Nucleic Acids Research, Vol 25, Issue 17 3532-3536, Copyright © 1997 by Oxford University Press
HF Rosenberg and KD Dyer
The human eosinophil-derived neurotoxin (hEDN) is a secretory effector
protein from eosinophilic leukocytes that is a member of the ribonuclease A
(RNase A) family of ribonucleases. EDN is a rapidly evolving protein,
accumulating non-silent mutations at a rate exceeding those of most other
functional coding sequences studied in primates. Although all primate EDNs
retain the structural and functional residues known to be prerequisites for
ribonuclease activity, we have shown previously that recombinant EDN
derived from a New World monkey sequence ( Saguinus oedipus ) had
significantly less catalytic activity than the human (hEDN) ortholog.In
this work, we have prepared recombinant proteins from EDN from sequences
derived from orangutan (Pongo pygmaeus, oEDN) and Old World monkey (Macaca
fascicularis, mcEDN) genomic DNAs, and from a second New World monkey
sequence (Aotus trivirgatus, omEDN) as well. The catalytic efficiencies [ k
cat/ K m (M- 1s-1)] determined for both oEDN and mcEDN were similar to that
determined previously for hEDN, while omEDN displayed approximately 100-
fold less catalytic activity. The relative ribonuclease activities of
hEDN/omEDN chimeras pointed to a C-terminal segment as crucial to the
enhanced catalytic activity hEDN, and substitution of Arg 132-Ile 133 of
hEDN with the Thr-Thr pair at the analogous position in omEDN resulted in
an approximately 10-fold reduction in hEDN's catalytic efficiency. However,
the reverse substitution, Arg-Ile for Thr-Thr in omEDN, did not enhance the
catalytic efficiency of this relatively inactive protein. These results
indicate that the Arg and/or Ile residues adjacent to the C-terminus are
necessary (but not sufficient) for enhanced ribonuclease activity among the
primate EDNs, and will permit prediction of the relative ribonuclease
activities based on differences in primary structure.
ARTICLES
Diversity among the primate eosinophil-derived neurotoxin genes: a specific C-terminal sequence is necessary for enhanced ribonuclease activity
Laboratory of Host Defenses, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA. hr2k@nih.gov
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