WO2004040304A2

WO2004040304A2 - Digoxin labelled proteins and production and uses thereof

Info

Publication number: WO2004040304A2
Application number: PCT/GB2003/004675
Authority: WO
Inventors: Gordan Lauc
Original assignee: Bio-Med Reagents Limited
Priority date: 2002-11-01
Filing date: 2003-10-30
Publication date: 2004-05-13
Also published as: AU2003276423A8; GB0225476D0; WO2004040304A3; AU2003276423A1

Abstract

The present invention provides a probe comprising a protein labelled with a digoxin moiety. In a preferred embodiment the protein is a lectin. The probe of the present invention is useful in, e.g. investigating glycoconjugaes in mixtures of biomolecules.

Description

Digoxin Labelled Proteins and Production and Uses Thereof

This invention relates to proteins such as lectins labelled with digoxin, a method of synthesis and uses thereof.

Glycoconjugates are biological macromolecule containing a carbohydrate moiety; the term therefore encompasses, amongst others, glycolipids, glycoproteins and proteoglycans .

Previous research has demonstrated the importance of the carbohydrate part of glycoconjugates in numerous physiological processes. These processes, however, cannot be fully understood without knowledge of the structure of both the protein and carbohydrate parts of the involved molecules.

Lectins are simple and versatile tools for the analysis of carbohydrate on glycoconjugates and have been used in many different technologies, such as affinity chromatography, histopathology, multiwell microassays and Western blots. Since lectins do not share a common and unique structural feature that allows easy detection with a secondary reagent (unlike antibodies, for example) , it is necessary to incorporate a suitable label into these molecules to enable subsequent identification and detection using a secondary reagent. For example, a streptavidin/biotin labelled enzyme system or an anti-label antibody. Known labels for lectins include biotin and digoxigenin.

The problem with using biotin as a label is the presence of endogenous biotin in various tissues which can lead to false-positives on application of the secondary reagent. This is shown below in Fig. 1.

Digoxigenin is a deglycosylated form of the cardiac glycoside digoxin, and is an excellent label for lectins because it eliminates the problems of false positives. As shown in Fig. 1, there is very little background presence of digoxin in normal tissues.

Digoxin itself was not used as a label for lectins as the high acid sensitivity of the glycosidic bonds between the digitoxoses, and the alkaline sensitivity of the lactone ring, has impeded chemical derivatization of this molecule and therefore its use as a label. In co-pending Application GB 0007513.5 (published as GB 2361699) it was found that an activated form of digoxin can be attached to a small ligand directed to a receptor. Thus, when probes are brought into contact with the receptor, the receptor binds the ligand. Illumination with ultraviolet light causes covalent crosslinking of the probe to the receptor. Although GB 2361699 discloses the use of digoxin as a label, it does not disclose direct labelling of a protein such as a lectin. It has now been found that direct attachment to large biological molecules like proteins can be achieved. Such labelled proteins can be used as effective biological tools.

According to the present invention there is provided a protein labelled with a digoxin moiety. Advantageously the protein is a lectin. It is preferred that the digoxin moiety is covalently bound to an amino group of the lectin. Advantageously the digoxin moiety is covalently bound directly (i.e. without intermediate or linking molecules) to the lectin.

Lectins from plant or animal origin are both suitable to be labelled according to the invention. Examples of lectins of animal origin include lectins such as: 1) Calnexin 2) M-type lectins 3) L-type lectins 4) P-type lectins 5 ) C-type lectins 6 ) Galectins 7) I-type lectins; and 8) R-type lectins.

Lectins from plant origin were used in the example below, which demonstrates labelling of lectins isolated from Sambucus nigra (Sambucus Nigra Agglutinin or SNA) , Tri ticum vulgare (Wheat Germ Agglutinin or WGA) , Galanthus nivalias_r or Canavalin envalin { Concanavalin A or Con A) although any lectin would be suitable. Table I contains a non-exhaustive list of other plants from which lectins can be isolated and used within the scope of the invention.

According to another embodiment of the present invention there is provided a method of labelling a protein, and more preferably a lectin, with a digoxin moiety, said method comprising the steps of; 1. reacting digoxin with CNBr to form activated digoxin; 2. reacting the protein to be labelled with the activated digoxin under suitable conditions to form a digoxin-labelled protein; and optionally 3. separating the digoxin-labelled protein from the reaction mixture.

Whilst CNBr is the reactant which has been shown to be particularly efficient in activating digoxin, the scope of the invention extends to functional equivalents .

According to a particularly preferred embodiment of the invention the protein is a lectin.

Preferably the activated digoxin is dissolved in a water-miscible organic solvent such as ethanol, methanol or acetonitrile prior to reacting with the protein.

It is further preferred that the activated digoxin to be reacted with the protein is added in 5 to 20 fold molar excess.

It is further preferred that the activated digoxin and protein be left to react for at least 5 hours, advantageously at least 16 hours.

According to yet a further embodiment of the present invention there is provided the use of a digoxin-labelled protein to analyse the properties of a macromolecule . Preferably the protein is a lectin. It is further preferred that the macromolecule to be analysed is a glycoconjugate.

The digoxin-labelled lectin can be used in any type of assay in which a peptide probe may generally be used. These include, but are not restricted to, blotting (e.g. Western blotting), histological staining and ELISAs. According to a further embodiment of the present invention there is provided a method of analysing glycoconjugates in a mixture of biomolecules, said method comprising:

1. exposing said mixture to a digoxin-labelled protein under suitable condition to form a digoxin-labelled protein/biomolecule complex. 2. removing the unbound digoxin-labelled protein; 3. determining the presence and/or amount of the retained digoxin-labelled protein/biomolecule complex.

Suitably the mixture of biomolecules is first separated using, for example, electrophoresis. Preferably the biomolecules are blotted onto a suitable membrane, such as Immobilon PVDF membrane or nitrocellulose, prior to exposure to the digoxin-labelled lectin.

Preferably the presence and/or amount of digoxin- labelled lectin is visualised by exposing the mixture to a digoxin specific probe containing a chromogenic, chemoluminescent or radioactive marker. Preferably an anti-digoxin antibody linked to alkaline phosphatase is used.

According to an alternative embodiment of the present invention there is provided a method of analysing glycoconjugates in a mixture of biomolecules, said method comprising: 1. adding digoxin-labelled proteins to a mixture of biomolecules under suitable conditions to form digoxin-labelled protein/glycoconjugate complexes; 2. exposing the complexes thus formed to an immobilised anti-digoxin antibody; and 3. removing unbound digoxin-labelled proteins.

Embodiments of the present invention will now be described, by way of example only, with reference to the accompanying figures in which:

Fig. 1 shows the results of a Western blot of various homogenates of rat tissues (1-cortex, 2- thy us, 3-serum, 4-spleen and 5-liver) which were developed with a streptavidin-biotinylated alkaline phosphatase complex (panel A) or an anti-digoxin antibody labelled with alkaline phosphatase (panel B) .

Fig. 2 shows the structures of digoxin and digoxigenin.

Fig. 3 shows the reaction scheme for the activation of digoxin with CNBr and subsequent linking of CNBr-activated digoxin with an amino group in a target compound. Fig. 4 shows SDS-PAGE separation of extracts from human cell line (HL60) . The extract has been probed with 3 lectins (ConA, WGA and SNA) labelled with either biotin or digoxin and developed with alkaline phosphatase conjugated with streptavidin and anti-digoxin respectively. The lanes are designated as follows: A - Con A/Biotin; B - Con A/Digoxin; C - WGA/Biotin; D - WGA/Digoxin; E - SNA/Biotin; and F - SNA/Digoxin.

Fig. 1 shows a comparison of the degree of background reading obtained when a biotin or a digoxin specific probe is used to analyse extracts from various rat tissues (in the absence of any exogenous biotin or digoxin) . As can be clearly seen, there are a significant number of bands visualised when the biotin specific probe is used (Panel A) . The converse can be seen when the probe for digoxin is used, the limited amount of background staining being solely visible in the liver extract (Panel B) . This demonstrates that use of digoxin as a label is preferable to use of biotin. In using digoxin there is reduced chance of obtaining erroneous results due to false positives, and also it is easier to identify bands of interest without the clutter of the background staining.

The current invention describes a generic method for the preparation of digoxin-labelled proteins by activating digoxin with cyanogen bromide (CNBr) and reacting the activated digoxin with the amino groups on the polypeptide backbone or side chains of the protein. The structures of digoxin and digoxigenin are shown in Fig. 2 and the reaction sequence is summarised in Fig. 3.

Several lectins, including those from Sambucus nigra (SNA) , Triticum vulgare (WGA) , and Concanavalin A (Con A) have been labelled in this way and successfully used to investigate the carbohydrate structure of glycoproteins . The preparation of digoxin-labelled Con A is described in detail below, but the same procedure could be applied to all lectins, and indeed to any proteins.

Activation of digoxin using CNBr

Digoxin (Aldrich Chem. Co., Milwaukee, WI) (100 μmol, 78.1 irtg) was dissolved in 3 ml 33% tetrahydrofuran, 66% 2 M potassium phosphate buffer, pH 12, to form a biphasic mixture containing 33 M digoxin. CNBr (Aldrich Chem. Co., Milwaukee, WI) (10-fold excess) was added to this mixture as a 5 M solution in tetrahydrofuran. The reaction mixture was stirred at 22°C for 30 - 60 min. Formation of the product was monitored by thin-layer chromatography (TLC) on 0.2 mm Silica Gel 60 F254 precoated on aluminium sheets (Merck, Darmstadt, Germany) , which was developed in chloroform : methanol : water (80:20:1, v:v:v) . Digoxin was detected by "charring" after spraying with 15% H₂S0₄ in 80% ethanol. "Activated" digoxin appeared as the major product with Rf slightly lower than the digoxin. The estimated product yield was usually 40 - 60%. If CNBr was excluded from the reaction mixture, TLC analysis revealed no change in digoxin for 60 min, suggesting that the lactone ring was stable under the alkaline conditions.

The reaction mixture was evaporated under reduced pressure, and the dried powder was redissolved in 20 ml mixture of chloroform and 1 M NaCl (1:1, v/v) . After vigorous shaking, the phases were separated and the water phase was extracted with additional 10 ml chloroform. Virtually all the digoxin derivative was found in the combined chloroform phases. The combined chloroform phases were briefly washed with 5 ml water to remove any residual water-soluble material, and dried under reduced pressure. Any remaining unreacted digoxin did not interfere with the reaction with the lectin. If the activated digoxin./unreacted digoxin was stored dry, the ratio of "activated - digoxin" to digoxin remained constant for several weeks at room temperature.

Labelling of Con A with digoxin

Pure Con A (Vector Laboratories, Burlingame, CA) (2 mg) was dissolved in 1 ml 50 mM sodium carbonate buffer, pH 9.5 and dialysed against two 1000ml changes of the same buffer at +4°C. CNBr-activated digoxin (2 μmol) was dissolved in 200 μl methanol and rapidly mixed with the Con A solution. After overnight incubation at room temperature, 2 μmol of glycine was added to inactivate any remaining CNBr- activated digoxin. The reaction mixture was dialysed against three 1000 ml changes of 10 mM sodium phosphate buffer, pH 7.5 at +4°C and freeze- dried.

The procedure for labelling other lectins is very similar to the one described for Con A, but since lectins vary in the availability of free amino groups, solubility and stability, it may be advantageous to optimise the labelling conditions for each lectin.

In addition, high levels of labelling of proteins such as lectins might hinder their reactivity and optimal conditions were found to depend upon particular concentration of DOX and incubation time. These optimal conditions can be easily determined by carrying out routine tests. Depending upon the lectin being labelled, it has been found that activated digoxin is preferably added to the lectin in 5- to 20-fold molar excess and incubated overnight.

It was further observed that the addition of a water-miscible organic solvent such as ethanol, methanol or acetonitrile at a final concentration of 10 - 30% can be advantageous to keep the activated digoxin in solution. The binding of digoxin-labelled lectins could be detected with either anti-digoxin or anti- digoxigenenin antibodies which can be commercially obtained or produced according to routine and well- known techniques.

Probing with digoxin-labelled lectins

Fig. 4 give the results obtained using three different lectins (ConA, WGA and SNA) to probe extracts from a human cell line (HL60) . Cells were collected, washed with PBS and lysed by sonication in 1% Triton X-100, 15 mM NaCl, 5 mM Tris/HCl pH 8.0. Proteins were separated electrophoretically in 12% SDS-polyacrylamide slab gels. After electrophoresis, proteins were transferred onto Immobilon PVDF membrane in a semi-dry apparatus. Blotting membranes were blocked overnight with 3% BSA, incubated for 60 min in 3 ml 1 μg/ml solution of lectin labelled with digoxin or biotin and developed with an anti-digoxigenin antibody (Roche Biochemicals) or streptavidin (Sigma) respectively conjugated with alkaline phosphatase. The binding was visualised with 0.02 mg/ml BCIP (Sigma) and 0.04 mg/ml NBT (Sigma) in 50 mM Tris/HCl, pH 8.5, 100 mM NaCl, 5 mM MgCl₂.

The results show that for ConA, SNA and WGA the patterns obtained for biotin and digoxin-labelled lectins are very similar. 1 Digoxin-labelled lectins can also be used to study 2 carbohydrate structures that are present on tissue 3 sections (histochemistry) and that are expressed on 4 proteins absorbed to base of multiwell assay plates 5 (ELISA) . 6 7 Plants from which suitable lectins may be obtained include the following:

9 10 Table 1 11

Claims

1. A probe comprising a protein labelled with a digoxin moiety.

2. A probe as claimed in claim 1 wherein the digoxin moiety is bound covalently to an amino group of the protein.

3. A probe as claimed in claim 1 or 2 wherein the protein is a lectin.

4. A probe as claimed in claim 3 in which the lectin is selected from the group comprising calnexin, M-type lectins, L-type lectins, P-type lectins, C-type lectins, galectins, l-type lectins and R-type lectins.

5. A probe as claimed in claim 4 wherein the lectin is any one of Sambucus nigra agglutinin, wheat germ agglutinin and concanavalin A.

6. A method of labelling a protein with a digoxin moiety, said method comprising the steps of; - reacting digoxin with CNBr to form activated digoxin; - reacting the protein to be labelled with the activated digoxin under suitable conditions to form a digoxin-labelled protein; and optionally - separating the digoxin-labelled protein from the reaction mixture.

7. The method of claim 6 wherein the protein is a lectin.

8. The method of claim 6 or 7 wherein the activated digoxin is dissolved in a water-miscible organic solvent such as ethanol, methanol or acetonitrile prior to reacting with the protein.

9. The method of any one of claims 6 to 8 in which the activated digoxin is used in 5 to 20 fold molar excess over the protein to be labelled.

10. The method of any one of claims 6 to 9 wherein the activated digoxin and protein are left to react for at least 5 hours.

11. The ^'method of claim 10 wherein digoxin and protein are left to react for at least 16 hours.

12. Use of a digoxin-labelled protein to analyse the properties of a macromolecule.

13. The use of claim 12 wherein the protein is a lectin.

14. The use of claim 12 or 13 wherein the macromolecule to be analysed is a glycoconjugate .

15. The use of any one of claims 12 to 14 wherein the digoxin-labelled protein is used in blotting, histological staining or an ELISA.

16. A method of analysing glycoconjugates in a mixture of biomolecules, said method comprising:

- exposing said mixture to a digoxin-labelled protein under suitable condition to form a digoxin-labelled protein/biomolecule complex. - removing the unbound digoxin-labelled protein; and - determining the presence and/or amount of the retained digoxin-labelled protein/biomolecule complex.

17. The method of Claim 16 wherein the protein is a lectin.

18. The method of claim 16 or 17 wherein the mixture of biomolecules has been previously separated by electrophoresis .

19. The method of any one of claims 16 to 18 wherein the biomolecules are blotted onto a suitable membrane prior to exposure to the digoxin- labelled lectin.

20. The method of claim 19 wherein the membrane is Immobilon PVDF membrane or nitrocellulose.

21. The method of any one of claims 16 to 20 wherein the presence and/or amount of digoxin-labelled lectin is determined by exposing the mixture to a digoxin specific probe containing a chromogenic, chemoluminescent or radioactive marker.

22. The method of claim 21 wherein the digoxin specific probe is an anti-digoxin antibody linked to alkaline phosphatase.

23. A method of analysing glycoconjugates in a mixture of biomolecules, said method comprising:

- adding digoxin-labelled proteins to said mixture of biomolecules under suitable conditions to form digoxin-labelled protein/glycoconjugate complexes; - exposing the complexes thus formed to an immobilised anti-digoxin antibody; and - removing unbound digoxin-labelled proteins.

24. The method of claim 23 wherein the protein is a lectin.