can be used as controls. This project will be divided into two phases which will coincide with the phases of the training program. Ultimately the goal of these efforts is to determine the relationship of GP110 to neutrophil function. Phase I is designed to familiarize the candidate with the technology and literature related to the project. Phase II involves more sophisticated concepts and technology and is planned to fulfil the requirements leading to the Ph.D. degree. Phase I of this proposal is the structural characterization of GP110 which will include isolation of GP110 from human neutrophils, LJP neutrophils and HL-60 cells by affinity chromatography using monoclonal antibody; determination of amino acid composition and partial sequences of GP110; nature of linkage between oligosaccharide and the protein core of GP110, composition of oligosaccharide of GP110 and determination of the extent of modification of this protein such as methylation, phosphorylation, acetylation or other post-translational modifications. The partial peptide sequences for GP110 will be used for synthesizing oligonucleotide probes. Phase II of this proposal is to determine if the decrease in the GP110 content of LJP neutrophils is genetic in origin. These studies include: creation of a cDNA library for differentiated HL-60 cells, identification and cloning of the gene for GP110 using oligonucleotide probes and monoclonal antibody. Southern blot analysis of the gene structure will be done using a large battery of restriction endonucleases. Thee experiments are aimed at describing normal gene structure and organization and to detect polymorphism. LJP neutrophil material will also be analyzed for the remote possibility of the detection of a deletion or mutation. LJP gene structure will be evaluated for deletion or mutation by RNAse A cleavage.
