Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1996-10-08
1999-02-09
Jones, W. Gary
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435912, 530300, 5303871, 536 241, 536 2431, 536 2433, C12Q 168, C12P 1934, A61K 3816, C07H 2104
Patent
active
058692479
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to a nucleotide sequence encoding a natural resistance-associated macrophage protein, the protein product thereof, nucleotide probes and primers thereto, polypeptide fragments of the protein and related antibodies.
BACKGROUND TO THE INVENTION
Macrophages are the main phagocytic cells of animals and play a key role in the immune system. Macrophages bind and injest particles recognised as foreign by the immune system. Such particles include microorganisms.
The three microorganisms Salmonella typhimurium, Leishmania donovani and Mycobacterium bovis (BCG) are all intracellular pathogens of macrophages. Three separate groups of scientists had previously identified genes capable of controlling resistance and susceptibility to each of these microorganisms. The genes were designated respectively Ity, Lsh and Bcg. Subsequent work has led the scientists to conclude that Ity/Lsh/Bcg is a single gene and is expressed at the macrophage level (Ref 1).
Recently, Vidal et al (Ref 2) cloned a murine gene as the most likely candidate to be Lsh/Ity/Bcg. This gene has been termed the natural resistance-associated macrophage protein (Nramp) gene. A cDNA for Nramp was isolated from a pre B-cell cDNA library and sequenced. The amino acid sequence for the protein product was deduced from the nucleotide sequence and predicts a 53 kDa protein. On the basis of the deduced amino acid sequence, Vidal et al proposed as a function of the Nramp protein the transport of nitrate across the membrane of the intracellular vacuole of the macrophage containing the microorganisms. In the acid environment of this vacuole, the nitrate could be converted via nitrite to toxic nitric oxide thereby to enhance killing of the microorganisms.
The present applicants have isolated and sequenced a macrophageexpressed Nramp cDNA. Contrary to the teaching of Vidal et al the present applicants have found a different nucleotide sequence including a region encoding an additional amino acid sequence at the N-terminus. Surprisingly, the additional amino acid sequence includes structural features which may be responsible for protein-protein interactions essential in signal transduction pathways thereby suggesting that Nramp controls early amplification of transmembrane signalling in disease resistant macrophages by binding the SH3 domain of tyrosine kinases or other molecules.
SUMMARY OF THE INVENTION
The present invention provides in one aspect a natural resistance-associated macrophage protein having an N-terminal region comprising an SH3 binding domain. When present in the macrophage, the protein is capable of controlling resistance to pathogenic microorganisms. SH3 (Src homology 3) domains are believed to mediate specific protein-protein interactions required in signal transduction (Ref 3) and have been identified as related sequences in a variety of proteins (Refs 4 and 5). In one embodiment of the present invention, the SH3 binding domain comprises the SH3 binding motif PGPAPQPXPXR (SEQ ID NO: 1), more particularly PGPAPQPAPCR (SEQ ID NO: 2). This motif is found in the protein obtainable from mice. In another embodiment of the present invention, the SH3 binding domain comprises the SH3 binding motif PXSPTSPXPXXAPPRXT (SEQ ID NO: 3), more particularly PTSPTSPGPQQAPPRET (SEQ ID NO: 4). This motif is found in the protein obtainable from humans. Typically, SH3 binding domains are rich in proline and sometimes serine. Preferably, the SH3 binding domain obtainable from mice further comprises the polypeptide segment (S,A)PP(R,K)XSRPXXXS(I,V)XSX (SEQ ID NO: 5) at the N-terminal end of the SH3 binding motif. More particularly, the polypeptide segment is SPPRLSRPSYGSISSL (SEQ ID NO: 6). The SH3 binding domain obtainable from humans preferably further comprises the polypeptide segment GPQRLSGSSYGSISS (SEQ ID NO: 7).
A further preferred feature of the N-terminal region is the presence of one or more consensus sequences for protein kinase C (PKC) phosphorylation. Preferably, the N-terminal region has two protein
REFERENCES:
Vidal et. al. Cell 73:469-485 (May 1993).
Sambrook et. al. Molecular Cloning : A Laboratory Manual. Cold Spring Harbor Press (1989) pp. 17.2 & 17.10-.44.
Baker et. al. J. Cell. Biochem 18p (suppl): 204 (Jan. 4, 1994).
Morgenstern et. al. Nod. Ac. Res. 18:3587-3596 (Jun 25, 1990).
Van Duijn et al in Protein Biotechnology ed. Franks (1993) The Humana Press Inc pp. 365-393.
Augubel et. al ed.s Snovt Protocols in Molecular Biology (1989) John Wiley & Sons. New York pp. 75-78,136-137, 141-142, 158-159, 177-178, 190-194, 201-231.
Barton Charles Howard
Blackwell Jenefer Mary
White Jacqueline Katie
Jones W. Gary
Shoemaker Debra
The Wellcome Trust Limited as Trustee to the Wellcome Trust
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