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Characterization of dendritic cell and macrophage cell surface proteins

University of British Columbia

Dendritic cells play a critical role in the induction of the immune response and execute unique functions in comparison to other antigen presenting cells such as macrophages and B cells. The research in this thesis was directed towards characterizing molecular components of dendritic cells and macrophages. The molecules chosen for study were CD45 and an uncharacterized protein, the F4/80 molecule. CD45 is an important receptor tyrosine phosphatase expressed as various isoforms due to highly regulated alternative splicing. Analysis of CD45 cell surface expression on purified, cultured splenic and thymic dendritic cells, and freshly isolated dendritic cells, demonstrates that dendritic cells express both the CD45R0 and CD45RB isoforms. Biochemical analysis of purified splenic dendritic cells confirms the expression of CD45R0 and CD45RB. In addition, purified splenic dendritic cell CD45 possesses intrinsic tyrosine phosphatase activity. Macrophage CD45 isoform expression is also limited largely to CD45R0 and small amounts of CD45RB or CD45RA depending on the macrophage source. CD45 from macrophages possesses comparable tyrosine phosphatase activity relative to dendritic cell CD45. The demonstration of CD45 phosphatase activity in cells lacking antigen receptors suggests a possible role for CD45 in leukocyte events such as recirculation, homing, heterophilic adhesion events, or signalling via non-antigen specific receptors. The F4/80 molecule was first characterized over a decade ago and is known to be expressed on dendritic cells and macrophages. However, few molecular details of the F4/80 molecule had been elucidated. This study documents that the F4/80 molecule contains N-linked carbohydrates, O-linked carbohydrates, sialic acids primarily in α2-6 linkages to galactose, and chondroitin sulfate modifications. Furthermore, the extensive N-linked carbohydrate modifications contain a dominant component of repeating N-acetylglucosamine or N acetyllactosamine units. The protein contains extensive intramolecular disulfide bonds, has a slow rate of intracellular transport (T[sub]1/2=60 minutes), and has a basic pi of 7.5 for the entire molecule in spite of the negative post-translational modifications. The modifications of the F4/80 molecule described may also account for certain functional properties. For example, chondroitin sulfate can mediate interactions with CD44 and fibronectin, whereas α2-6 linked sialic acids are involved in the binding to CD22. Thus, a substantial description of the biochemical nature of the F4/80 molecule is provided, which leads to direct inferences for potential functions of the molecule. In characterizing CD45 and the F4/80 molecule, this work provides a better understanding of two proteins of dendritic cells and macrophages. This information should assist both the basic understanding of dendritic cell and macrophage biology and in developing experimental and clinical targets for functional modulation of dendritic cells and macrophages.

Thesis/Dissertation

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2009-02-20

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http://hdl.handle.net/2429/4842

Microbiology and Immunology

University of British Columbia

UBCV

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