Pro-protein converting enzyme

Details Pro-protein converting enzyme Pro-protein converting enzyme

1999-09-13

2002-04-30

LeGuyader, John L. (Department: 1635)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Carbohydrate doai

C435S006120, C435S325000, C435S375000, C536S023100, C536S024500

Reexamination Certificate

active

06380171

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to protein processing enzymes or pro-hormone convertases (PCs), specifically to PC5, more specifically to the human PC5.
BACKGROUND OF THE INVENTION
Pro-hormone convertases (PCs) belong to a family of enzymes responsible for the maturation of proteic precursors into active proteins or enzymes. Up to now, many human enzymes of that family have been identified, namely furin, PC1, PC2, PC4 and PC7. Each enzyme has a tissular distribution which may be restricted (for example, PC4 is restricted to male germ cells) or ubiquitous (furin is such an example). Although all these enzymes share the properties of cleaving precursor proteins at basic or dibasic residues, they nevertheless have differing cleavage specificities. The action of a specific pro-hormone convertase is therefore governed by the cleavage sequence of a given protein substrate, and/or by the location of that enzyme in a tissue expressing or responding to a given proteic substrate growth factor or hormone.
Renin is an aspartyl protease which makes an important contribution to cardiovascular physiology and pathophysiology through its key role in the synthesis of the vasoactive octapeptide angiotensin II (AII). While the kidney is the primary source of circulating active renin, several additional tissues, including the pituitary and adrenal glands, placenta, uterus, ovary, testes, heart, vasculature and brain express the renin gene (reviewed in
1-4
). The presence of additional components of the RAS (renin-angiotensin system) in these tissues, including angiotensin converting enzyme (ACE) and angiotensin II receptors, has led to the proposal that certain tissues might contain a locally active tissue renin-angiotensin system (tRAS) although the actual function of the various tRAS is still largely a matter of conjecture.
Renin is first synthesized as an enzymatically inactive precursor, prorenin, which is converted to active renin by the proteolytic removal of a 43 amino acid amino-terminal prosegment. The activity of the RAS within any given tissue would, therefore, be dependent on the existence of proteolytic enzymes capable of converting prorenin to active renin and on the expression of such prorenin processing enzymes (PPEs) in the same cells that express prorenin. The identity of the enzyme(s) responsible for the proteolytic activating human prorenin in vivo is still uncertain. Furthermore, it is possible that multiple PPEs exist in humans and these may differ among renin-producing tissues. Biochemical and microscopic studies of renin in the kidney suggest that candidate PPEs should be selective for cleavage of human prorenin at Lys
42
, Arg
43
of the prosegment
5
and would be active in secretory granules of the juxtaglomerular (JG) cells.
6
The lysosomal enzyme cathepsin B has been co-localized with human renin/prorenin in the secretory granules of JG cells and human pituitary lactotrophs
7,8
and has been shown to cleave human prorenin in vitro with a high affinity and selectivity for the proper cleavage site.
9
The prohormone convertase PC1 has also been shown to cleave human prorenin with the correct site-and organelle specificity in transfected cells
10
and to co-localize with renin in the adrenal medulla and derived tumors
11
, but not in JG cells.
12
In an effort to identify novel PPEs, we recently determined the distribution of processing enzymes in an established renin-expressing tissue culture cell line derived from an oncogene-induced mouse tumor (As4.1 cells
13
). One such enzyme, the mouse prohormone convertase PC5, was found. Mouse PC5 is capable of partially cleaving human prorenin.
Miranda et al. (38) describe the cDNA and protein sequences for a human PC6 enzyme obtained by PCR from CD4
+
T lymphocytes. PC5 and PC6 are different names given to what appears to be the same enzyme. However, the sequences of Miranda et al. comprise a plurality of substitutions when compared to the present PC5 sequences. Moreover, the size of messenger RNA encoding PC6 and PC5 are similar but not identical. Since the present PC5 sequences were obtained from human adrenals, both enzymes may be isoforms, differentially expressed in tissues and they may have different activities.
Prorenin and HIV gp160 are most probably not the only proteic precursors to be recognized and cleaved by PC5. Many growth factors responsible for cell proliferation are cleaved by one or more PCs: they include platelet-derived-growth factors A and B (PDGF's), epidermal-growth-factor (EGF), insulin-like growth factors I and II (IGF's), transforming growth factors &agr; and &bgr; (TGF's). Each of these named growth factors has the typical cleavage site motif K/R—(X)
n
—R↓ (where n=0,2,4,6). Full biological potency is conferred to these growth factors only after cleavage at these sites, by one or more of the PC enzyme family. There is therefore a possibility that manipulating the expression of the PCs would affect cell proliferation via deficient growth factor activation.
Out of the >450,000 patients/year in the U.S. and Canada who undergo percutaneous transluminal coronary angioplasty (PTCA), 30-50% of them will restenose their coronaries within 3-6 months. This flare-up of endothelial and smooth-muscle cells proliferation is due to the activation of numerous regulatory growth factors. Therefore, knowing which enzyme(s) is (are) responsible for this activation, and manipulating the level of expression of this or theses enzyme(s) would be particularly useful to prevent restenosis.
STATEMENT OF THE INVENTION
The present invention relates to the human PC5 (hPC5). We demonstrate that hPC5 isolated form human adrenals proteolytically activates human prorenin with the expected site- and organelle- specificity and that it is co-expressed with prorenin in the zona glomerulosa of the adrenal cortex. Therefore, PC5 is a prorenin-processing enzyme (PPE). Silencing the expression of PC5 would find a specific application in inhibiting the production of renin, and a method of inhibiting the production of renin is an object of the invention. Since the production of renin is one of targets of the RAS involved in hypertension. Furthermore, we demonstrate that hPC5 is overexpressed in atherosclerotic coronary arteries. Antisense oligonucleotides have been designed, amongst which one has been shown to successfully silence the expression of hPC5 in smooth muscle cells in culture. This antisense inhibited carotid stenosis in a in vivo rabbit carotid injury model. These results indicate that a method of silencing the expression of PC5 would find a specific application in preventing restenosis.
PC5 is known to be expressed in CD4
+
T cells, along with furin and PC7. The three enzymes are capable of converting HIV gp160 into its fusiogenic form. Therefore, antisense constructs, particularly the oligonucleotide that successfully inhibited restenosis, will find a use in inhibiting expression of the activity of PC5 towards HIV gp160.
The complete amino acid and nucleotide sequence of hPC5 is described hereinbelow and are another object of this invention. Recombinant vectors and hosts comprising as a new insert, whole or part of hPC5, are also an object of the invention.
Oligopeptides derived from the proteic sequence of hPC5 are also an object of the invention.
Antibodies directed against the whole protein hPC5 or a part thereof are also an object of the invention.
Diagnostic methods and kits comprising oligonucleotides or antibodies binding PC5 nucleic acids or protein or peptides are also an object of the invention.
This invention will be described hereinbelow by way of specific embodiments, examples and figures which purpose is to illustrate the contemplated aspects of the invention, and not to limit the scope of the invention.


REFERENCES:
patent: 5866351 (1999-02-01), Franzusoff et al.
patent: WO94 15945 (1994-06-01), None
C. Haléne et al., “Specific Regulation of Gene Expression by Antisense, Sense and Antigene Nucleic Acids”, Biochimica et Biophysica Acts, Jan. 1990.
T. Nakaga

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