Variants of apolipoprotein A-I

Details Variants of apolipoprotein A-I Variants of apolipoprotein A-I

1998-06-05

2001-07-10

LeGuyader, John L. (Department: 1633)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

C435S320100, C530S350000, C536S023100, C536S023500

Reexamination Certificate

active

06258596

ABSTRACT:

This application is the U.S. National phase of International Application No. PCT/FR96/00747, filed May 20, 1996, which claimed priority to French Application No. FR/95/06061 filed May 22, 1995.
The present invention relates to a new variant of apolipoprotein A-I. It also relates to any nucleic acid coding for this new variant. It relates, in addition, to the use of this protein or these nucleic acids for therapeutic purposes. More especially, the invention relates to a new variant of apolipoprotein A-I containing, in particular, a mutation at position 151.
Apolipoprotein A-I (apoA-I) is the major constituent of the high density lipoproteins (HDL), which are macromolecular complexes composed of cholesterol, phospholipids and triglycerides. ApoA-I is a protein consisting of 243 amino acids, synthesized in the form of a preproprotein of 267 residues, having a molecular mass of 28,000 daltons. The prepro form of apoA-I is synthesized in man by both the liver and the intestine. This form of protein is then cleaved to a proprotein which is secreted into the plasma. In the vascular compartment, proapoA-I is then converted to the mature protein (243 amino acids) by the action of a calcium-dependent protease. ApoA-I has a structural role and an active role in lipoprotein metabolism: apoA-I is, in particular, a cofactor of lecithin:cholesterol acyltransferase (LCAT), responsible for the esterification of plasma cholesterol.
The level of cholesterol contained in the HDL fraction and the plasma concentration of apoA-I are negative risk factors for the development of atherosclerosis in man. Epidemiological studies have, in effect, demonstrated an inverse correlation between the concentrations of HDL cholesterol and apoA-I and the incidence of cardiovascular diseases (E. G. Miller et al., Lancet, 1977:965-968). In contrast, a longevity would appear to be associated with a high level of HDL cholesterol. Recently, the protective role of apoA-I has been demonstrated in a model of transgenic mice expressing human apolipoprotein A-I (Rubin et al., Nature). Similarly, the infusion of HDL in rabbits induces a regression of the lesions (Badimon et al., J. Clin. Invest. 85, 1234-41, 1990). Different mechanisms have been proposed to explain the protective effect of HDL, and in particular a role of HDL in the reverse transport of cholesterol (Fruchart et al., Circulation, 87: 22-27, 1993) and an antiioxidant action of HDL (Forte T., Current Opinion in Lipidology, 5: 354-364, 1994)).
The gene coding for apoA-I has been cloned and sequenced (Sharpe et al., Nucleic Acids Res. 12(9) (1984) 3917). This gene, 1863 bp in length, comprises 4 exons and 3 introns. The cDNA coding for apoA-I has also been described (Law et al., PNAS 81 (1984) 66). This cDNA comprises 840 bp (see SEQ ID No. 1). Besides the wild-type form of apoA-I, different natural variants have been described in the prior art, the differences between these variants and the wild-type protein being given in the table below:
Variant:
Mutation
Variant
Mutation
Milano
Arg173Cys
Norway
Glu136Lys
Marburg
Lys107Ø
Pro165Arg
Munster2B
Ala158Glu
Pro3His
Giessen
Pro143Arg
Arg10Leu
Munster3A
Asp103Asn
Gly26Arg
Munster3B
Pro4Arg
Asp89Glu
Munster3C
Pro3Arg
Lys107Met
Munster3D
Asp213Gly
Glu139Gly
Munster4
Glu198Lys
Glu147Val
Yame
Asp13Tyr
Ala158Glu
Asp213Gly
Glu169Gln
Arg177His
The present invention is the outcome of the demonstration of a new series of variants of apolipoprotein A-I. This series of variant possesses, in particular, a replacement of the arginine residue at position 151 by a cysteine residue. The apoA-I variant according to the invention displays noteworthy therapeutic properties. In particular, it possesses especially important properties of anti-atherogenic protection. Thus, in a situation of extremely low levels of cholesterol in the HDL fraction, associated with a hypertriglyceridaemia, the presence of this variant prevents the development of any atherosclerosis, testifying to a very potent protective role, specific to this mutated apoA-I. In addition, the presence of a cysteine in the apoA-I according to the invention gives rise to the formation of dimers and other complexes linked via a disulphide bridge. This apoA-I occurs in free form in the plasma, bound as a diner to itself or combined with apolipoprotein A-II, which is another important protein associated with HDL and which also possesses a cysteine in its sequence. Moreover, the loss of the charge associated with the arginine at position 151 gives rise to the visualization of this mutant by isoelectric focusing of the plasma proteins followed by an immunological disclosure of the apoA-I.
In view of its especially noteworthy anti-atherogenic properties, this new protein according to the invention affords a substantial therapeutic advantage in the treatment and prevention of cardiovascular pathologies.
A first subject of the invention hence relates to a series of variants of human apolipoprotein A-I comprising a cysteine at position 151. The amino acid sequence of the reference apoA-I is described in the literature (see Law, cited above). This sequence, including the prepro region (residues 1 to 24), is presented in the sequence SEQ ID No. 2. A feature of the variants according to the invention hence lies in the presence of a cysteine at position 151 of th e mature apoA-I (corresponding to position 175 in the sequence SEQ ID No. 2), replacing the arginine present in the reference sequence. A preferred variant according to the invention comprises the peptide sequence SEQ ID No. 13, and still more preferably the peptide sequence lying between residues 68 and 267 of the sequence SEQ ID No. 2, residue 175 being replaced by a cysteine.
The variants according to the invention are represented, in particular, by apoA-I Paris, that is to say an apoA-I possessing a cysteine at position 151 relative to the native apoA-I. The variants according to the invention may also carry other structural modifications relative to the reference apolipoprotein A-I, and in particular other mutations, deletions and/or additions. According to a particular embodiment, the variants of the invention also comprise other mutations leading to the replacement of residues by cysteines. Thus, another particular variant combines the mutation present in the variant apoA-I Paris and apoA-I milano. Other mutations may also be present, affecting residues which do not significantly modify the properties of apoA-I. The activity of these variants may be verified, in particular, by a cholesterol efflux test.
The variants according to the invention may be obtained in different ways. They may, in the first place, be synthesized chemically by means of the techniques known to a person skilled in the art using peptide synthesizers. They may also be obtained from the reference apoA-I, by mutation(s). Advantageously, the proteins in question are recombinant, that is to say obtained by expression of a corresponding nucleic acid in a cell host, as described later.
As mentioned above, the variants according to the invention may be in monomeric form or in dimer form. The presence of at least one cysteine in the sequence of the variants of the invention makes it possible, in effect, for dimers to be produced by disulphide bonding. The dimers can be homodimers, that is to say dimers comprising two variants according to the invention (for example diApoA-I Paris); or heterodimers, that is to say dimers comprising a variant according to the invention and another molecule possessing a free cysteine (for example ApoA-I Paris:ApoA-II).
Another subject of the invention lies in a nucleic acid coding for an apolipoprotein A-I variant as defined above. The nucleic acid of the present invention can be a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA). Among DNAs, a complementary DNA (cDNA), a genomic DNA (gDNA), a hybrid sequence or a synthetic or semi-synthetic sequence may be used. The nucleic acid may, in addition, be one which is chemically modified, for example for the purpose of increasing its resistance to nucleases, its cell penetration

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