Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
2000-09-07
2003-07-01
Patterson, Jr., Charles L. (Department: 1652)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C435S219000, C435S348000, C536S023200
Reexamination Certificate
active
06586197
ABSTRACT:
BACKGROUND OF THE INVENTION
Aminopeptidase N (APN) is an exopeptidase that hydrolyses neutral amino acids from the amino (N)-termini of different proteins. In different cell types, APN is expressed as a soluble cytoplasmic enzyme and a membrane-bound ectoenzyme. This enzyme is found on the surface of diverse cell types including lung, kidney, intestine and brain cells of many animals (Kenny et al., 1987). The ectoenzyme form is attached to epithelial cells of intestinal brush borders and respiratory tracts of vertebrates by a hydrophobic N-terminal stalk (Kenny et al., 1987 and Takasaki et al., 1991). In insects, however, ectoenzyme attachment is via a glycosyl-phosphatidylinositol (GPI) anchor (Tomita et al., 1994; Garczynski and Adang, 1995; Luo et al., 1996a; Luo et al., 1996b; Luo et al., 1997a; and Luo et al., 1 997b). GPI-anchored proteins are relatively mobile on the membrane surface and can be clustered in microdomains with other proteins and specific lipids. The base of the GPI-anchor interacts with the intracellular environment and has been implicated in physiological functions, intracellular sorting and transmembrane signaling (McConville and Ferguson, 1993).
In intestinal epithelial cells, APN is important for the final hydrolysis step of ingested proteins. APN also has several important physiological roles in other tissues. For example, APN is implicated in tumor cell invasion and inhibition of aminopeptidase activity can suppress tumor cell spread (Fujii et al., 1995). In brain cells, APN serves a role in the breakdown and inactivation of peptide neurotransmitters (Kenny et al., 1987). In bovine renal brush border membrane vesicles (BBMV), partially purified APN was found to be associated with a Na
+
-dependent amino acid co-transporter (Plakidou-dymock et al., 1993).
APN molecules function as adventitious receptors for viruses. Human, feline, canine, and porcine coronaviruses utilize APN as their cellular receptors (Delmas et al., 1992; Yeager et al., 1992; and Tresnan et al., 1996). Cells refractory to coronaviruses from a particular animal species can be made susceptible by expression of an APN cDNA from that species (Benbacer et al., 1997). Human APN was shown to mediate human cytomegalovirus infection by increasing virus binding (McLaughlin and Aderem, 1995). Human, porcine and feline APNs have been cloned and expressed in different cell lines (Delmas et al.,. 1992; Yeager et al., 1992; Kolb et al., 1996; and Tresnan et al., 1996). Each of these vertebrate APNs were expressed on the cell surface as the N-terminal stalked form and bound a coronavirus.
Isoforms of APN located in the epithelial cells of insect midguts bind specifically to
Bacillus thuringiensis
Cry1 &dgr;-endotoxins. Toxin-binding APNs are reported for several lepidopteran species (see, e.g., Knight et al., 1994; Sangadala et al., 1994; Gill et al., 1995; Valaitis et al., 1995; Luo et al., 1996; and Yaoi et al., 1997). For example, Cry1Aa, Cry1Ab and Cry1Ac, but not Cry1C or Cry1E toxins bind to a purified 115 kDa APN from
Manduca sexta
(Masson et al., 1995). Also partially purified preparations of APN catalyze toxin-induced pore formation in membrane vesicles (Sangadala et al., 1994) and planar lipid bilayers (Schwartz et al., 1997).
Several APN isoforms have been purified and cloned from different insect species (see, e.g., Knight et al., 1995; Gill et al., 1995; Valaitis et a, 1995; Luo et al., 1996; Yaoi et al., 1997; Denolf et al., 1997; and Hua et al., 1998). However, there has been limited success in expressing insect APN cDNA in insect cells. The only example to date involved the expression of
Plutella xylostella
105 kDa APN in Sf9 cells using a baculovirus vector (Denolf et al., 1997). While the transformed cells of this study produced APN localized to the cell membrane, the APN was unable to bind to
B. thuringiensis
Cry1A toxins. Further, Denolf et al were unsuccessful in expressing two 120 kDa APNs from
Manduca sexta
using the same vector.
The complete structural and functional characterization of insect APN will require the successful expression of insect APN in insect cells. Successful expression of insect APN in insect cells as described in Luo et al. (1999) would also facilitate study of APN-toxin interactions, as well as provide a screening system for obtaining novel pesticide agents.
BRIEF SUMMARY OF THE INVENTION
The subject invention pertains to cells expressing a polynucleotide encoding an Aminopeptide N (APN), and methods of using the same to identify pesticide agents. One aspect of the invention pertains to an isolated polynucleotide which encodes a full length APN from
Manduca sexta
(M sexta) (SEQ ID NO: 1). Another aspect pertains to a fragment of said fill length polynucleotide which is sufficient to encode a functional protein.
In another aspect, the subject invention pertains to a cell or cells transfected with a polynucleotide encoding an APN or fragments thereof, such that a functional polynucleotide is expressed. Preferably, the polynucleotide is expressed forming a protein which is localized at the cell membrane of said cell or cells. A further aspect pertains to descendent generations of said cells which express APN that is localized on the cell membrane.
In a further aspect, the subject invention is directed to a method of identifying pesticide agents comprising obtaining a cell or cells transfected with a polynucleotide encoding an APN or fragments thereof, such that said polynucleotide is expressed to produce a protein localized at the cell membrane of said cell or cells, and screening one or more pesticide agents for their ability to produce an observable effect on said cell or cells.
In yet another aspect, the subject invention is drawn to novel pesticide agents obtained according to the subject methods.
In a still further aspect, the subject invention is drawn to an expression vector comprising a polynucleotide encoding APN or a functional fragment thereof.
An alternative aspect of the subject invention pertains to a method of identifying novel aminopeptidase inhibitors comprising obtaining cells having APN localized on the cell membranes thereof; and screening one or more compounds of interest for their ability to inhibit aminopeptidase activity.
REFERENCES:
Ayres, M.D., Howard, S.C., Kuzio, J., Lopez-Ferber, M. and Dossec, R.D. (1994) “The complete DNA sequence ofAutograph californicanuclear polyhedrosis virus,”Virology202:586-605.
Benbacer, L., Kut, E., Besnardeau, L., Laude, H. and Delmas, B. (1997) “Interspecies aminopeptidase-N chimeras reveal species-specific receptor recognition by canine coronavirus, feline infectious peritonitis virus, and transmissible gastroentritis virus,”J. Virol.71:734-737.
Chaudhri, M., Steverding, D., Kittelberger, D., Tjia, S. and Overath, P. (1994) “Expression of glycosylphosphadylinositol-anchoredTrypanosoma bruceitransferrin-binding protein complex in insect cells,”Proc. Natl. Acad. Sci. U.S.A.91:6443-6447.
Davies, A. and Morgan, B. P. (1993) “Expression of the glycosylphosphatidlinositol-linked complement-inhibiting protein CD59 antigen in insect cells using a baculovirus vector,”Biochemical Journal295:889-896.
Delmas, B., Gelfi, J., Haridon, R.L., Vogel, L.K., Sjostrom, H., Noren, O. and Laude, H. (1992) “Aminopeptidase N is a major receptor for the enteropathogenic coronavirus TGEV,”Nature357:417-419.
Denolf, P., Hendrickx, K., Van Damme, J., Jansens, S., Peferoen, M., Degheele, D. and Van Rie, J. (1997) “Cloning and characterization ofManduca sextaandPlutella xylostellamidgut aminopeptidase N enzymes related toBacillus thuringiensistoxin-binding proteins,”Eur. J. Biochem.248:748-761.
Ferre, J., Real, M.D., Van Rie, J., Jansens, S. and Peferoen, M. (1991) “Resistance to theBacillus thuringiensisbioinsecticide in a field population ofPlutella xylostellais due to a change in a midgut membrane receptor,”Proc. Natl. Acad. Sci. USA88:5119-5123.
Garczynski, S.F. and Adang, M.J. (1995) “Bacillus thuringiensisCryIA(c) d-endotoxin binding aminopeptidase in theManduca sextamidgut has a glycosyl-phosphatidyl
Adang Michael J.
Luo Ke
Patterson Jr. Charles L.
Saliwanchik Lloyd & Saliwanchik
University of Georgia Research Foundation Inc.
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