Functional dissociation between proforms and mature forms of proteinase 3, azurocidin, and granzyme B in regulation of granulopoiesis.
OBJECTIVE
We previously demonstrated that secreted proform(s) of the neutrophil serine protease PR3 (proteinase 3) can down-modulate the fraction of normal human colony-forming unit granulocyte-macrophage (CFU-GM) in S-phase, whereas PR3 extracted from mature neutrophils lacks this ability. The objective of this study was to characterize the structural and functional dissociation between secreted proforms and granule-stored mature forms and to extend the investigation to other related hematopoietic serine proteases.


METHODS
Conditioned media containing secreted proteases from transfectant cell lines with stable expression of human PR3, neutrophil elastase, cathepsin G, azurocidin, and granzymes A, B, H, K, and M were tested for their ability to reduce the fraction of normal human CFU-GM in S phase. Furthermore, recombinant PR3, azurocidin, and granzyme B with defined N-terminal propeptides, and the respective mature forms without propeptide, were functionally characterized.


RESULTS
In addition to PR3, secreted proforms of azurocidin and granzymes A, B, H, K, and M, but not cathepsin G or neutrophil elastase, have S-phase reducing activity. This activity is restricted to the dipeptide proforms, whereas mature forms without propeptide have no S-phase reducing activity. On the other hand, only the mature forms of PR3 and granzyme B could bind the serine protease inhibitor diisopropylfluorophosphate (DFP), or aprotinin in the case of azurocidin. We also demonstrate that granulocyte colony-stimulating factor-stimulated CD34+ cells and interleukin-2-stimulated lymphocytes secrete active proforms of PR3 and granzyme B, respectively.


CONCLUSIONS
These results demonstrate distinctive functional and conformational differences between proforms and mature forms of these hematopoietic serine proteases and suggest novel growth regulatory mechanisms in granulopoiesis.
Department of Hematology, Institution for Laboratory Medicine, Biomedical Center, Lund University, Lund, Sweden.
Stefan
S
Sköld
Lennart
L
Zeberg
Urban
U
Gullberg
Tor
T
Olofsson
D023181:Antimicrobial Cationic Peptides; D007611:Aprotinin / Q000494:pharmacology; D001773:Blood Cells / Q000187:drug effects; D001798:Blood Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D001854:Bone Marrow Cells / Q000187:drug effects / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D002403:Cathepsins / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured / Q000187:drug effects; D003114:Colony-Forming Units Assay; D017077:Culture Media, Conditioned / Q000494:pharmacology; D003594:Cytoplasmic Granules / Q000378:metabolism; D016179:Granulocyte Colony-Stimulating Factor / Q000494:pharmacology; D006098:Granulocytes / Q000166:cytology*; D053804:Granzymes; D006410:Hematopoiesis / Q000502:physiology*; D006412:Hematopoietic Stem Cells / Q000201:enzymology* / Q000378:metabolism / Q000648:ultrastructure; D006801:Humans; D007376:Interleukin-2 / Q000494:pharmacology; D007531:Isoflurophate / Q000494:pharmacology; D007668:Kidney / Q000166:cytology; D019272:Leukocyte Elastase / Q000378:metabolism; D053146:Myeloblastin; D011487:Protein Conformation; D011498:Protein Precursors / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000502:physiology; D016196:S Phase / Q000187:drug effects; D017384:Sequence Deletion; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D014162:Transfection
C062991:AZU1 protein, human; D023181:Antimicrobial Cationic Peptides; D001798:Blood Proteins; D002352:Carrier Proteins; D017077:Culture Media, Conditioned; D007376:Interleukin-2; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D015842:Serine Proteinase Inhibitors; D007531:Isoflurophate; D016179:Granulocyte Colony-Stimulating Factor; D007611:Aprotinin; D002403:Cathepsins; C509213:GZMB protein, human; D053804:Granzymes; D012697:Serine Endopeptidases; D019272:Leukocyte Elastase; D053146:Myeloblastin