The N-terminal tetrapeptide of neutrophil proteinase 3 causes S-phase arrest in granulopoietic progenitors.
OBJECTIVE
Secreted enzymatically inactive proforms of hematopoietic serine proteases proteinase 3 (PR3), azurocidin, and granzymes A, B, H, K, and M are able to reduce the fraction of granulopoietic progenitors (CFU-GM) in S-phase, whereas human leukocyte elastase (HLE) and cathepsin G lack this ability. The objective of the present study was to map the specific sequence(s) of PR3 and other hematopoietic serine proteases responsible for the downmodulation of S-phase.


METHODS
Synthetic peptides corresponding to N-terminal sequences of PR3, purified recombinant PR3, and HLE, as well as hybrid proteins constructed by interchanging the N-terminal regions of PR3 and HLE, thus creating PR3/HLE and HLE/PR3, respectively, were tested for their ability to reduce the fraction of human marrow CFU-GM killed by cytosine arabinoside. In addition, we measured the effect of synthetic peptides on bromodeoxyuridine (BrdU) incorporation in common myeloid progenitors (CMP) and granulocyte/macrophage progenitors (GMP) isolated by cell sorting.


RESULTS
The common N-terminal motif of PR3 and other serine proteases (i.e., IVGG or IIGG) downmodulate the S-phase of CFU-GM at 40 to 80 nM concentration. Tetrapeptide IVGG, but not IVGR, significantly reduces BrdU incorporation in GMP within the CD34+ population. When the N-terminal of HLE is presented by the HLE/PR3 hybrid protein it is fully active.


CONCLUSIONS
These findings demonstrate that the downmodulatory effect on CFU-GM in S-phase is an S-phase arrest mediated by the first four N-terminal amino acids of PR3, and also suggest that this activity is dependent on the configuration of the proform providing the correct presentation of this N-terminal motif.
Sköld
Stefan
S
Division of Hematology & Transfusion Medicine, Department of Laboratory Medicine, Biomedical Center, Lund University, Lund, Sweden.
Rosberg
Bodil
B
Olofsson
Tor
T
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002460:Cell Line; D006098:Granulocytes / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D006410:Hematopoiesis / Q000187:drug effects; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D006801:Humans; D053146:Myeloblastin; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000494:pharmacology*; D010449:Peptide Mapping; D016196:S Phase; D012697:Serine Endopeptidases / Q000737:chemistry / Q000502:physiology*
D010446:Peptide Fragments; D012697:Serine Endopeptidases; D053146:Myeloblastin