IDENTIFICATION AND SEQUENCE ANALYSIS OF THE OVINE FORMS OF
ELAFIN AND SECRETORY LEUCOPROTEASE INHIBITOR
T. Brown1, D. Collie2 and J-M. Sallenave1;
1Rayne
Laboratory, Medical School, University of Edinburgh; 2Wellcome Trust
Centre for Research in Comparative Respiratory Medicine, Easter Bush
Veterinary Centre, Roslin, Edinburgh.
The development of a large animal model for chronic neutrophil mediated lung
disease will have many benefits with regards therapeutic strategies. Endogenous
antiproteases are thought to play an important role in the control of inflammation
in the lung. Our aim was to clone the ovine orthologs of the genes for the
antiproteases
elafin
(elastase
specific
inhibitor)
and
SLPI
(secretory
leukoprotease inhibitor) and to insert the coding cDNAs into adenoviral vectors
for use in an ovine model of chronic lung disease.
To achieve this we used RACE PCR to identify the cDNAs in question and
then formulated primers to allow screening of an ovine BAC library from which
gene sequence would be obtained. Multiple ovine organs were used to extract
RNA which was used in Northern blot analysis and RT-PCR for tissue
distribution. The cDNAs were then inserted into shuttle vectors to allow the
construction
of
replication
deficient
adenoviral
vectors
coding
for
the
antiproteases of interest. The possibility of augmenting gene expression in vitro
was examined using lipopolysaccharide, polyethylenimine, EGTA in hypotonic
solution and calcium phosphate precipitates.
We isolated two distinct forms of ovine elafin cDNA, coding for proteins of 196
and 220 amino acids. Both consisted of a hydrophobic signal sequence of 23
amino acids followed by a region of hexapeptide repeats known as a
transglutaminase
substrate
domain,
and
finally
a
C
terminal
portion
corresponding to a whey acidic protein domain. The SLPI cDNA coded for a
protein of 132 amino acids consisting of a hydrophobic signal sequence of 24
amino acids followed by two whey acidic protein domains. Positive BAC clones
allowed us to obtain sequence for the entire genes. Elafin mRNA was found by
Northern blot analysis in tongue, trachea, small and large intestine and skin. RTPCR confirmed this and showed SLPI mRNA to be found in tongue, trachea,
kidney, bladder and skin. CDNAs for the long form of elafin and for SLPI were
inserted into shuttle vector pIRES-GFP#3. The elafin shuttle vector has been
used to construct a replication deficient adenovirus. This virus has been used in
vitro to infect cells and the protein produced inhibits human neutrophil elastase.
Likewise, the SLPI shuttle vector has been used to transfect cells and the ovine
SLPI produced proved to be an active anti-elastase. Augmentation of protein
production after Ad-elafin infection of A549 cells was shown with LPS, PEI,
hypotonic EGTA, and most strikingly calcium phosphate precipitate.
The identification of the ovine forms of elafin and SLPI valuable in the
development of a sheep model of chronic lung disease. The availability of the
replication deficient adenoviral vectors will allow us to use these promising
antiproteases in the in vivo setting.