Modified proteinase inhibitors

Details Modified proteinase inhibitors Modified proteinase inhibitors

1997-05-21

2004-01-20

McElwain, Elizabeth F. (Department: 1638)

Multicellular living organisms and unmodified parts thereof and

Method of introducing a polynucleotide molecule into or...

The polynucleotide confers pathogen or pest resistance

C800S278000, C800S298000, C435S069100, C435S069200, C435S419000, C435S468000, C435S320100, C435S410000, C536S023600, C536S024100, C530S370000

Reexamination Certificate

active

06680424

ABSTRACT:

The invention relates to proteinase inhibitors and in particular novel proteinase inhibitors, methods for producing such inhibitors and products and processors including such inhibitors.
Proteinases are enzymes that break down proteins, their substrate specificity varies considerably and therefore does not form a basis for the purpose of classification. Rather, typically, these enzymes are classified according to the nature of the catalytic reaction that each undertakes. Thus proteinases are divided into four groups termed serine proteinases, cysteine proteinases, aspartic proteinases and metalloproteinases. Serine proteinases and cysteine proteinases are both widespread and diverse and are found in both prokaryotic and eukaryotic organisms, including plants and animals. In contrast, aspartic proteinases seem to be found only in eukaryotic organisms. Since these enzymes are used to break down protein the origin and/or the location of the enzymes determines whether they are beneficial or detrimental to a given organism. For example, where the enzymes are used by pathogens or parasites or pests they are typically used to break down host cell tissue and are therefore detrimental.
Pathogens, parasites or pests such as bacteria, fungi, plants, insects, nematode worms etc produce proteinases which break down host cell tissue to the detriment of the host.
For example, annual global crop losses caused by fungi exceed a thousand million pounds. The pathogen,
Botrytis cinerea
is of major economic importance because it causes disease in thirty crop plant species, with serious losses incurred in the glass house, in viticulture and as a result of post-harvest disease of fruit and vegetables. This major pathogen can be overcome with fungicides but unfortunately, there are disadvantages associated with the use of fungicides in order to control it. These include a financial burden associated with use of the fungicide, the potential environmental hazard arising from the use of toxic fungicides, with attendant consumer concern, and major problems of pathogen resistance to fungicides. In addition, many fungicides are effective against only a limited range of pathogenic fungi.
The above disadvantages are also common to the use of synthetic agents manufactured against other pathogens, parasites or pests such as insects, or specific insects, bacteria or specific bacteria and other eukaryotic organisms including, but not limited to: protozoa such as amoebas, intestinal flagellates and ciliates, haemoflagellates, such as leishmania or trypanosomes, sporozoa, such as those responsible for malaria, arthropod-borne organisms; helminths such as trematodes or flukes, cestoidea, acanthocephala, nematodes, trichuris, trichinella, hook worms, filariae, spiruroids; arthropods such as acarina or mites, ticks heteroptera, lice, flees, diptera such as disease-carrying flies including mosquitos, maggots and myiasis.
Inhibition of proteinases is known to occur naturally following pathogen infection. For example, it has been shown that following infection by
Phytophthora infestans
varieties of tomato able to resist the fungus show increased levels of proteinase inhibitors (1). This relationship between resistance and the capacity to produce proteinase inhibitors has been used to good effect in the control of pathogen, pests and parasitic diseases. For example, in the most relevant prior art known to the applicant, plant pests are controlled by recombinantly introducing a proteinase inhibitor, animal-derived egg white cystatin, into a selected monocotyledon such as a cereal, forage or turf grass, or a dicotyledon such as a vegetable, tube, or sugar crop, (EP 0 348 348). Similarly, plant nematode pests have been controlled using a proteinase inhibitor, plant-derived cowpea trypsin inhibitor, which has been recombinantly introduced into tobacco, tomato, cotton, oilseed rape, vegetable crop or ornamental plants (EP 0 502 730). In addition, it has been suggested that proteinase inhibitors can be used as anti-parasitic proteins which ideally can be administered to a host species either in a medicament or a food (UK Patent Application No. 94 03819.7).
It is therefore known to use proteinase inhibitors to neutralise the effects of proteinases and so combat the effects of pathogens, parasites or pests. In particular, it is known to transgenically produce plants which are provided with a specific proteinase inhibitor, such as a cysteine proteinase inhibitor.
However, it is the object of the present invention to provide a modified proteinase inhibitor which has greater efficacy than that of its unmodified counterpart or the natural proteinase inhibitor; or alternatively to synthetically manufacture and improve a proteinase inhibitor so as to provide, in one embodiment a hybrid proteinase inhibitor.
In one aspect of our invention we have focused on the group of proteinase inhibitors known as cystatins. The protein sequences of approximately 25 cystatins are known. It is possible to undertake alignment studies of these sequences in order to provide a basis for identifying structural similarities. It has been suggested that there are sufficient differences between plant and animal cystatins to justify separate classification of the two, indeed, a comparison of a plant cystatin, Oryzacystatin I Oc-I [DNA sequence structure shown in
FIG. 3
, SEQ ID NO:30], and an animal cystatin, egg white cystatin, reveals a significant number of differences showing that overall amino acid conservation is not high. Moreover, there are significant differences in the binding properties of animal and plant cystatins. Thus the dissociation constant Ki varies, for example, egg white cystatin has a Ki of 5×10
−12
M, whereas the plant cystatin, Oc-I, (derived from rice), has a Ki of 3×10
−8
M.
Alignment data of a number of cystatins (SEQ ID NO:1-28) is shown in FIG.
1
. The amino acids are numbered 1-181. It can be seen that there is a conserved inhibitory site at alignment amino acids 100-104, represented by the motif QVVAG (SEQ ID NO:56) or QLVAG (SEQ ID NO:57). In addition, it can be seen that there is a conserved PW motif at alignment amino acids 160-161.
This conservation occurs in approximately two thirds of the known sequence structures and is thought from structural studies to be involved in the functioning of the protein and thus for inhibition of proteinases. However, some cystatins with low Ki values do not possess this PW motif therefore its importance in cystatin function is unclear.
Other works have recombinantly manufactured novel cystatins. For example, the human cysteine proteinase inhibitor cystatin C, which participates in the intracellular catabolism of proteins and peptides, in the proteolytic conversion of prohormones, in the extracellular degregation of collagen and in the penetration of normal tissues with malignant cells, has been altered. Workers have modified cystatin C so that one or more amino acids at positions 5-17, 55-59 and/or 68 have been replaced by other amino acids thus retaining the total 120 amino acids in the sequence structure. Modifications were undertaken in order to provide an animal-derived cystatin C considered to have constant activity. (WO 88/09384).
We have found, surprisingly, that site-directed modification of a plant cystatin such as, for example, Oryzacystatin I (Oc-I) can improve its binding properties and thus improve the efficacy of the enzyme in inhibiting proteinases. The site-directed modification involves elimination of the amino acid aspartic acid at position 86 of the amino acid sequence structure of the plant cystatin, this elimination improves the Ki 13 fold, that is to 2.3×10
−9
M. This modification is represented by elimination of aspartic acid (symbol D) at position 163 of the alignment amino acids shown in FIG.
1
.
Clearly, this improvement in plant cystatin Ki does not exceed animal cystatin Ki, and particularly egg white cystatin. However, there is growing concern about the liberal approach to cross species transgenics. That is to

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