Lsr receptor, its activity, its cloning, and its applications to the diagnosis, prevention and/or treatment of obesity and related risks or complications   

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 11/862,842, filed Sep. 27, 2007, which is a continuation of U.S. application Ser. No. 10/650,507, filed Aug. 27, 2003, now U.S. Pat. No. 7,291,709, which is a divisional of U.S. application Ser. No. 09/269,939, filed May 28, 1999, now U.S. Pat. No. 6,635,431, which is the national stage of international application No. PCT/IB98/01257, filed Aug. 6, 1998.

The present invention relates to a new complex receptor polypeptide LSR (Lipolysis Stimulated Receptor), characterized by its functional activities, the cloning of the cDNAs complementary to the messenger RNAs encoding each of the subunits of the multimeric complex, vectors and transformed cells, methods of diagnosis and of selection of compounds which can be used as medicament for the prevention and/or treatment of pathologies and/or of pathogeneses such as obesity and anorexia, hyperlipidemias, atherosclerosis, diabetes, hypertension, and more generally the various pathologies associated with abnormalities in the metabolism of cytokines.

Obesity is a public health problem which is both serious and widespread: in industrialized countries, a third of the population has an excess weight of at least 20% relative to the ideal weight. The phenomenon continues to worsen, in regions of the globe whose economies are being modernized, such as the Pacific islands, and in general. In the United States, the number of obese people has passed from 25% at the end of the 70s to 33% at the beginning of the 90s.

Obesity considerably increases the risk of developing cardiovascular or metabolic diseases. It is estimated that if the entire population had an ideal weight, the risk of coronary insufficiency would decrease by 25% and that of cardiac insufficiency and of cerebral vascular accidents by 35%. Coronary insufficiency, atheromatous disease and cardiac insufficiency are at the forefront of the cardiovascular complications induced by obesity. For an excess weight greater than 30%, the incidence of coronary diseases is doubled in subjects under 50 years. Studies carried out for other diseases are equally eloquent. For an excess weight of 20%, the risk of high blood pressure is doubled. For an excess weight of 30%, the risk of developing a non-insulin-dependent diabetes is tripled. That of hyperlipidemias is multiplied by 6.

The list of diseases whose onset is promoted by obesity is long: hyperuricemia (11.4% in obese subjects, against 3.4% in the general population), digestive pathologies, abnormalities in hepatic functions, and even certain cancers.

Whether the physiological changes in obesity are characterized by an increase in the number of adipose cells, or by an increase in the quantity of triglycerides stored in each adipose cell, or by both, this excess weight results mainly from an imbalance between the quantities of calories consumed and those of the calories used by the body. Studies on the causes of this imbalance have been in several directions. Some have focused on studying the mechanism of absorption of foods, and therefore the molecules which control food intake and the feeling of satiety. Other studies have been related to the basal metabolism, that is to say the manner in which the body uses the calories consumed.

The treatments for obesity which have been proposed are of four types. Food restriction is the most frequently used. The obese individuals are advised to change their dietary habits so as to consume fewer calories. This type of treatment is effective in the short-term. However, the recidivation rate is very high. The increase in calorie use through physical exercise is also proposed. This treatment is ineffective when applied alone, but it improves, however, weight loss in subjects on a low-calorie diet. Gastrointestinal surgery, which reduces the absorption of the calories ingested, is effective but has been virtually abandoned because of the side effects which it causes. The medicinal approach uses either the anorexigenic action of molecules involved at the level of the central nervous system, or the effect of molecules which increase energy use by increasing the production of heat. The prototypes of this type of molecule are the thyroid hormones which uncouple oxidative phosphorylations of the mitochondrial respiratory chain. The side effects and the toxicity of this type of treatment make their use dangerous. An approach which aims to reduce the absorption of dietary lipids by sequestering them in the lumen of the digestive tube is also in place. However, it induces physiological imbalances which are difficult to tolerate: deficiency in the absorption of fat-soluble vitamins, flatulence and steatorrhoea. Whatever the envisaged therapeutic approach, the treatments of obesity are all characterized by an extremely high recidivation rate.

The molecular mechanisms responsible for obesity in humans are complex and involve genetic and environmental factors. Because of the low efficiency of the treatments known up until now, it is urgent to define the genetic mechanisms which determine obesity, so as to be able to develop better targeted medicaments.

More than 20 genes have been studied as possible candidates, either because they have been implicated in diseases of which obesity is one of the clinical manifestations, or because they are homologues of genes involved in obesity in animal models. Situated in the 7q31 chromosomal region, the OB gene is one of the most widely studied. Its product, leptin, is involved in the mechanisms of satiety. Leptin is a plasma protein of 16 kDa produced by the adipocytes under the action of various stimuli. Obese mice of the ob/ob type exhibit a deficiency in the leptin gene; this protein is undetectable in the plasma of these animals. The administration of leptin obtained by genetic engineering to ob/ob mice corrects their relative hyperphagia and allows normalization of their weight. This anorexigenic effect of leptin calls into play a receptor of the central nervous system: the ob receptor which belongs to the family of class 1 cytokine receptors. The ob receptor is deficient in obese mice of the db/db strain. The administration of leptin to these mice has no effect on their food intake and does not allow substantial reduction in their weight. The mechanisms by which the ob receptors transmit the signal for satiety are not precisely known. It is possible that neuropeptide Y is involved in this signalling pathway. It is important to specify at this stage that the ob receptors are not the only regulators of appetite. The Melanocortin 4 receptor is also involved since mice made deficient in this receptor are obese (Gura, 1997).

The discovery of leptin and the characterization of the leptin receptor at the level of the central nervous system have opened a new route for the search for medicaments against obesity. This model, however, rapidly proved disappointing. Indeed, with only one exception (Montague et al., 1997), the genes encoding leptin or its ob receptor have proved to be normal in obese human subjects. Furthermore and paradoxically, the plasma concentrations of leptin, the satiety hormone, are abnormally high in most obese human subjects. Most of the therapeutic research efforts in this direction have centered on the characterization of the effect of leptin at the level of the central nervous system.

SUMMARY

The present invention results from a focusing of the research effort on the discovery of the mechanisms of leptin elimination. The most widely accepted working hypothesis is that the plasma levels of leptin are high in obese subjects because this hormone is produced by the adipose tissue and that the fatty mass is increased in obese subjects. The inventors have formulated a different hypothesis and have postulated that the concentrations of leptin are increased in obese individuals because the clearance of this hormone is reduced. This deficiency causes a leptin resistance syndrome and the obese individual develops a suitable response to the high concentrations of leptin. In this perspective, the treatment of obese subjects ought to consist not in an increase in the leptin levels but in a normalization thereof. At this stage, it is essential to recall that the ob type receptors are signalling type receptors. These receptors can bind leptin at the level of the plasma membrane but cannot cause the protein to enter inside the cell for it to be degraded therein. The ob receptors are not endocytosis receptors.

The inventors have characterized a receptor, in particular hepatic, called LSR receptor, whose activity is dual. The LSR receptor allows, on the one hand, endocytosis of lipoproteins, when it is activated by the free fatty acids, thus serving as a pathway for the clearance of lipoproteins. This pathway serves mainly, but not exclusively, for the clearance of particles high in triglycerides of intestinal origin (Mann et al., 1995). This activity, expressed most particularly at the hepatic level, is dependent on the presence of free fatty acids which, by binding to the receptor, induce a reversible change in the conformation of this complex and allow it to bind, with a high affinity, various classes of lipoproteins such as those containing apoprotein B or apoprotein E.

On the other hand, under normal conditions, in the absence of free fatty acids, the complex receptor LSR does not bind lipoproteins, but is capable of binding a cytokine, in particular leptin, and then of internalizing it and of degrading it.

The present invention therefore relates to a purified LSR receptor, in particular of hepatic cells, characterized in that it is capable, in the presence of free fatty acids, of binding lipoproteins, and in the absence of free fatty acids, of binding a cytokine, preferably leptin.

According to the invention, this LSR receptor is, in addition, characterized in that the bound lipoproteins or the bound cytokine are incorporated into the cell and then degraded, the bound lipoproteins containing in particular apoprotein B or E.

It should be understood that the invention does not relate to the LSR receptors in a natural form, that is to say that they are not taken in their natural environment but obtained by purification from natural sources, or alternatively obtained by genetic recombination, or alternatively by chemical synthesis and capable, in this case, of containing non-natural amino acids, as will be described below. The production of a recombinant LSR receptor, which may be carried out using one of the nucleotide sequences according to the invention, is particularly advantageous because it makes it possible to obtain an increased level of purity of the receptor.

More particularly, the invention relates to a purified rat LSR receptor, characterized in that it comprises at least one subunit having a molecular weight of about 66 kDa and a subunit having a molecular weight of about 58 kDa.

Preferably, the purified rat LSR receptor of the present invention is characterized in that it contains an α subunit comprising the amino acid sequence of SEQ ID NO:2 or a sequence homologous thereto, or an α′ subunit comprising the amino acid sequence of SEQ ID NO:4 or a sequence homologous thereto, and one, preferably three, β subunits comprising the amino acid sequence of SEQ ID NO:6 or a sequence homologous thereto.

The invention also relates to a purified mouse LSR receptor, characterized in that it comprises at least one subunit having a molecular weight of about 66 kDa and a subunit having a molecular weight of about 58 kDa.

Preferably, the purified mouse LSR receptor of the present invention is characterized in that it contains an α subunit comprising the amino acid sequence of SEQ ID NO:16 or a sequence homologous thereto, or an α′ subunit comprising the amino acid sequence of SEQ ID NO:17 or a sequence homologous thereto, and one, preferably three, 3 subunits comprising the amino acid sequence of SEQ ID NO:18 or a sequence homologous thereto.

The invention also relates to a purified human LSR receptor, characterized in that it comprises at least one subunit having a molecular weight of about 72 kDa and a subunit having a molecular weight of about 64 kDa.

Preferably, the purified human LSR receptor of the present invention is characterized in that it contains an α subunit comprising the amino acid sequence of SEQ ID NO:8 or a sequence homologous thereto, or an α′ subunit comprising the amino acid sequence of SEQ ID NO:10 or a sequence homologous thereto, and one, preferably three, 3 subunits comprising the amino acid sequence of SEQ ID NO:12 or a sequence homologous thereto.

A particularly preferred embodiment of the LSR receptors of the present invention is a recombinant LSR receptor obtained by expressing, in a recombinant host, one or more nucleotide sequences according to the invention. This preferred recombinant receptor consists of an α or α′ subunit and one, preferably three, β subunits, in particular an α or α′ subunit and three β subunits of a human LSR receptor.

The invention relates to polypeptides, characterized in that they are a constituent of an LSR receptor according to the invention.

It should be understood that the invention does not relate to the polypeptides in a natural form, that is to say that they are not taken in their natural environment. Indeed, the invention relates to the peptides obtained by purification from natural sources, or alternatively obtained by genetic recombination, or alternatively by chemical synthesis, and capable, in this case, of containing non-natural amino acids, as will be described below. The production of a recombinant polypeptide, which may be carried out using one of the nucleotide sequences according to the invention or a fragment of one of these sequences, is particularly advantageous because it makes it possible to obtain an increased level of purity of the desired polypeptide.

The invention therefore relates to a purified, isolated or recombinant polypeptide comprising a sequence of at least 5, preferably at least 10 to 15, consecutive amino acids of an LSR receptor, as well as the homologues, equivalents or variants of the said polypeptide, or one of their fragments. Preferably, the sequence of at least 10 to 15 amino acids of the LSR receptor is a biologically active fragment of an LSR receptor.

More particularly, the invention relates to purified, isolated or recombinant polypeptides comprising a sequence of at least 10 to 15 amino acids of a rat LSR receptor, of a mouse LSR receptor or of a human LSR receptor.

In the present description, the term polypeptide will be used to also designate a protein or a peptide.

The subject of the present invention is also purified nucleic acid sequences, characterized in that they encode an LSR receptor or a polypeptide according to the invention.

The invention relates to a purified nucleic acid, characterized in that it comprises at least 8, preferably at least 10 and more particularly at least 15 consecutive nucleotides of the polynucleotide of a genomic, cDNA or RNA sequence of the LSR receptor, as well as the nucleic acid sequences complementary to this nucleic acid.

More particularly, the invention relates to the purified, isolated or recombinant nucleic acids comprising a sequence of at least 8, preferably at least 10 and more particularly at least 15 consecutive nucleotides of the polynucleotide of a nucleic sequence of a mouse LSR receptor or of a human LSR receptor.

The invention also relates to the variant, mutated, equivalent or homologous nucleic sequences of the nucleic sequences according to the invention, or one of their fragments. It finally relates to the sequences capable of hybridizing specifically with the nucleic sequences according to the invention.

The invention therefore also relates to the nucleic acid sequences contained in the gene encoding the LSR receptor, in particular each of the exons of the said gene or a combination of exons of the said gene, or alternatively a polynucleotide extending over a portion of one or more exons. Preferably, these nucleic acids encode one or more biologically active fragments of the human LSR receptor.

The present invention also relates to the purified nucleic acid sequences encoding one or more elements for regulating the expression of the LSR gene. Also included in the invention are the nucleic acid sequences of the promoter and/or regulator of the gene encoding the receptor according to the invention, or one of their allelic variants, the mutated, equivalent or homologous sequences, or one of their fragments.

The invention also relates to the purified nucleic sequences for hybridization comprising at least 8 nucleotides, characterized in that they can hybridize specifically with a nucleic sequence according to the invention.

Preferably, nucleic acid fragments or oligonucleotides, having as sequences the nucleotide sequences according to the invention can be used as probes or primers.

The invention also comprises methods for screening cDNA and genomic DNA libraries, for the cloning of the isolated cDNAs and/or the genes coding for the receptor according to the invention, and for their promoters and/or regulators, characterized in that they use a nucleic sequence according to the invention.

The nucleic sequences, characterized in that they are capable of being obtained by one of the preceding methods according to the invention or the sequences capable of hybridizing with the said sequences, form part of the invention.

The invention also comprises the cloning and/or expression vectors containing a nucleic acid sequence according to the invention.

The vectors according to the invention, characterized in that they comprise elements allowing the expression and/or the secretion of the said sequences in a host cell, also form part of the invention.

The invention comprises, in addition, the host cells, in particular the eukaryotic and prokaryotic cells, transformed with the vectors according to the invention, as well as the mammals, except man, comprising one of the said transformed cells according to the invention.

Among the mammals according to the invention, there will be preferred animals such as mice, rats or rabbits, expressing a polypeptide according to the invention, the phenotype corresponding to the normal or variant LSR receptor, in particular mutated of human origin.

These cells and animals can be used in a method of producing a recombinant polypeptide according to the invention and can also serve as a model for analysis and screening.

The invention also relates to the use of a cell, of a mammal or of a polypeptide according to the invention for studying the expression and the activity of the receptor according to the invention, and the direct or indirect interactions between the said receptor and chemical or biochemical compounds which may be involved in the activity of the said receptor.

The invention also relates to the use of a cell, of a mammal or of a polypeptide according to the invention for screening a chemical or biochemical compound capable of interacting directly or indirectly with the receptor according to the invention, and/or capable of modulating the expression or the activity of the said receptor.

Production of Polypeptides Derived from the LSR Receptor

The invention also relates to the synthesis of synthetic or recombinant polypeptides of the invention, in particular by chemical synthesis or using a nucleic acid sequence according to the invention.

The polypeptides obtained by chemical synthesis and capable of comprising non-natural amino acids corresponding to the said recombinant polypeptides are also included in the invention.

The method of producing a polypeptide of the invention in recombinant form is itself included in the present invention, and is characterized in that the transformed cells are cultured under conditions allowing the expression of a recombinant polypeptide having a polypeptide sequence according to the invention, and in that the said recombinant polypeptide is recovered.

The recombinant polypeptides, characterized in that they are capable of being obtained by the said method of production, also form part of the invention.

The mono- or polyclonal antibodies or fragments thereof, chimeric or immunoconjugated antibodies, characterized in that they are capable of specifically recognizing a polypeptide or a receptor according to the invention, form part of the invention.

There may be noted in particular the advantage of antibodies specifically recognizing certain polypeptides, variants or fragments, which are in particular biologically active, according to the invention.

The invention also relates to methods for the detection and/or purification of a polypeptide according to the invention, characterized in that they use an antibody according to the invention.

The invention comprises, in addition, purified polypeptides, characterized in that they are obtained by a method according to the invention.

Moreover, in addition to their use for the purification of polypeptides, the antibodies of the invention, in particular the monoclonal antibodies, may also be used for the detection of these polypeptides in a biological sample.

More generally, the antibodies of the invention may be advantageously used in any situation where the expression, normal or abnormal, of a polypeptide of the LSR receptor, normal or mutated, needs to be observed.

Also forming part of the invention are the methods for the determination of an allelic variability, a mutation, a deletion, a loss of heterozygosity or a genetic abnormality, characterized in that they use a nucleic acid sequence or an antibody according to the invention.

These methods relate to, for example, the methods for the diagnosis of the predisposition to obesity, to the associated risks, or to pathologies associated with abnormalities in the metabolism of cytokines, by determining, in a biological sample from the patient, the presence of mutations in at least one of the sequences described above. The nucleic acid sequences analysed may be either the genomic DNA, the cDNA or the mRNA.

Nucleic acids or antibodies based on the present invention can also be used to allow a positive and differential diagnosis in a patient taken in isolation, or a pre-symptomatic diagnosis in an at risk subject, in particular with a familial history.

In addition, the detection of a specific mutation may allow an evolutive diagnosis, in particular as regards the intensity of the pathology or the probable period of its appearance.

Also included in the invention are the methods for selecting chemical or biochemical compounds capable of interacting, directly or indirectly, with the receptor or the polypeptide or nucleotide sequences according to the invention, and/or allowing the expression or the activity of the LSR receptor to be modulated.

The invention relates in particular to a method for selecting chemical or biochemical compounds capable of interacting with a nucleic acid sequence contained in a gene encoding an LSR receptor, the said method being characterized in that it comprises bringing a host cell expressing an LSR receptor or a fragment of the said receptor into contact with a candidate compound capable of modifying the expression or the regulation of the expression of the said nucleic sequence, and detecting, directly or indirectly, a modification of the expression or of the activity of the LSR receptor.

The invention also relates to a method for selecting chemical or biochemical compounds capable of interacting with the LSR receptor, the said method being characterized in that it comprises bringing an LSR receptor or a fragment of the said receptor, or a host cell expressing an LSR receptor or a fragment of the said receptor, into contact with a candidate compound capable of modifying the LSR activity, and detecting, directly or indirectly, a modification of the activity of the LSR receptor or the formation of a complex between the candidate compound and the said LSR receptor or the said polypeptide.

The invention comprises the compounds capable of interacting directly or indirectly with an LSR receptor as well as the compounds capable of interacting with one or more nucleic sequences of the LSR receptor. It also comprises the chemical or biochemical compounds allowing the expression or the activity of the receptor according to the invention to be modulated. The compounds, characterized in that they were selected by one of the methods according to the present invention, also form part of the invention.

In particular, among these compounds according to the invention, there are preferred the antibodies according to the invention, the polypeptides according to the invention, the nucleic acids, oligonucleotides and vectors according to the invention, or a leptin or one of its derived compounds, preferably one of its protein variants, or leptins which are chemically modified or are obtained by genetic recombination, or the protein gC1qR or one of its analogues, or one of their fragments.

The invention comprises, finally, compounds capable of modulating the expression or the activity of the receptor according to the invention, as medicament for the prevention of pathologies and/or of pathogeneses such as obesity and anorexia, hyperlipidemias, atherosclerosis, diabetes, hypertension, and more generally the various pathologies associated with abnormalities in the metabolism of cytokines.

DETAILED DESCRIPTION

The invention relates to a purified LSR receptor (“Lipolysis Stimulated Receptor”), preferably hepatic, consisting of at least one α or α′ subunit and at least one β subunit. The α subunit has a molecular weight of about 66 kDa in rats and in mice and of about 72 kDa in humans. The α′ subunit has a molecular weight of about 64 kDa in rats and in mice and of about 70 kDa in humans. The β subunit has a molecular weight of about 58 kDa in rats and in mice and of about 64 kDa in humans.

The inventors have formulated the hypothesis according to which the most abundant, and probably the most active, form of the LSR receptor is that in which an α or α′ subunit and three β subunits exist. It appears, however, possible that the α and α′ subunits, on the one hand, and the β subunit, on the other, have distinct biological functions and that these functions can be performed in a cell independently of their assembly in the form of a receptor.

The inventors have also observed that a complex can form between the LSR receptor and the gC1qR receptor having a molecular weight of about 33 kDa, or a homologous protein. It appears that the gC1qR receptor is transiently combined with the LSR receptor and that the presence of a C1q protein or of homologous proteins makes it possible not only to dissociate gC1qR from the LSR receptor but also to activate the LSR receptor, including in the absence of fatty acids.

The present invention therefore relates to a receptor, in particular of hepatic cells, characterized in that it is capable, in the presence of free fatty acids, of binding lipoproteins, and in the absence of free fatty acids, of binding a cytokine, preferably the bound leptin, lipoproteins and cytokine being incorporated and then degraded by the cell, it being possible for the said receptor, in addition, to bind the gC1qR protein or one of its analogous proteins.

The LSR receptor represents the principal pathway for the elimination of lipoproteins of intestinal origin and of particles high in triglycerides, in particular VLDLs and chylomicrons. The LSR receptor can also serve as a pathway for the elimination of LDLs, particles high in cholesterol, which are for the most part removed by the LDL receptor pathway, but of which about 30% are eliminated at the hepatic level by pathways different from the LDL receptor.

The inventors have in fact demonstrated that the LSR receptor is capable of binding lipoproteins, in particular the lipoproteins high in triglycerides, and then of internalizing and degrading them. This lipoprotein clearance activity by the receptor requires the presence of free fatty acids, for example oleate, and is inhibited in the presence of antibodies directed against LSR or against peptides derived from LSR.

The inventors have also demonstrated that in the absence of free fatty acids, for example oleate, the LSR receptor is capable of binding cytokines, preferably leptin. The leptin clearance function is, however, only possible if the receptor has not bound fatty acids produced by the hepatic lipase or by the hormone-sensitive lipase of the adipose tissue. Once the cytokines have been bound, the LSR receptor internalizes them and degrades them. This cytokine, preferably leptin, degradation activity is inhibited by antibodies directed against LSR or against peptides derived from LSR.

The inventors have shown that it is the α subunit of the LSR receptor which is most particularly involved in the binding of cytokines, and preferably of leptin.

Furthermore, the inventors have shown, with the aid of mice, that, in vivo, the LSR receptors carry out the hepatic capturing of cytokines, preferably of leptin.

The high levels of leptin in all obese human subjects can be explained by several molecular mechanisms which are capable of reducing the hepatic clearance of leptin, including in particular:

a) alteration of one or more genes for LSR, and/or of their promoters; b) facilitation, by post-transcriptional modifications, of the allosteric rearrangement allowing the passage from the cytokine-competent conformation to the lipoprotein receptor conformation; c) deficiency in the transport of vesicles containing LSR from, or towards, the plasma membrane (this function depends on the integrity of the cytoskeleton); d) increase in the degradation of LSR; e) increase in the lipid calorie ration which, by diverting the receptor towards the clearance of lipoproteins, reduces in part its capacity to degrade leptin.

Finally, the inventors have demonstrated that cytokines, preferably leptin, modulate the activity of the LSR receptor in the presence of free fatty acids. More particularly, the cytokines increase the lipoprotein clearance activity of the LSR receptor and more precisely, the binding, internalization and degradation of the VLDLs and LDLs. This increase in the LSR activity could be the result of the increase in the apparent number of LSR receptor at the surface of the cells following an increase in protein synthesis and following a mobilization of endocytosis vesicles. In addition, the inventors have shown, with the aid of mice, that, in vivo, cytokines, preferably leptin, are capable of reducing postprandial lipaemic response.

Leptin, and probably other cytokines, are therefore regulators of the activity of LSR. A syndrome of resistance to leptin, or to other cytokines, can lead to a hypertriglyceridemia, which is either permanent or limited to the postprandial phase.

The role played by LSR in the clearance of leptin makes it possible to formulate a physiopathological model which requires a revision of the strategies used for treating obesity. It is indeed essential to reduce the concentrations of leptin in obese human subjects in order to restore the physiological fluctuations of this hormone.

Accordingly, it is possible to envisage using compounds for the treatment of obesity allowing modulation of the number of LSR receptors, of their recycling rate, or of the change in their conformation, and/or allowing in particular:

1. leptinemia, and therefore the sensations of satiety and of hunger, to be controlled; 2. normal leptin concentrations to be restored and normal regulation of dietary habit by the normal perception of the sensations of hunger and of satiety 3. triglyceridemia to be controlled; 4. the plasma concentrations of residues of chylomicrons, highly atherogenic particles, to be regulated.

The role played by the LSR receptor in the hepatic clearance of lipoproteins of intestinal region makes it possible to envisage using compounds capable of modulating the expression and/or the activity of LSR in order to modulate the distribution of lipids of dietary origin between the peripheral tissues, in particular the adipose tissues, and the liver. A treatment of obesity will consist in promoting the hepatic degradation of lipoproteins, and thereby reducing their storage in the adipose tissue, and regulating their plasma concentrations. The latter effect makes it possible to envisage the use of such compounds to reduce the risks associated with obesity, in particular the atherogenic risks.

It is possible to envisage using methods of regulating the activities of LSR to introduce treatments which make it possible to overcome the vicious circle which characterizes anorexia nervosa. By reducing the number of receptors, it should be possible to promote weight gain in anorexic or undernourished subjects.

Under these conditions, it is advantageous to selectively inhibit the clearance of leptin by using synthetic peptides or pharmacological molecules which either reduce the synthesis of LSR or block its capacity to bind leptin and/or lipoproteins, or alternatively increase the catabolism of the receptor.

Analysis of the primary structure of the α subunit of LSR, as described below, shows a site homologous to the cytokine binding sites present on their receptors, as well as two routing signals which allow endocytosis and rapid degradation of ligands in the lysozomes. This observation is new in the sense that the cytokine receptors do not allow the internalization and the degradation of ligands. These receptors have been characterized on the basis of their intracellular signalling properties.

Thus, in addition to it having the property of allowing the proteolytic degradation of lipoproteins and of leptin, it is highly probable that the LSR receptor also carries out the degradation of other cytokines. This function can be studied by virtue of the anti-LSR antibodies and of transfected CHO cells expressing the α subunit of LSR as described in Example 4. The involvement of LSR in the clearance of cytokines is essential because these molecules play an important role in the regulation of the metabolism of lipids, of the metabolism of glucose, and in the regulation of food intake and of weight gain.

The molecular mechanisms by which the cytokines modulate the physiological functions involved in obesity and its complications are numerous and complex. It is worth noting, however, the fact that abnormalities in the metabolism of cytokines are associated with hypertriglyceridemia which frequently accompanies viral, bacterial or protozoal infections. Moreover, cytokines, and more particularly Tumor Necrosis Factor (TNF), induce a transient hypertriglyceridemia similar to that observed in certain forms of obesity-related diabetes.

The reduction in the number of LSR receptors expressed in the liver of obese mice could explain a deficiency in the elimination of some cytokines, this deficiency causing metabolic disruptions such as those found in obesity. The use of hepatic cells in culture, and of the various models of obese animals cited below, will make it possible to determine, among all the cytokines and more particularly those which induce weight loss (IL-6, LIF, OSM, CNTF, IL-11, IL-12α, as well as TNFα and TNFβ), those which modulate the expression and/or the activity of LSR. The determination of such cytokines can, for example, be carried out using methods such as those presented in Examples 4 to 6.

Finally, analysis of the primary structure of the α LSR reveals potential phosphorylation sites. This opens the perspective of a regulation of cellular activity by the LSR receptor. A particularly important example would be the involvement of LSR in the regulation of the production of “Acute Phase Proteins” under the impetus of various stimuli, including cytokines.

The involvement of LSR in the clearance and the degradation of cytokines may, in addition, not be limited to the liver. Indeed, while it has been demonstrated that the expression of LSR is predominantly hepatic, it is also certain that the expression of this receptor is not limited to this organ. Preliminary Northern-blot analysis on various human tissues has been able to reveal, in addition to the hepatic products, expression products in the kidney and in the testicle. A more thorough analysis will make it possible to show the different tissues expressing LSR in humans. In this perspective, LSR could be involved in the degradation of cytokines not only at the hepatic level, but also at the level of the peripheral tissues. A deficiency in this activity could be involved in the pathogenesis of autoimmune diseases, of multiple sclerosis and of rheumatoid arthritis. Accumulation of cytokines is frequently found in the pathogenesis of these diseases.

The invention relates to polypeptides, characterized in that they are a constituent of an LSR receptor according to the invention. The invention relates more particularly to the polypeptides characterized in that they constitute the α, α′ or β subunits of the LSR receptor.

The invention relates more particularly to a purified, isolated or recombinant polypeptide comprising a sequence of at least 5, preferably of at least 10 to 15 consecutive amino acids of an LSR receptor, as well as the homologues, equivalents or variants of the said polypeptide, or one of their fragments. Preferably, the sequence of at least 10 to 15 amino acids of the LSR receptor is a biologically active fragment of an LSR receptor.

Preferably, the invention relates to purified, isolated or recombinant polypeptides comprising a sequence of at least 10 to 15 amino acids of a rat LSR receptor, of a mouse LSR receptor or of a human LSR receptor.

In a first preferred embodiment of the invention, the polypeptide is characterized in that it comprises a sequence of at least 10 to 15 consecutive amino acids of a sequence chosen from the group comprising the sequences of SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6, as well as the variants, equivalents or homologues of this polypeptide, or one of their fragments. Preferably, the polypeptide is a homologue or a biologically active fragment of one of the abovementioned sequences.

In a second preferred embodiment of the invention, the polypeptide is characterized in that it comprises a sequence of at least 10 to 15 consecutive amino acids of a sequence chosen from the group comprising the sequences of SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18, as well as the variants, equivalents or homologues of this polypeptide, or one of their fragments. Preferably, the polypeptide is a homologue or a biologically active fragment of one of the abovementioned sequences.

In a third preferred embodiment of the invention, the polypeptide is characterized in that it comprises a sequence of at least 10 to 15 consecutive amino acids of a sequence chosen from the group comprising the sequences of SEQ ID NO:8, SEQ ID NO:10 and SEQ ID NO:12, as well as the variants, equivalents or homologues of this polypeptide, or one of their fragments. Preferably, the polypeptide is a homologue or a biologically active fragment of one of the abovementioned sequences.

Among the preferred polypeptides of the invention, there will be noted particularly the polypeptides having the human sequence SEQ ID NO:8, SEQ ID NO:10 or SEQ ID NO:12, as well as those having the rat sequence SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6, or those having the mouse sequence SEQ ID NO:16, SEQ ID NO:17 or SEQ ID NO:18. The fragments corresponding to the domains represented in FIGS. 1 to 6, whose positions on the sequences corresponding to SEQ ID NO:2, 8 or 16, are indicated in Tables 1, 3 and 4.

Finally, the invention also relates to the polypeptides of SEQ ID NO:29 and SEQ ID NO:30.

The present invention also relates to polypeptides comprising the polypeptides described above, as well as their homologous, equivalent or variant polypeptides, as well as the fragments, preferably biologically active, of the said polypeptides.

Among the polypeptides according to the invention, also preferred are the polypeptides comprising or consisting of an amino acid sequence chosen from the amino acid sequences as described above, characterized in that the said polypeptides are a constituent of the receptor according to the invention.

The systematic analysis of the products of the 3 rat cDNAs described in the present application is schematically represented in FIG. 1. The α subunit of the rat LSR receptor, a protein encoded by the longer cDNA (LSR-Rn-2097), has the following characteristics.

Potential glycosylation sites are found at positions 12-14 and 577-579. A potential site of attachment of glycosaminoglycans is found at position 14-17.

Several phosphorylation sites are located at the level of the NH2-terminal end (positions 193-196, 597-600, 169-171, 172-174, 401-403, 424-426, 464-466, 467-469, 185-188, 222-225, 436-439, 396-399, 504-507, 530-533, 624-627, 608-615), suggesting that the latter is oriented towards the intracellular region.

Moreover, the protein has, on the NH2-terminal side, a hydrophobic amino acid sequence separated into two parts by 2 amino acids inducing a hairpin structure in which the two arms would consist of hydrophobic amino acids. It is reasonable to assume that this region represents the fatty acid binding site of LSR. The glove-finger structure thus produced can accommodate an aliphatic hydrocarbon chain. The two amino acids are, more precisely in the case of rat LSR, two Prolines situated at positions 31 and 33 of the polypeptide sequence of the α subunit.

Still on the NH2-terminal side is a consensus sequence for binding to clathrin, a protein which lawns the inner surface of the “coated pits” (Chen et al., 1990). These specific regions of the plasma membrane allow rapid endocytosis of membrane proteins. Such a consensus sequence is found at the level of the LRP-α2-macroglobulin receptor, of CRAM and of the LDL receptor (Herz et al., 1988; Lee et al., 1990; Goldstein et al., 1995). The consequence of a mutation at this level is a substantial delay in the internalization of the LDLs and induces familial hypercholesterolemia (Davis et al., 1986).

The receptor then possesses a hydrophobic amino acid sequence which constitutes a potential transmembrane domain. The length of this segment allows only one passage across the phospholipid bilayer (Brendel et al., 1992).

Between this clathrin binding signal and the hydrophobic chain corresponding to the single transmembrane segment are 2 motifs LI et LL (Letourneur et al., 1992). These two motifs are found in the following proteins: glut 4 glucose carrier (Verhey et al., 1994); the nonvariant chain and the histocompatibility complex class II (Zhong et al., 1997; Parra-Lopez et al., 1997). These signals control endocytosis and the intracellular addressing of proteins in the peripheral membrane system.

On the C-terminal side, there is then a cysteine-rich region which exhibits homology with the cytokine receptors and more particularly: the TNF 1 and 2 (Tumor Necrosis Factor 1 and 2) receptors; the low-affinity NGF (Nerve Growth factor) receptor; the Shope fibroma virus TNF soluble receptor; CD40, CD27 and CD30, receptors for the cytokines CD40L, CD27L and CD30L; the T cell protein 4-1 BB, receptor for the putative cytokine 4-1BBL, the FAS antigen (APO 1), receptor for the FASL protein involved in apoptosis, the T cell 0X40 antigen, receptor for the cytokine 0X40L, and the vaccinia virus A53 protein (Cytokines and their receptors, 1996; Banner et al., 1993).

In addition to this cysteine-rich segment, there is a region of amino acids which are alternately charged + and − (Brendel et al., 1992). This region provides a potential binding site for the apoprotein ligands Apo B and Apo E.

This region contains, in addition, an RSRS motif found in lamin (Simos et al., 1994) and in SF2′ (Krainer et al., 1991).

The LSR α′ form encoded by the LSR-Rn-2040 cDNA possesses all the domains described above based on the LSR α sequence encoded by the LSR-Rn-2097 cDNA, with the exception of the LI/LL element, whose Leucine doublet is removed by alternative splicing. Although possessing sequences which are very similar, the subunits α encoded by LSR-Rn-2097 and α′ encoded by LSR-Rn-2040 could therefore differ in their recycling rate and their addressing. The β form encoded by LSR-Rn-1893 does not possess a transmembrane domain or a region rich in cysteines and homologous to the cytokine receptors. However, it possesses at the NH2-terminal level the hydrophobic region separated by a repetition of prolines, the region rich in charged amino acids and the RSRS motif. This constituent is probably positioned entirely outside the cell where it is bound via disulphide bridges either to the product of LSR-Rn-2040, or to that of LSR-Rn-2097.

Table 1 below lists the different domains or motifs described above, indicates whether or not they belong to each of the subunits of the LSR receptor, as well as the positions of the start and end of the said domains or motifs, or of regions carrying the said domains or motifs, as indicated in the sequence of SEQ ID NO:2.

TABLE 1 Position on SEQ ID NO: 2 Presence on: Domain or motif Start End α α′ β Potential fatty acid binding site 23 41 X X X Potential clathrin binding site 104 107 X X X Signal for transport: LI 183 184 X X X LL 195 196 X Transmembrane domain 204 213

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