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- Bülow, Elinor
(författare)
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Sorting and processing of neutrophil granule proteins
- 2002
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)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.
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| 2. |
- Bülow, Elinor, et al.
(författare)
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Sorting for storage in myeloid cells of nonmyeloid proteins and chimeras with the propeptide of myeloperoxidase precursor.
- 2002
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Ingår i: Journal of Leukocyte Biology. - 1938-3673. ; 71:2, s. 279-288
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Tidskriftsartikel (refereegranskat)abstract
- During formation of polymorphonuclear neutrophils, proteins are synthesized for storage in granules. Whereas sorting of proteins into distinct subtypes of cytoplasmic granules may reflect the coordinated expression of the proteins contained in them, still the mechanism(s) for the retrieval of proteins from the constitutive secretion is unknown. To investigate the mechanisms of retrieval, nonmyeloid secretory proteins were expressed in myeloid cell lines, and their subcellular fate was assessed. The contribution of the propeptide (MPOpro) of the myeloperoxidase (MPO) precursor was investigated by determining the fate of chimeras containing MPOpro. The nonmyeloid protein alpha(1)-microglobulin (alpha(1)-m) was targeted to storage organelles in 32D cells and colocalized with the lysosomal marker LAMP-1, whereas soluble TNF receptor 1 (sTNFR1) was secreted without granule targeting. Fusion of MPOpro to alpha(1)-m delayed exit from endoplasmic reticulum (ER), but subsequent targeting to dense organelles was indistinguishable from that of alpha(1)-m alone. Fusion proteins between MPOpro and sTNFR1 or green fluorescent protein expressed in myeloid 32D, K562, or PLB-985 cells did not associate stably with calreticulin or calnexin, molecular chaperones that normally interact transiently with the MPO precursor, but were still efficiently retained in the ER followed by degradation. We conclude that normally secreted, nonmyeloid proteins can be targeted efficiently to storage organelles in myeloid cells, that myeloid cells selectively target some proteins for storage but not others, and that MPOpro may contribute to the prolonged ER retention of the MPO precursor independent of the ER-molecular chaperones calreticulin and calnexin.
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| 3. |
- Bülow, Elinor, et al.
(författare)
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Sorting of neutrophil-specific granule protein human cathelicidin, hCAP-18, when constitutively expressed in myeloid cells.
- 2002
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Ingår i: Journal of Leukocyte Biology. - 1938-3673. ; 72:1, s. 147-153
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Tidskriftsartikel (refereegranskat)abstract
- Neutrophil granulocytes carry storage organelles, e.g., azurophil and specific granules. Poorly understood are the mechanisms for retrieval from constitutive secretion followed by sorting for storage. Therefore, we asked whether the specific granule protein human cathelicidin (hCAP-18) could be sorted for storage in other granules when the biosynthetic window is widened to allow this. We observed that hCAP-18 was targeted for storage in lysosome-related organelles when expressed constitutively in the rat basophilic leukemia and the mouse promyelocytic (MPRO) cell lines. In addition, premature release of the antibiotic C-terminal peptide LL-37 was observed. Retention of hCAP-18 was diminished by induction of differentiation of MPRO cells. In conclusion, a specific granule protein with native conformation may be sorted for storage in lysosome-related organelles of myeloid cells and converted prematurely to a supposedly biologically active form.
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