Sorting and processing of neutrophil granule proteins

Abstract: The neutrophil granulocytes have a critical role in innate immunity in particular through phagocytosis of microorganisms. Their cytoplasmic granule subsets store digestive enzymes and antibiotic proteins that are released during this event. During the passage in the secretory pathway the granule proteins are retrieved from constitutive secretion to become sorted for storage. The aim of this thesis was to understand retrieval and sorting for storage through studying processing and sorting of MPO, BPI and hCAP-18 and exogenous secretory proteins. We transfected cDNA of these human proteins to the murine myeloid RBL and 32D cell lines. Mutations of cDNA were made before transfection. A propeptide- deleted MPO precursor was not processed to mature MPO but degraded or secreted, indicating that the propeptide is a prerequisite for final processing and targeting of proMPO to granules. Chimeras between the MPO propeptide and secretory proteins prolonged the ER retention compared with that of the native proteins. Thus, the propeptide may mediate the long ER-residence of proMPO. The amino-terminal half of BPI, when expressed alone, was targeted for storage, while the carboxy-terminal half was not, but, it increased the stability of BPI and chimeric proteins, indicating that this half is important for stability while the amino-terminal half may be important for sorting. The non-myeloid LBP and alpha1-microglobulin were targeted to, but unstable, in granules, indicating cell-specific rather than protein-specific sorting in the cells. But not all proteins were sorted, e g sTNFR1 and propeptide-deleted MPO were secreted, suggesting that physico-chemical properties affect sorting. hCAP-18 was targeted, i e missorted, to the lysosome-related organelles of RBL cells with subsequent prematurely processing and degradation. Thus, the results of the thesis indicate cell-specific retention mechanisms for sorting for storage in myeloid cells.