Compounds having lectinic properties and their biological...

Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Glycoprotein – e.g. – mucins – proteoglycans – etc.

Reexamination Certificate

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C530S357000, C530S412000, C514S008100, C514S012200, C514S021800, C424S185100

Reexamination Certificate

active

06774220

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to compounds having lectinic properties, and to their biological applications.
More particularly, it relates to proteins or polypeptides of the sarcolectin type.
2. Description of the Related Art
It is known that this term has been used to designate the lectins identified in human osteogenic sarcomas or those induced in the hamster (after inoculation with Moloney sarcoma virus) where they are present in large quantities. However, sarcolectins are also found in a wide variety of normal or tumoral tissues in vertebrates. (primates, rodents, fowl).
They may also be detected on the surface of normal or transformed cells of human or animal origin.
Purified preparations of sarcolectins have already been described and their properties reported.
The most regularly characterised physical-chemical properties are:
sensitivity to proteases (trypsin, PRONASE® (proteolytic enzyme), but also resistance to controlled treatments, under certain conditions, with pepsin;
resistance to temperature variations (−20° C. to +100° C.);
resistance to pH variations (2 to 8);
resistance to detergents such as SDS and di-thiothreitol;
migration in SDS-PAGE gel of the protein in the molecular weight region of 65-55 kd.
Their biological properties are of three orders (see references (1) to (8) given at the end of the specification, with the other references mentioned below, in the document “Bibliographic references”):
agglutination of normal or transformed cells (activity at cell membrane level). Cytoagglutination may be inhibited due to the affinity of sarcolectins for simple sugars.
stimulation of the growth of human T and B lymphocytes, Daudi lymphoid cells, and cells adhering to the substrate such as L929 murine cells, transformed rat fibroblasts (Fr3T3) and human fibroblasts (FS4). Sarcolectins thus act as promoters of cell growth, of undetermined specificity;
reduction or suppression of the antiviral state pre-established by interferon (IFN) and restoration in the cell of the initial sensitivity to the virus. After antiviral resistance has been established, SCLs inhibit the continuation of the synthesis of interferon-dependent proteins, for example, protein kinase and 2-5A synthetase. Restoration of the initial state depends on the doses of SCL and may be more or less complete. When the cell is restored, it is possible either to stimulate growth by SCLs or by other growth factors, or on the other hand to re-treat the cells with IFN in order to develop antiviral resistance again.
SUMMARY OF THE INVENTION
A The invention is based on obtaining highly purified preparations of SCL which have made it possible to develop strategies leading to the isolationof cDNA clones coding for a 55 kd protein, the study of which has revealed unexpected biological properties.
It should be noted that all previous attempts prior to the invention retained the 65 kd protein as the molecule having the biological properties of SCL. During identifications based on electrophoresis on acrylamide gel followed by Western blot, the major band in the 65 kd region had in fact been retained as holding all the biological properties: the 55 kd band is not constantly observed and moreover appears minor in all cases. Now, in a surprising manner, it appears that the 65 kd band corresponds to an artifact which results from the fixation of a few SCL molecules on albumin, but that the molecule possessing sarcolectin-type properties is in reality the 55 kd protein which contains all the genetic information responsible for the biological expression of the molecule.
The object of the invention is, therefore, to provide various products binding to 55 kd SCL, namely in particular, proteins, polypeptides or fragments thereof, DNA sequences coding for these proteins or these polypeptides or fragments thereof, or on the other hand, inhibiting their expression, and antibodies directed against these proteins, polypeptides or fragments thereof.
The term sarcolectin or SCL, as used hereinafter in the specification, will designate without distinction proteins, polypeptides and fragments of these compounds, or their derivatives, as long as they possess lectinic properties as defined according to the invention.
The invention also relates to processes for obtaining these various products.
According to another aspect, the invention also relates to biological applications for these products.
The sequences of nucleotides according to the invention are sequences isolated from their natural environment and are characterised in that they contain at least part of the sequence SEQ ID NO: 1, one or more nucleotides being modified if necessary, it being understood that these sequences are capable of coding for sarcolectins, i.e. proteins, polypeptides or fragments of these compounds, or derivatives, having lectinic properties.
The sequence SEQ ID NO: 1 is given at the end of the specification, with the other sequences mentioned below, in the document entitled “List of sequences”.
The term lectinic properties means the ability of the SCLs to agglutinate normal or transformed cells, their stimulating effect on cell growth and their effect of inhibiting the antiviral effect induced by interferon, under the conditions described in (8).
Such sequences used according to conventional recombinant DNA techniques are capable of coding for proteins or polypeptides, or fragments thereof, having lectinic activity, comprising at least one chain of amino acids as indicated in SEQ ID NO: 1, in which one or more amino acids are modified, if necessary.
These sequences are further characterised in that they are capable of hybridising with at least one fragment of SEQ ID NO: 1 carrying at least part of the genetic information for a sarcolectin. This hybridisation may be carried out under stringent conditions, but also under relaxed conditions as described in (10).
The invention relates in particular to the nucleotide sequence corresponding to the open reading frame running from position 62 to position 1429 in SEQ ID NO: 1.
In this sequence, the domains of the 5′ ends and/or 3′ ends are particularly preferred given that they contain the genetic information for the fragments possessing the said lectinic properties.
From this point of view, the sequence of about 405 bp running from position 62 to 467 in SEQ ID NO: 1, as represented in SEQ ID NO: 2, is quite particularly preferred.
These 5′ and 3′ domains are characterised in that they contain a large proportion of amino acids capable of being phosphorylated, such as serine, threonine and tyrosine.
The various sequences mentioned above may be genome sequences or of the genome type since certain chains of nucleotides may be separated by introns which will be excised to lead to the expression of mature SCLS.
The corresponding mRNA sequences, or the antisense sequences corresponding to the sequences defined above, and the complementary sequences of these various sequences, also come within the scope of the invention.
These various sequences of nucleotides are further characterised in that they contain no DNA of mammals, infectious agents, prions and other materials of natural products.
As indicated above, the invention also relates to the sequences derived from SEQ ID NO: 1.
These derived sequences are obtained by modification, substitution, alteration, mutation or genetic and/or chemical deletion of one or more nucleotides of SEQ ID NO: 1 or of a fragment of SEQ ID NO: 1, it being understood that they possess genetic information for coding for an SCL retaining at least in part the lectinic activity presented by the polypeptide coded by SEQ ID NO: 1, this activity being increased, if necessary.
If desired, these modifications make it possible to adapt the sequences defined above to the expression in a type of vector or host, or to facilitate cell penetration of the coded polypeptide or to increase its activity.
The invention also relates to the expression vectors containing at least one of the nucleotide sequences defined above, under the

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