2 Results and Discussion
2.4 Identification of the carbohydrate binding site in galectins
2.4.4 Identification of the carbohydrate binding site in human galectin-4
There is no crystallographic data available for complexes of chicken galectin-3 with lactose or blood groups saccharides, however the mass spectrometric results obtained indicate a binding structure closely matching that of human galectin-3. All chicken galectin sequences binding to lactose or blood group saccharides could be aligned with the corresponding human sequences (Figure 58), suggesting a similar carbohydrate-binding behavior of the two proteins.
hGal-3[130-144] hGal-3[152-162] hGal-3[177-183]
130MLITILGTVKPNANR IALDFQR GNDVAFHFNPR FNENNRRVIVCNTK LDNNWGR E184 cGal-3[120-134] cGal-3[142-152] cGal-3[160-173]
120LLITITGTVNSNPNR FSLDFKR GQDIAFHFNPR FKEDHKR VIVCNSMFQNNWGK E174
A-tri A-tetra
Lac B-tri A-tri A-tetra Lac
B-tri A-tri A-tetra
hGal-3[130-144] hGal-3[152-162] hGal-3[177-183]
130MLITILGTVKPNANR IALDFQR GNDVAFHFNPR FNENNRRVIVCNTK LDNNWGR E184 cGal-3[120-134] cGal-3[142-152] cGal-3[160-173]
120LLITITGTVNSNPNR FSLDFKR GQDIAFHFNPR FKEDHKR VIVCNSMFQNNWGK E174
A-tri A-tetra
Lac B-tri A-tri A-tetra Lac
B-tri A-tri A-tetra
Figure 58. Sequence alignment of the MS-identified carbohydrate-binding peptides from human and chicken galectin-3 and the corresponding ligands to which they showed affinity. Peptides cGal-3[120-134] and hGal-3[130-144] bound only to blood group A oligosaccharides.
2.4.4 Identification of the carbohydrate binding site in human galectin-4
Galectin-4 consists of two CRDs (N-terminal Gal-4N and C-terminal Gal-4C) joined by a linker peptide and is therefore considered a tandem-repeat galectin. Both domains bind lactose with similar affinities, comparable to those of other galectins, but they have different binding specificities for more complex carbohydrates [175-178].
The proteolytic extraction-MS approach was employed to identify the lactose binding site of intact human galectin-4 (hGal-4) and of the two separate CRDs: hGal-4N and hGal-4C. The sequence numbering for hGal-4C was kept identical as in the intact galectin-4. Two different sets of experiments were performed, one using trypsin and the other using clostripain. The reason for using clostripain, a protease preferring arginine residues, was to generate partial peptides (containing uncleaved lysine sites)
and thus obtain more information on the lactose binding site. Peptide hGal-4[46-67]
(peptide 16) was the only lactose-binding peptide identified by mass spectrometry of the elution fraction from the tryptic extraction of hGal-4N on a lactose column (Figure 59a). The mass spectrum of the elution fraction from the lactose column on which the clostripain digest was added showed ions of peptides hGal-4[46-67] and hGal-4[68-89] (peptides 16 and 17). The latter fragment contained two missed cleavage sites at Lys73 and Lys83 (Figure 59b).
a) b)
c)
1200 1600 2000 2400 2800 m/z
2497.90[46-67]
46FFVNFVVGQDPGSDVAFHFNPR67
1 MAYVPAPGYQ PTYNPTLPYY QPIPGGLNVG MSVYIQGVAS EHMKRFFVNF
51 VVGQDPGSDV AFHFNPRFDG WDKVVFNTLQ GGKWGSEERK RSMPFKKGAA
101 FELVFIVLAE HYKVVVNGNP FYEYGHRLPL QMVTHLQVDG DLQLQSINFI
151 GG
1200 1600 2000 2400 2800 m/z
2497.89[46-67]
2556.93[68-89]
46FFVNFVVGQDPGSDVAFHFNPR67
68FDGWDKVVFNTLQGGKWGSEER89
a) b)
c)
1200 1600 2000 2400 2800 m/z
2497.90[46-67]
46FFVNFVVGQDPGSDVAFHFNPR67
1200 1600 2000 2400 2800 m/z
2497.90[46-67]
46FFVNFVVGQDPGSDVAFHFNPR67
1 MAYVPAPGYQ PTYNPTLPYY QPIPGGLNVG MSVYIQGVAS EHMKRFFVNF
51 VVGQDPGSDV AFHFNPRFDG WDKVVFNTLQ GGKWGSEERK RSMPFKKGAA
101 FELVFIVLAE HYKVVVNGNP FYEYGHRLPL QMVTHLQVDG DLQLQSINFI
151 GG
1200 1600 2000 2400 2800 m/z
2497.89[46-67]
2556.93[68-89]
46FFVNFVVGQDPGSDVAFHFNPR67
68FDGWDKVVFNTLQGGKWGSEER89
Figure 59. (a) MALDI-TOF mass spectrum of the elution fraction from the tryptic extraction of hGal-4N on a lactose column, showing the signal of monoprotonated peptide [46-67]; (b) MALDI-TOF mass spectrum of the elution fraction from the clostripain extraction of hGal-4N on a lactose column, containing signals of peptides [46-67] and [68-89]; (c) Sequence of hGal-4N, in which the identified peptides are highlighted in red.
The mass spectrum of the elution fraction from the tryptic extraction of the C-domain on a lactose column (Figure 60a) showed signals of peptides hGal-4[249-261] (peptide 18) and hGal-4[227-240] (peptide 19). The extraction experiment with clostripain led to the recovery of two additional peptides: hGal-4[220-240] (peptide 19a), which contained a missed cleavage site at Lys226, and hGal-4[249-279] (peptide 18a), with two missed cleavage sites at Lys261 and Lys262 (Figure 60b).
227VGSSGDIALHINPR240
249NSLLNGSWGSEEK261
1000 1100 1200 1300 1400 1500 1600 1700 m/z 1436.83
1100 1500 1900 2300 2700 3100 m/z 2244.77
3478.21 [249-279]
[220-240]
220SFAINFKVGSSGDIALHINPR240
151 --- --- ---SLP TMEGPPTFNP PVPYFGRLQG
201 GLTARRTIII KGYVPPTGKS FAINFKVGSS GDIALHINPR MGNGTVVRNS
251 LLNGSWGSEE KKITHNPFGP GQFFDLSIRC GLDRFKVYAN GQHLFDFAHR
301 LSAFQRVDTL EIQGDVTLSY VQI
227VGSSGDIALHINPR240
249NSLLNGSWGSEEK261
1000 1100 1200 1300 1400 1500 1600 1700 m/z 1436.83
1000 1100 1200 1300 1400 1500 1600 1700 m/z 1436.83
1100 1500 1900 2300 2700 3100 m/z 2244.77
1100 1500 1900 2300 2700 3100 m/z 2244.77
3478.21 [249-279]
[220-240]
220SFAINFKVGSSGDIALHINPR240
151 --- --- ---SLP TMEGPPTFNP PVPYFGRLQG
201 GLTARRTIII KGYVPPTGKS FAINFKVGSS GDIALHINPR MGNGTVVRNS
251 LLNGSWGSEE KKITHNPFGP GQFFDLSIRC GLDRFKVYAN GQHLFDFAHR
301 LSAFQRVDTL EIQGDVTLSY VQI
Figure 60. (a) MALDI-TOF mass spectrum of the elution fraction from the tryptic extraction of hGal-4C on a lactose column, showing the signals of peptides [249-261] and [227-240]; (b) MALDI-TOF mass spectrum of the elution fraction from clostripain extraction of hGal-4C on a lactose column, containing signals of the same two peptides [249-261] and [227-240]
and additional ions of fragments [220-240] and [249-279]; (c) Sequence of hGal-4C, in which the identified peptides are highlighted in red.
Tryptic extraction-MS of the intact galectin-4 provided three lactose-binding peptides, consistent with the overall results obtained by the same method on the separate N- and C- terminal domains. Two tryptic peptides, namely hGal-4[249-261]
(peptide 18) and hGal-4[227-240] (peptide 19) originated from the C-domain and one fragment was part of the N-domain, namely hGal-4[46-67] (peptide 16) (Figure 61a).
The extraction with clostripain produced three additional peptides, one from the N-domain and two from the C-N-domain, also in agreement with the previous results on the isolated domains. All additional peptides contained uncleaved lysine residues and produced peaks with lower intensity (Figure 61b). To conclude, the lactose-binding peptides identified for the individual N- and C-domains are, taken together, identical to those determined for the intact galectin-4. The apparent lack of competition between N- and C-domain peptides might be explained by comparable affinities for lactose.
a) b)
c) 1 MAYVPAPGYQ PTYNPTLPYY QPIPGGLNVG MSVYIQGVAS EHMKRFFVNF
51 VVGQDPGSDV AFHFNPRFDG WDKVVFNTLQ GGKWGSEERK RSMPFKKGAA
101 FELVFIVLAE HYKVVVNGNP FYEYGHRLPL QMVTHLQVDG DLQLQSINFI
151 GGQPLRPQGP PMMPPYPGPG HCHQQLNSLP TMEGPPTFNP PVPYFGRLQG
201 GLTARRTIII KGYVPPTGKS FAINFKVGSS GDIALHINPR MGNGTVVRNS
251 LLNGSWGSEE KKITHNPFGP GQFFDLSIRC GLDRFKVYAN GQHLFDFAHR
301 LSAFQRVDTL EIQGDVTLSY VQI
1300 1700 2100 2500 m/z
1436.73
1100 1500 1900 2300 2700 3100 m/z 2556.91
c) 1 MAYVPAPGYQ PTYNPTLPYY QPIPGGLNVG MSVYIQGVAS EHMKRFFVNF
51 VVGQDPGSDV AFHFNPRFDG WDKVVFNTLQ GGKWGSEERK RSMPFKKGAA
101 FELVFIVLAE HYKVVVNGNP FYEYGHRLPL QMVTHLQVDG DLQLQSINFI
151 GGQPLRPQGP PMMPPYPGPG HCHQQLNSLP TMEGPPTFNP PVPYFGRLQG
201 GLTARRTIII KGYVPPTGKS FAINFKVGSS GDIALHINPR MGNGTVVRNS
251 LLNGSWGSEE KKITHNPFGP GQFFDLSIRC GLDRFKVYAN GQHLFDFAHR
301 LSAFQRVDTL EIQGDVTLSY VQI
1300 1700 2100 2500 m/z
1436.73
1300 1700 2100 2500 m/z
1436.73
1100 1500 1900 2300 2700 3100 m/z 2556.91
1100 1500 1900 2300 2700 3100 m/z 2556.91
Figure 61. (a) MALDI-TOF mass spectrum of the elution fraction from the tryptic extraction of intact hGal-4 on a lactose column, showing signals of peptides [46-67], [249-261] and [227-240];
(b) MALDI-TOF mass spectrum of the elution fraction from clostripain extraction of hGal-4 on a lactose column, showing signals of the same three peptides and additional ions of fragments [68-89], [220-240] and [249-279]; (c) Sequence of full-length hGal-4, in which the identified peptides are highlighted in red.
There are no NMR or X-ray structure data currently available for galectin-4 in complex with carbohydrates. The MS results on galectin-4 were compared with the data obtained for galectin-1 and -3 (Figure 62), which were presented in the previous chapters of this thesis. The HxNxR galactose motif common to all studied galectins is represented by a set of peptides containing His63, Asn65, Arg67 from the N-domain (peptide 16) and His236, Asn238, Arg240 in the C-domain (peptides 19 and 19a). A tryptophan residue, essential for the galactose specificity, was found along with other galactose- and glucose-contacting amino acids in peptides 17 (Trp83), 18 and 18a (Trp257). This tryptophan residue stacks against C4-C5-C6 of galactose and is conserved among all studied galectins.
Gal-1[37-48] Gal-1[64-73]
hGal1: 36K DSNNLCLHFNPR---DGGAWGTEQR E74
hGal-3[152-162] hGal-3[177-183]
hGal3: 151R GNDVAFHFNPR---LDNNWGREER Q184
hGal-4[46-67] hGal-4[68-89]
hGal4N: 45R FFVNFVVGQDPGSDVAFHFNPR67 68FDGWDKVVFNTLQGGKWGSEER F90
hGal-4[227-240] hGal-4[249-261]
hGal4C: 226K VGSSGDIALHINPR---NSLLNGSWGSEEK K262 Figure 62. Sequence alignment of lactose-binding peptides from galectin-1, -3 and -4, identified by
mass spectrometry. The canonical carbohydrate-binding motifs are conserved between all peptides.