Abstract
The present invention relates to mutants of S100A12 having at least one mutation in the high-affinity calcium binding hand or the low-affinity calcium binding hand or the zinc binding region. The present invention also relates to methods of detecting S100A12 dimers in a sample as well as methods of diagnosis using the S100A12 mutant of the invention, as well as to diagnostic compositions and kits comprising such an S100A12 mutant. The present invention further relates to a method of generating an antibody that specifically binds to an S100A12 dimer using the S100A12 mutant of the invention, as well as to an antibody that specifically binds to an S100A12 dimer.
Claims
1. A S100Al2 mutant, wherein the mutant comprises at least one mutation at an amino acid corresponding to amino acid His16, Ser19, Lys22, His24, Asp26, Thr27, Glu32, Asp62, Asn64, Asp66, Glu73, His86, or His90 of human S100Al2 as set forth in SEQ ID NO: 01.
2. The mutant of claim 1, wherein the mutant is capable of forming S100Al2 dimers.
3. The mutant of claim 1, wherein the mutant does not significantly form S100Al2 tetramers or S100Al2 hexamers.
4. The mutant of claim 1, wherein the mutant has a sequence identity of at least about 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% as compared to any one of the sequences of SEQ ID NO: 05, SEQ ID NO: 06, or SEQ ID NO: 07 wherein the mutant is capable of forming S100Al2 dimers and does not significantly form S100Al2 tetramers or S100Al2 hexamers.
5. A kit comprising a S100Al2 mutant of claim 1.
6. A method of generating an antibody against the S100Al2 mutant of claim 1, comprising administering to a mammal an antibody-generating peptide from human S100Al2 as set forth in SEQ ID NO: 01, the peptide comprising one or more mutation(s) at an amino acid corresponding to amino acid His16, Ser19, Lys22, His24, Asp26, Thr27, Glu32, Asp62, Asn64, Asp66, Glu73, His86, or His90, and purifying the antibody.
7. The method of claim 6 wherein the one or more mutations is a glycine or alanine at said amino acid(s).
8. The method of claim 6 wherein the peptide comprises one, two, three, four, or five mutation(s) at an amino acid corresponding to amino acid Glu32, Asp62, Asn64, Asp66, or Glu73.
9. The mutant of claim 3, wherein the mutant has no more than 1% (w/w) impurity of the S100Al2 tetramers or S100Al2 hexamers.
10. The mutant of claim 4, wherein the mutant has no more than 1% (w/w) impurity of the S100Al2 tetramers or S100Al2 hexamers.
11. The mutant of claim 4, wherein the mutant has sequence identity of at least 95% as compared to any one of the sequences of SEQ ID NO: 05, SEQ ID NO: 06, or SEQ ID NO: 07.
12. The mutant of claim 11, wherein the mutant has sequence identity of at least 99% as compared to any one of the sequences of SEQ ID NO: 05, SEQ ID NO: 06, or SEQ ID NO: 07.
13. The mutant of claim 12, wherein the mutant has the following sequence: SEQ ID NO: 05, SEQ ID NO: 06, or SEQ ID NO: 07.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1) FIG. 1: Sequence alignment between human S100A12 (SEQ ID NO: 01) and human S100A9 (SEQ ID NO: 08). Positions involved in the complexation of Ca.sup.2+ are highlighted.
(2) FIG. 2: Multiple sequence alignment between human S100A12 (SEQ ID NO: 01), canine S100A12 (SEQ ID NO: 02), equine S100A12 (SEQ ID NO: 03), and bovine S100A12 (SEQ ID NO: 04). Positions involved in the complexation of Ca.sup.2+ are highlighted in grey, positions involved in the complexation of Zn.sup.2+ are depicted in bold italic letters.
(3) FIG. 3: Mutants of human S100A12. FIG. 3A shows the amino acid sequence of a Glu73.fwdarw.Ala mutant of human S100A12 (SEQ ID NO: 05). FIG. 3B shows the amino acid sequence of an Asn64.fwdarw.Ala mutant of human S100A12 (SEQ ID NO: 06). FIG. 3C shows the amino acid sequence of an Asn64.fwdarw.Ala, Glu73.fwdarw.Ala mutant of human S100A12 (SEQ ID NO: 07).
(4) FIG. 4: Molecular masses determined by ESI-MS. The molecular masses of the recombinant S100A8 and S100A9 proteins are determined by ESI-MS under denaturing conditions and compared with their theoretical calculated masses. S100A8 refers to human S100A8 protein of Uniprot accession no. P05109 (version 1 as of 1 Jan. 1988, SEQ ID NO: 09). S100A9 refers to human S100A9 protein of Uniprot accession no. P06702 (version 1 as of 1 Jan. 1988, SEQ ID NO: 08). S100A9 (N69A) refers to a mutant of human S100A9 having a single Asn69.fwdarw.Ala substitution. S100A9 (E78A) refers to a mutant of human S100A9 having a single Glu78.fwdarw.Ala substitution. S100A9 (N69A+E78A) refers to a mutant of human S100A9 having a double Asn69.fwdarw.Ala and Glu78.fwdarw.Ala substitution.
(5) FIG. 5: MALDI mass spectra in the absence and presence of calcium under native solvent conditions. (a) MALDI mass spectra of recS100A8/S100A9 wt complexes in the absence (left) and presence (right) of calcium using 2,6-dihydroxy-acetophenone as matrix. The mass spectra between m/z 20,000 and 100,000 together with an inset between m/z 46,000 and 50,000 are shown. T.sup.+, singly charged; T.sup.2+, doubly charged; T.sup.3+, triply charged tetramer. (b) MALDI mass spectra of recS100A8/S100A9(N69A) mutant complexes in the absence (left) and presence (right) of calcium. RecS100A8/S100A9(E78A) and recS100A8/S100A9(N69A+E78A) showed almost identical results (data not shown).
(6) FIG. 6: ESI mass spectra in the absence and presence of calcium under native solvent conditions. (a) ESI mass spectra of recS100A8/S100A9 wild-type complexes. The mass spectra between m/z 1000 and 4000 together with an inset between m/z 2800 and 4000 are shown. In the absence of calcium (left) the main signals observed correspond to heterodimers with charge states 10+ and 9+. In the presence of calcium (right) signals corresponding to tetramers occurred at charge states 16+, 15+, 14+ and 13+ in the range between m/z 3000-3800. (b) ESI mass spectra of recS100A8/S100A9(N69A) mutant complexes. In the absence and presence of calcium the main signals correspond to heterodimers with charge states 10+ and 9+, no tetramers were found. recS100A8/S100A9(E78A) and recS100A8/S100A9(N69A+E78A) showed almost identical results (data not shown).
(7) FIG. 7: Density gradient centrifugation of S100A8/S100A9 proteins. wt and mutant recS100A8/S100A9 complexes were loaded on a glycerol gradient in the presence of either 1 mM EGTA or 100 μM Ca2+. After centrifugation, successive fractions of the gradients were analyzed by SDS-PAGE. In the presence of EGTA wt and mutant complexes showed an almost identical distribution centered in the low density fractions of the gradient. Addition of calcium induced a marked shift for recS100A8/S100A9 wt to higher glycerol densities, whereas for the mutant complexes recS100A8/S100A9(N69A) and recS100A8/S100A9(E78A) no shift was observed.
(8) FIG. 8: Glycerol centrifugation of S100A12 proteins. Wild type (wt) and mutant recS100A12 complexes were loaded on a 15% glycerol solution in the presence of either EGTA or Ca.sup.2+ or Zn.sup.2+ or Ca.sup.2+/Zn.sup.2+. Buffer conditions were 20 mM HEPES, 140 mM NaCl, pH 7.4. After centrifugation, successive fractions of the gradients were analyzed by the colorimetric assay Bradford. In the presence of EGTA wt and mutant complexes showed an almost identical distribution centered in the low density fractions of the gradient. Addition of calcium or zinc alone induced no shift for wtS100A12 to higher glycerol fractions. Addition of calcium plus zinc induced a marked shift for wtS100A12 to higher glycerol fractions, whereas for the mutant complex recS100A12(E73A) no shift was observed. FIG. 8A: Protein concentrations of collected fractions determined by Bradford assay. Sample A: S100A12 wild type+2 mM EGTA, sample B S100A12 wild type+5 mM CaCl.sub.2, sample C: S100A12 wild type+1 mM Zn, sample D S100A12 wild type+200 μM Zn, sample E: S100A12 wild type+5 mM CaCl.sub.2+200 μM Zn, sample F: S100A12 wild type+2.5 mM CaCl.sub.2+100 μM Zn FIG. 8B: Summary of collected and analysed fractions of wtS100A12 (see FIG. 8A) in the presence or absence of bivalent cations as indicated in the figure. FIG. 8C Summary of collected and analysed fractions of wtS100A12 and recS100A12E73A mutant in the presence (5 mM CaCl.sub.2+200 μM Zn, 2.5 mM CaCl.sub.2+100 μM Zn) or absence (2 mM EGTA) of bivalent cations as indicated in the figure.
EXAMPLES
(9) The following examples illustrate the invention. These examples should not be construed as to limit the scope of this invention. The examples are included for purposes of illustration and the present invention is limited only by the claims.
Example 1: Differentiation Between S100A8/S100A9 Heterodimers and Teteramers Using MALDI-MS and ESI-MS
(10) Electrospray ionization mass spectrometry (ESI-MS) confirms the theoretically calculated masses without the N-terminal methionine (SwissProt) for mutated and non-mutated recombinant S100A8/S1009 proteins (FIG. 4).
(11) S100A8 and S100A9 exist as heterodimers in the absence of calcium, and these heterodimeric complexes associate to (S100A8/S100A9).sub.2 tetramers upon calcium-binding. FIG. 5(a) shows the matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) spectra of the recS100A8/S100A9 wt proteins. In the absence of calcium, samples show intense signals of singly charged heterodimers. In contrast, in the presence of Ca2+ wt S100A8/S100A9 show a base peak in first shot spectra that corresponds to a singly charged heterotetramer (T+: 48 kDa) composed of two molecules recS100A8 and two molecules of recS100A9, respectively. Other prominent signals are detected at molecular masses of around 24 kDa and 16 kDa, representing doubly charged (T2+) or triply charged (T3+) tetramers in accordance with results reported earlier for the native proteins purified from human granulocytes. The number of Ca2+ bound to the tetramers was calculated from the difference between the observed masses of the tetramers and the sum of the calculated theoretical molecular masses of the monomeric components.
(12) The oligomerization properties of the recS100A8/S100A9 mutant complexes can be determined by MALDI-MS. As shown exemplarily for the N69A mutant in FIG. 5(b), all mutant S100A9 proteins display signals for singly charged heterodimers in the absence of calcium. In contrast to the results obtained with the recS100A8/S100A9 wt proteins no heterotetramers can be observed for the mutant complexes in the presence of calcium (FIG. 5). The base peaks under these conditions exclusively represent S100A8/S100A9(N69A), S100A8/S100A9(E78A) and S100A8/S100A9(N69A+E78A) heterodimers. All MALDI-MS experiments presented here were also confirmed by ESI-MS measurements (see FIG. 6).
(13) Density gradient centrifugation can be employed in order to confirm the different complex formation patterns obtained in the mass spectrometric studies (FIG. 7). In EGTA-containing samples the recS100A8/S100A9 wt and mutant complexes are found in the same range of fractions of the glycerol gradient (19(±2)%), indicating that in the absence of calcium the formation of heterodimers is preferred in wt proteins and all S100A9 mutants. In the presence of calcium, wt complexes shifted to fractions of significantly higher glycerol concentrations (23(±2)%), as observed earlier for S100A8/S100A9 purified from granulocytes. This shift reflects the calcium-induced formation of high-molecular (S100A8/S100A9).sub.2 tetramers. In contrast, after addition of calcium, the mutant complexes recS100A8/S100A9(N69A) and recS100A8/S100A9(E78A) show no shift to higher glycerol concentrations, confirming that heterotetramer formation is disturbed.
Example 2: Differences in Oligomerization States of S100A12 Wild Type and S100A12 Mutants
(14) Glycerol centrifugation can be employed in order to confirm the different complex formation patterns as previously observed for S100A8/S100A9 complexes. In EGTA-containing samples the recS100A12 wt and mutant rec S100A12E73A complexes are found in the same range of glycerol fractions 4-10 indicating that in the absence of calcium/zinc the formation of homodimers is preferred in wt proteins and the tested S100A12 mutant. In the presence of calcium or zinc alone wtS100A12 was found in the same fractions as observed before under EGTA-conditions, indicating that for S100A12 in contrast to S100A8/S100A9 calcium alone is not sufficient to induce oligomerization. However, in the presence of calcium and zinc, wild type S100A12 complexes shifted to fractions of significantly higher numbers (9-15). This shift reflects the calcium/zinc-induced formation of high-molecular S100A12 tetramers and hexamers. In contrast, after addition of calcium and zinc, the mutant complex recS100A12(E73A) shows no shift to higher glycerol fractions, confirming that tetramer/hexamer formation is disturbed (FIG. 8).
