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J Biol Chem, Vol. 273, Issue 47, 31086-31091, November 20, 1998
From the This study reports properties of site-directed
mutants of human protein C that display enhanced calcium and/or
membrane binding properties. Mutants containing the S11G modification
all showed increased affinity for membranes at saturating calcium
concentration. Ser-11 is unique to human protein C, whereas all other
vitamin K-dependent proteins contain glycine. This site is
located in a compact region of the protein, close to a suggested
membrane contact site. Additional changes of H10Q or S12N resulted in
proteins with lower calcium requirement for membrane contact but
without further increase in membrane affinity at saturating calcium.
Mutations Q32E and N33D did not, by themselves, alter membrane affinity to a significant degree. These mutations were included in other mutant
proteins and may contribute somewhat to higher function in these
mutants. This family of mutants helped discriminate events that are
necessary for protein-membrane binding. These include calcium binding
to the free protein and subsequent protein-membrane contact. Depending
on conditions of the assay used, the mutants displayed increased
activity of the corresponding activated protein C (APC) derivatives.
The degree of enhanced activity (up to 10-fold) was dependent on the
concentration of phospholipid and quality of phospholipid (± phosphatidylethanolamine) used in the assay. This was expected, because
APC is active in its membrane-associated form, which can be regulated
by changes in either the protein or phospholipid. As expected, the
largest impact of the mutants occurred at low phospholipid
concentration and in the absence of phosphatidylethanolamine. The
anticoagulant activity of all proteins was stimulated by protein S,
with the greatest impact on the enhanced mutants. Whereas plasma
containing Factor V:R506Q was partially resistant to all forms of APC,
the enhanced variants were more active than normal APC. Protein C
variants with enhanced function present new reagents for study of
coagulation and may offer improved materials for biomedical applications.
Enhancement of Human Protein C Function by Site-directed
Mutagenesis of the
-Carboxyglutamic Acid Domain
,, and Department of Clinical Chemistry,
Malmö General Hospital and the University of Lund,
S-20502 Malmö, Sweden and the ¶ Department of Biochemistry,
University of Minnesota, St. Paul, Minnesota 55108
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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