Ganz is with Will Rogers Pulmonary etc.
The pro region of human neutrophil defensin
contains a motif that is essential for normal
subcellular sorting
L Liu and T Ganz
Will Rogers Pulmonary Research Laboratory, University of California, Los Angeles School of Medicine 90024-1736.
Human defensins (human neutrophil peptides) HNP 1-3 are 29-30 amino acid antibiotic and cytotoxic peptides highly
abundant in the cytoplasmic granules of polymorphonuclear leukocytes. The peptides are produced from 94 amino acid
(aa) prepropeptides by proteolytic cleavage of the signal sequence and stepwise removal of the 44-45 aa anionic propiece.
To study the role of the propiece, we constructed five in- frame deletions in preproHNP-1 cDNA between the signal
peptidase site and the amino-terminus of the mature defensin region (aa 21-64). The wild type HNP-1 cDNA and the
deletion mutants were ligated into the pBabe-Neo retroviral vector, expressed in GP+E86 packaging derivative of NIH
3T3 cells, then transduced into the 32D cl3 granulocytic cell line. For each construction and both cell lines, we measured
the accumulation of the various defensin forms in cells and media by 24- hour labeling or pulse-chase with 35S-cysteine-
and immunoprecipitation/sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Deletions in the
amino-terminal two-fifths of the propiece, delta 21-28 and delta 21-38, had only minor effects on defensin biosynthesis
in both cell lines and did not interfere with the accumulation of mature defensin in the granules of 32D cl3 cells. Deletions
in the carboxyterminal three-fifths of the propiece (delta 21- 51 and delta 21-64) diminished net defensin synthesis,
blocked constitutive secretion of prodefensin in both cell lines, and interfered with defensin accumulation in cytoplasmic
granules of 32D cl3 cells. These effects were reproduced by the smaller deletion delta 40-51, which contains highly
conserved secondary structure. The propiece segment 40-51 appears to be essential for the subcellular trafficking and
sorting of HNP-1 defensin.
Volume 85, Issue 4, pp. 1095-1103, 02/15/1995
Copyright © 1995 by The American Society of Hematology |
