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Blood, Vol. 92 No. 4 (August 15), 1998:
pp. 1415-1422
On the Role of the Proform-Conformation for Processing and
Intracellular Sorting of Human Cathepsin G
Daniel Garwicz,
Anders Lindmark,
Ann-Maj Persson, and
Urban Gullberg
From the Department of Hematology, Lund University, Lund, Sweden.
The serine protease cathepsin G is synthesized during the
promyelomonocytic stage of neutrophil and monocyte differentiation. After processing, including removal of an amino-terminal propeptide from the catalytically inactive proform, the active protease acquires a
mature conformation and is stored in azurophil granules. To investigate
the importance of the proform-conformation for targeting to granules, a
cDNA encoding a double-mutant form of human preprocathepsin G lacking
functional catalytic site and amino-terminal prodipeptide (CatG/Gly201/ Gly19Glu20) was
constructed, because we were not able to stably express a mutant
lacking only the propeptide. Transfection of the cDNA to the rat
basophilic leukemia RBL-1 and the murine myeloblast-like 32D cl3 cell
lines resulted in stable, protein-expressing clones. In contrast to
wild-type proenzyme,
CatG/Gly201/Gly19Glu20 adopted a
mature conformation cotranslationally, as judged by the early
acquisition of affinity to the serine protease inhibitor aprotinin,
appearing before the carboxyl-terminal processing and also in the
presence of the Golgi-disrupting agent brefeldin A. The presence of a
mature amino-terminus was confirmed by amino-terminal radiosequencing.
As with wild-type proenzyme,
CatG/Gly201/Gly19Glu20 was
proteolytically processed carboxyl-terminally and glycosylated with
asparagine-linked carbohydrates that were converted into complex forms.
Furthermore, it was targeted to granules, as determined by subcellular
fractionation. Our results show that the initial proform-conformation
is not critical for intracellular sorting of human cathepsin G. Moreover, we demonstrate that double-mutant cathepsin G can achieve a
mature conformation before carboxyl-terminal processing of the proform.
© 1998 by The American Society of Hematology.
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