2 Results and Discussion
2.5 Interaction studies of synthetic CRD peptides with carbohydrates
2.5.1 Characterization of interactions of synthetic galectin peptides with
2.5 Interaction studies of synthetic CRD peptides with carbohydrates
2.5.1 Characterization of interactions of synthetic galectin peptides with carbohydrates by affinity-mass spectrometry
To ascertain the specificity of ligand-contacting peptides identified by mass spectrometry, affinity experiments were carried out with a number of corresponding synthetic peptides: hGal-1[64-73], DGGAWGTEQR (peptide 1), identified in the elution fraction for galectin-1 in both excision and extraction approaches; hGal-1[64-73]m, GGQRETGWDA (peptide 1a), a mutated version of peptide 1; hGal-1[58-74], IVCNSKDGGAWGTEQRE (peptide 1b), a prolonged version of peptide 1 that includes the amino acids Val59 and Asn61; hGal-1[37-48], DSNNLCLHFNPR (peptide 2), identified in the elution fraction for galectin-1 in both proteolytic excision and extraction experiments; hGal-1[37-48]m, FDNSPRLCNNHL (peptide 2a), a mutated version of peptide 2; hGal-3[177-183], LDNNWGR (peptide 4), identified in the elution fraction for galectin-3 in both proteolytic excision and extraction;
hGal-3[177-183]m1, LDNNFGR (peptide 4a), a W/F mutated version of peptide 4;
hGal-3[177- 183]m2, LDNNLGR (peptide 4b), a W/L mutated version of peptide 4;
hGal-3[177-183]m3, AAAAWAA (peptide 4c), a mutated version of peptide 4;
hGal-3[178-182], DNNWG (peptide 4d), as a truncated version of peptide 4;
hGal-3[152-162], GNDVAFHFNPR (peptide 5), identified in the elution fraction for galectin-3; hGal-3[157-163], FHFNPRF (peptide 6), a shorter version of peptide 5, used to pinpoint the minimal recognition structure; hGal-3[157-163]m, FPFNHFR (peptide 6a), a mutated version of peptide 6; hGal-3[157-175], FHFNPRFNENNRRVIVCNT (peptide 7), a longer version of peptide 6, used to assess the importance of Val171 and Asn174; hGal-3[157-175]m, RTHVNFNRFINNPNRCEFV (peptide 7a), a mutated version of peptide 7;
cGal-3[142-152], GQDIAFHFNPR (peptide 9); cGal-3[160-173], VIVCNSMFQNNWGK (peptide 10). All peptides were synthesized in carboxamidated form. The sequences are summarized in Table 3. The mutated sequences 1a, 2a, 6a and 7a served as controls and were obtained by random scrambling of peptide sequences 1, 2, 6 and 7.
Table 3. Synthetic hGal-1, hGal-3 and cGal-3 peptides selected for affinity studies.
No. Peptide Sequence [M+H+]+calc.
1 hGal-1[64-73] DGGAWGTEQR 1077.09
1a hGal-1[64-73]m GGQRETGWDA 1077.09
1b hGal-1[58-74] IVCNSKDGGAWGTEQRE 1850.99
2 hGal-1[37-48] DSNNLCLHFNPR 1430.57
2a hGal-1[37-48]m FDNSPRLCNNHL 1430.57
4 hGal-3[177-183] LDNNWGR 873.94
4a hGal-3[177-183]m1 LDNNFGR 834.91
4b hGal-3[177- 183]m2 LDNNLGR 800.89
4c hGal-3[177-183]m3 AAAAWAA 630.72
4d hGal-3[178-182] DNNWG 604.60
5 hGal-3[152-162] GNDVAFHFNPR 1273.39
6 hGal-3[157-163] FHFNPRF 964.11
6a hGal-3[157-163]m FPFNHFR 964.11
7 hGal-3[157-175] FHFNPRFNENNRRVIVCNT 2377.68 7a hGal-3[157-175]m RTHVNFNRFINNPNRCEFV 2377.68
9 cGal-3[142-152] GQDIAFHFNPR 1302.42
10 cGal-3[160-173] VIVCNSMFQNNWGK 1640.91
All peptides were analyzed by affinity-mass spectrometry on micro-columns with Sepharose-immobilized (i) lactose, (ii) sucrose and (iii) maltose, as well as with unmodified Sepharose. The columns with unmodified Sepharose, immobilized sucrose and maltose were used as three different sets of controls. The columns with unmodified Sepharose were employed to assess the unspecific binding of the peptides to the affinity matrix. Sucrose and maltose (Figure 71) do not contain β-galactose, which is essential for binding to galectins. Therefore, these columns were used to investigate the specificity of the identified peptides for galactose.
contains β-galactose, which is essential for recognition by galectins.
The peptides were dissolved in binding buffer and added over the columns.
After incubation and washing, the affinity-bound peptides were eluted and all recovered fractions analyzed by MALDI-MS. Galectin-1 peptides 1, 1b, 2 (Figure 72), human galectin-3 peptides 4, 5, 6, 7 (Figure 73) and chicken galectin-3 peptides 9, 10 (Figure 74) were found in the elution fractions from the lactose columns, but not from the control columns, which demonstrated their specificity for lactose. Notably, peptide 6, although very short, still bound to lactose, which underscores the importance of the HxNxR motif for galactose binding. The scrambled peptides 1a, 2a, 6a and 7a, although having the same composition as the test peptides, did not bind to any of the columns, this result showing that the structure of the peptides is also critically important for their affinity. Trp181 is known to be important for galactose binding in human galectin-3. Therefore, mutants of peptide 4 were designed to evaluate the importance of Trp181 in peptides derived from galectin-3. The W/F and W/L mutants of hGal-3[177-183] (peptides 4a and 4b) did not bind to lactose, showing that tryptophan is an essential amino acid residue for affinity. Peptide 4c, in which Trp181 was conserved, while all other amino acids were replaced with alanine, did not bind to lactose, indicating that the other amino acids are also important for affinity. Peptide 4d, obtained by reducing the size of peptide 4 to pinpoint the minimal recognition structure, showed no binding to lactose. None of the test or control peptides bound to
maltose, sucrose or to the unmodified Sepharose. The results of the affinity studies are summarized in Table 4 and the complete mass spectrometric data (supernatant, washing and elution spectra) are shown in the Appendix (Figures A 8-16).
a) b)
c)
hGal-1[58-74]
58IVCNSKDGGAWGTEQRE74 hGal-1[64-73]
64DGGAWGTEQR73 1076.2
1000 1400 1800 2200 m/z
hGal-1[37-48]
37DSNNLCLHFNPR48
900 1300 1700 2100 m/z
1429.3
a) b)
c)
hGal-1[58-74]
58IVCNSKDGGAWGTEQRE74 hGal-1[64-73]
64DGGAWGTEQR73 1076.2
1000 1400 1800 2200 m/z
1076.2
1000 1400 1800 2200 m/z
hGal-1[37-48]
37DSNNLCLHFNPR48
900 1300 1700 2100 m/z
1429.3
900 1300 1700 2100 m/z
1429.3
Figure 72. MALDI-TOF mass spectra of the elution fractions from the interaction of synthetic hGal-1 peptides with lactose. (a) Elution fraction of peptide 1, hGal-1[64-73]; (b) Elution fraction of peptide 1b, hGal-1[58-74]; (c) Elution fraction of peptide 2, hGal-1[37-48]; All three peptides exhibited affinity for lactose, thus confirming the results of proteolytic excision and extraction.
hGal-3[177-183]
177LDNNWGR183
hGal-3[152-162]
152GNDVAFHFNPR162
hGal-3[157-163]
157FHFNPRF163
hGal-3[157-175]
157FHFNPRFNENN RRVIVCNT175
a) b)
c) d)
hGal-3[177-183]
177LDNNWGR183
hGal-3[152-162]
152GNDVAFHFNPR162
hGal-3[157-163]
157FHFNPRF163
hGal-3[157-175]
157FHFNPRFNENN RRVIVCNT175
a) b)
c) d)
Figure 73. MALDI-TOF mass spectra of the elution fractions from the interaction of synthetic hGal-3 peptides with lactose. (a) Elution fraction of peptide 4, hGal-3[177-183]; (b) Elution fraction of peptide 5, hGal-3[152-162]; (c) Elution fraction of peptide 6, hGal-3[157-163]; (d) Elution fraction of peptide 7, hGal-3[157-175]. All four peptides showed affinity for lactose.
cGal3-[160-173]
160VIVCNSMFQNNWGK173
a) b) 1639.20
900 1100 1300 1500 1700 1900 m/z 1301.38
1000 1200 1400 1600 1800 2000 m/z cGal-3[142-152]
142GQDIAFHFNPR152 cGal3-[160-173]
160VIVCNSMFQNNWGK173
a) b) 1639.20
900 1100 1300 1500 1700 1900 m/z 1301.38
1000 1200 1400 1600 1800 2000 m/z 1301.38
1000 1200 1400 1600 1800 2000 m/z cGal-3[142-152]
142GQDIAFHFNPR152
Figure 74. MALDI-TOF mass spectra of the elution fractions from the interaction of synthetic cGal-3 peptides with lactose. (a) Elution fraction of peptide 9, cGal-3[142-152]; (b) Elution fraction of peptide 10, cGal-3[160-173]. Both peptides showed affinity for lactose.
Table 4. Summary of the results from affinity-mass spectrometry of synthetic galectin peptides.*
No. Peptide Sequence Affinity
for lactose
Affinity for sucrose
Affinity for maltose
Affinity for Sepharose
1 hGal-1[64-73] DGGAWGTEQR + − − −
1a hGal-1[64-73]m GGQRETGWDA − − − −
1b hGal-1[58-74] IVCNSKDGGAWGTE
QRE + − − −
2 hGal-1[37-48] DSNNLCLHFNPR + − − −
2a hGal-1[37-48]m FDNSPRLCNNHL − − − −
4 hGal-3[177-183] LDNNWGR + − − −
4a hGal-3[177-183]m1 LDNNFGR − − − −
4b hGal-3[177-183]m2 LDNNLGR − − − −
4c hGal-3[177-183]m3 AAAAWAA − − − −
4d hGal-3[178-182] DNNWG − − − −
5 hGal-3[152-162] GNDVAFHFNPR + − − −
6 hGal-3[157-163] FHFNPRF + − − −
6a hGal-3[157-163]m FPFNHFR − − − −
7 hGal-3[157-175] FHFNPRFNENNRRVI
VCNT + − − −
7a hGal-3[157-175]m RTHVNFNRFINNPNR
CEFV − − − −
9 cGal-3[142-152] GQDIAFHFNPR + − − −
10 cGal-3[160-173] VIVCNSMFQNNWGK + − − −
* Affinity is indicated by "+" and the lack of affinity by "−".
Despite their small size, the lactose-binding peptides (1, 1b, 2, 4, 5, 6, 7, 9 and 10) displayed high specificity for lactose. This was well demonstrated by their lack of affinity to maltose, which does not contain β-galactose. The place of the β-galactose subunit of lactose is taken in maltose by α-glucose. Glucose and galactose are C4 epimers and an important role in the galactose/glucose differentiation by galectins is played by the orientation of the 4-OH group. While in galactose the 4-OH group is axial, in glucose it occupies an equatorial position. The axial orientation enables C–
H/π interactions between galactose and a tryptophan residue (Trp68 in galectin-1, Trp181 in human galectin-3, etc.). This interaction is not possible with glucose, due to the equatorial positioning of the 4-OH group. Plant lectins also differentiate between the axial and equatorial orientations of the 4-OH group and thus are able to distinguish galactose from glucose and mannose [189]. While binding to galectins the 4-OH galactose group also forms hydrogen bonds with the HxNxR motif (His158, Asn160 and Arg162 in galectin-3). Furthermore, the different type of linkage, β1-4 in lactose vs. α1-4 in maltose, might also have a contribution to lactose/maltose differentiation.
Two additional galectin-1 peptides were synthesized to study the effect of alkylation on lactose recognition. The identification of peptide hGal-1[1-18] from alkylated hGal-1, but not from the native protein, in the elution fractions from the proteolytic excision-MS experiments suggested a correlation between alkylation and affinity to lactose. The native hGal-1[1-18] peptide (peptide 3) was prepared by solid phase peptide synthesis and half of the synthesized peptide was treated with DTT and iodoacetamide to generate the alkylated form, hGal-1[1-18]a (peptide 3a). As a result, both cysteine residues Cys2 and Cys16 were found alkylated by mass spectrometry.
Two parallel experiments were then performed, employing affinity columns with immobilized lactose. After separately incubating peptides 3 and 3a with the affinity matrix, the columns were drained and washed. After performing the elution, the recovered elution fractions were analyzed by MALDI-MS. Only the alkylated peptide was identified in the elution fraction, while the native peptide did not bind (Figure 75).
This demonstrated that the affinity of the alkylated peptide [1-18] was indeed caused by carbamidomethylation of the Cys2 and Cys16 residues.
a) b)
c) d)
e) f)
hGal-1[1-18]
1ACGLVASNLNLKPGECLR18
Supernatant
Wash
Elution
Supernatant
Wash
Elution CH2CONH2
hGal-1[1-18]a
1ACGLVASNLNLKPGECLR18
CH2CONH2
a) b)
c) d)
e) f)
hGal-1[1-18]
1ACGLVASNLNLKPGECLR18
Supernatant
Wash
Elution
Supernatant
Wash
Elution CH2CONH2
hGal-1[1-18]a
1ACGLVASNLNLKPGECLR18
CH2CONH2
CH2CONH2
hGal-1[1-18]a
1ACGLVASNLNLKPGECLR18
CH2CONH2
Figure 75. Affinity-MS of hGal-1[1-18] synthetic peptides in native and alkylated form. (a) MALDI-TOF mass spectrum of the supernatant fraction showing the ion signal of the native peptide at m/z 1858.1; (b) MALDI-TOF mass spectrum of the supernatant fraction showing the ion signal of the alkylated peptide at m/z 1972.7; (c, d) The washing fractions from both columns were clean; (e) MALDI-TOF mass spectrum of the native peptide elution fraction did not contain any peptide signals, indicating lack of affinity. (f) MALDI-TOF mass spectrum of the alkylated peptide elution fraction shows the peptide signal, indicating affinity.
An additional affinity-MS experiment was performed with an equimolar mixture of peptides 3 and 3a by adding the mixture on a lactose column and proceeding in the same manner as for the previous samples. While the mass spectrum of the supernatant contained the signals of both peptides (Figure 76a), only the alkylated peptide was observed in the elution fraction (Figure 76b), thus supporting the previous results on the alkylation-induced affinity of peptide [1-18].
Elution
a)
b)
CH2CONH2
hGa-l1[1-18]a
1ACGLVASNLNLKPGECLR18
CH2CONH2
Supernatant
hGa-l1[1-18]
1ACGLVASNLNLKPGECLR18
CH2CONH2
hGal-1[1-18]a
1ACGLVASNLNLKPGECLR18
CH2CONH2
Elution
a)
b)
CH2CONH2
hGa-l1[1-18]a
1ACGLVASNLNLKPGECLR18
CH2CONH2
CH2CONH2
hGa-l1[1-18]a
1ACGLVASNLNLKPGECLR18
CH2CONH2
Supernatant
hGa-l1[1-18]
1ACGLVASNLNLKPGECLR18
CH2CONH2
hGal-1[1-18]a
1ACGLVASNLNLKPGECLR18
CH2CONH2
CH2CONH2
hGal-1[1-18]a
1ACGLVASNLNLKPGECLR18
CH2CONH2
Figure 76. Affinity-MS of an equimolar mixture of native and alkylated galectin-1 [1-18] synthetic peptides. (a) MALDI-TOF mass spectrum of the supernatant fraction, showing both peptides. (b) MALDI-TOF mass spectrum of the elution fraction showing only the alkylated peptide.
Further affinity-MS experiments were performed to evaluate the differences in binding to lactose/B-tri vs. A-tri/A-tetra observed for human and chicken galectin-3 (see Chapters 2.4.2.1 and 2.4.3.1). A first set of experiments was carried out with synthetic peptide 8 (hGal-3[130-144]), which was identified by proteolytic excision-MS of hGal-3 on A-tri and A-tetra columns, but not on lactose or B-tri columns (see Chapter 2.4.2.1). The peptide was dissolved in binding buffer and added on separate columns containing either immobilized lactose or A-tri. In agreement with the results of the proteolytic excision, peptide 8 was identified only in the elution fraction from the A-tri column, but not in that from the lactose column (Figure 77).
500 1000 1500 2000 2500 3000 m/z 1640.19
Supernatant hGal-3[130-144]
130MLITILGTVKPNANR144
500 1000 1500 2000 2500 3000 3500 m/z
Elution
500 1000 1500 2000 2500 3000 m/z 1640.54
500 1000 1500 2000 2500 3000 m/z 1641.11
Lac column A-tri column
No affinity Affinity
a) b)
c) d)
500 1000 1500 2000 2500 3000 m/z 1640.19
Supernatant hGal-3[130-144]
130MLITILGTVKPNANR144
500 1000 1500 2000 2500 3000 3500 m/z
Elution
500 1000 1500 2000 2500 3000 m/z 1640.54
500 1000 1500 2000 2500 3000 m/z 1641.11
500 1000 1500 2000 2500 3000 m/z 1640.19
500 1000 1500 2000 2500 3000 m/z 1640.19
Supernatant hGal-3[130-144]
130MLITILGTVKPNANR144
500 1000 1500 2000 2500 3000 3500 m/z 500 1000 1500 2000 2500 3000 3500 m/z
Elution
500 1000 1500 2000 2500 3000 m/z 1640.54
500 1000 1500 2000 2500 3000 m/z 1640.54
500 1000 1500 2000 2500 3000 m/z 1641.11
500 1000 1500 2000 2500 3000 m/z 1641.11
Lac column A-tri column
No affinity Affinity
a) b)
c) d)
Figure 77. Affinity-MS of synthetic peptide hGal-3[130-144] on lactose and A-tri columns. (a, b) MALDI-TOF mass spectra of the supernatant fractions, showing the signal of hGal-3[130-144]; (c) The elution fraction from the lactose column did not produce any peptide signals, indicating the lack of affinity of hGal-3[130-144] for lactose; (d) The elution fraction from the A-tri column showed the monoprotonated ion of hGal-3[130-144], indicating its affinity for A-tri.
A second affinity test employed all three synthetic peptides: hGal-3[177-183], hGal-3[152-162] and hGal-3[130-144]. An equimolar mixture of the peptides was added on two columns, one with immobilized A-tri and the other with B-tri. MALDI-MS of the elution fractions (Figure 78) showed that only hGal-3[177-183] and hGal-3[152-162] bound to B-tri. In addition to these two peptides hGal-3[130-144]
was identified from the A-tri column. Both Affinity-MS experiments confirmed that peptide hGal-3[130-144] did not bind to B-tri or lactose, but is specific only for A-tri and A-tetra.
a) b)
c) d)
hGal3[177-183]
hGal3[152-162]
hGal-3[130-144]
850 1050 1250 1450 1650 1850m/z
1273.21
1639.90
873.75
850 1050 1250 1450 1650 1850m/z
1273.44
1640.12
874.05
850 1050 1250 1450 1650 1850 m/z
873.96
1273.36
850 1050 1250 1450 1650 1850 m/z
874.18
850 1050 1250 1450 1650 1850m/z
1273.21
1639.90
873.75
850 1050 1250 1450 1650 1850m/z
1273.44
1640.12
874.05
850 1050 1250 1450 1650 1850 m/z
873.96
1273.36
850 1050 1250 1450 1650 1850 m/z
874.18
850 1050 1250 1450 1650 1850m/z
1273.21
1639.90
873.75
850 1050 1250 1450 1650 1850m/z
1273.21
1639.90
873.75
850 1050 1250 1450 1650 1850m/z
1273.44
1640.12
874.05
850 1050 1250 1450 1650 1850m/z
1273.44
1640.12
874.05
850 1050 1250 1450 1650 1850 m/z
873.96
1273.36
850 1050 1250 1450 1650 1850 m/z
850 1050 1250 1450 1650 1850 m/z
873.96
1273.36
850 1050 1250 1450 1650 1850 m/z
874.18
1640.04
1273.40
850 1050 1250 1450 1650 1850 m/z
850 1050 1250 1450 1650 1850 m/z
874.18 columns. (a, b) MALDI-TOF mass spectra of the supernatant fractions, both showing ion signals of peptides hGal-3[177-183], hGal-3[152-162] and hGal-3[130-144]; (c) The elution fraction from the A-tri column contained all three peptides; (d) Only peptides Gal-3[177-183] and hGal-3[152-162] were recovered from the B-tri column, indicating that hGal-3[130-144] is specific for A-tri and not for B-tri.
Further affinity-MS experiments were performed with synthetic peptide cGal-3[120-134] (peptide 11) on separate columns with immobilized lactose and blood group oligosaccharides. cGal-3[120-134] was previously identified by proteolytic excision-MS of cGal-3 on A-tri and A-tetra columns, but not on lactose or B-tri columns (see Chapter 2.4.3.1). The peptide was dissolved in binding buffer and added on separate columns containing either immobilized lactose, A-tri, B-tri or A-tetra. In agreement with the results of the proteolytic excision, peptide 11 was identified exclusively in the elution fraction from the A-tri and A-tetra columns, but not in that from the lactose or B-tri columns (Figure 79).
Lac A-tri
B-tri A-tetra
900 1300 1700 2100 m/z
900 1300 1700 2100 m/z
1612.65
900 1300 1700 2100 m/z
1612.80
900 1300 1700 2100 m/z
cGal-3[120-134]
120LLITITGTVNSNPNR134
a) b)
c) d)
Lac A-tri
B-tri A-tetra
900 1300 1700 2100 m/z
900 1300 1700 2100 m/z
900 1300 1700 2100 m/z
900 1300 1700 2100 m/z
1612.65
900 1300 1700 2100 m/z
1612.65
900 1300 1700 2100 m/z
1612.80
900 1300 1700 2100 m/z
1612.80
900 1300 1700 2100 m/z
cGal-3[120-134]
120LLITITGTVNSNPNR134
a) b)
c) d)
Figure 79. Affinity MS of synthetic peptide cGal-3[120-134] on lactose and blood group oligosaccharides columns. MALDI-TOF mass spectra of the elution fractions from affinity columns with immobilized (a) lactose, (b) A-tri, (c) B-tri and (d) A-tetra. Peptide cGal-3[120-134] was identified only in the elution fractions from the A-tri and A-tetra columns.
Additional Affinity-MS experiments were performed by incubating equimolar mixtures of the three synthetic cGal-3 peptides [120-134], [142-152] and [160-173]
with immobilized A-tri and B-tri. After elution with a aqueous ACN solution, MALDI-MS of the recovered fractions (Figure 80) indicated that cGal-3[120-134]
bound to A-tri, but not to B-tri.
1200 1400 1600 1800 m/z 1301.22
1612.10 1639.16
Supernatant
1200 1400 1600 1800 m/z
1301.41 1612.84 1639.30
Elution
1200 1400 1600 1800 m/z
1301.71
1614.09 1640.02
1200 1400 1600 1800 m/z
1301.99
1200 1400 1600 1800 m/z
1301.22
1612.10 1639.16
1200 1400 1600 1800 m/z
1200 1400 1600 1800 m/z
1301.22
1200 1400 1600 1800 m/z
1301.41 1612.84 1639.30
1200 1400 1600 1800 m/z
1200 1400 1600 1800 m/z
1301.41 1612.84 1639.30
1301.41 1612.84 1639.30
Elution
1200 1400 1600 1800 m/z
1301.71
1614.09 1640.02
1200 1400 1600 1800 m/z
1200 1400 1600 1800 m/z
1301.71
1200 1400 1600 1800 m/z
1301.99
1639.56
1200 1400 1600 1800 m/z
1200 1400 1600 1800 m/z
1301.99 columns. (a, b) MALDI-TOF mass spectra of the supernatant fractions, both showing ion signals of peptides cGal-3[142-152], cGal-3[160-173] and cGal-3[120-134]; (c) The elution fraction from the A-tri column contained all three peptides; (d) Only peptides cGal-3[142-152] and cGal-3[160-173] were recovered from the B-tri column, indicating that cGal-3[120-134] is specific for A-tri and not for B-tri.
2.5.2 Determination of dissociation constants for synthetic galectin