2 Results and Discussion
2.4 Identification of the carbohydrate binding site in galectins
2.4.6 Identification of the carbohydrate binding site in human galectin-8
Complexes of rGal-5 with carbohydrates have not yet been characterized by NMR or X-ray crystallography. Based on sequence similarities with other galectins it can be noticed that the two peptides identified by mass spectrometry are within the canonical carbohydrate-recognition domain of galectins and contain the key amino acid residues for ligand recognition. Peptide rGal-5[49-60] contains the HxNxR galactose-binding motif also identified for the other galectins investigated in this thesis. Peptide rGal-5[69-81] contains the key tryptophan residue found in all galectins, which is known to recognize and bind galactose through CH-π interactions.
This peptide also contains two amino acid residues which interact with lactose through hydrogen bonds, namely Glu79 and Arg81.
2.4.6 Identification of the carbohydrate binding site in human galectin-8
The lactose binding site was identified for the intact human galectin-8 (hGal-8S) and for the two individual CRDs, designated hGal-8N and hGal-8C. The sequence numbering for hGal-8C was kept identical as in the intact galectin-8. Both proteolytic excision and extraction approaches were employed for all three proteins. For hGal-8N, extraction using trypsin resulted in the identification of a single lactose-binding peptide, hGal-8[60-69] (peptide 12) (Figure 65a). The excision approach using a 12 hours trypsin digestion provided the same peptide (Figure 65b). The incubation time with trypsin was reduced in a subsequent experiment to 3 hours, in order to achieve an incomplete digestion. This led to the identification of an additional peptide, hGal-8[73-88] (peptide 13), containing a missed trypsin cleavage site at Lys85 (Figure 65c). The proteolytic extraction was repeated using clostripain (Arg-C), which resulted in the identification of both peptides (12 and 13) in the elution fraction (Figure 65d).
a) b)
c) d)
e)
1000 1200 1400 1600 1800 m/z
1174.18
60ADVAFHFNPR69 [60-69]
73AGCIVCNTLINEKWGR88
900 1100 1300 1500 1700 1900 m/z
60ADVAFHFNPR69
1778.32 1174.03[60-69]
[73-88]
60ADVAFHFNPR69
800 1000 1400 1800 m/z
1174.10[60-69]
73AGCIVCNTLINEKWGR88
60ADVAFHFNPR69
[73-88]
[60-69]
1000 1200 1400 1600 1800 m/z
1174.12
1778.01
1 MMLSLNNLQN IIYNPVIPFV GTIPDQLDPG TLIVIRGHVP SDADRFQVDL
51 QNGSSMKPRA DVAFHFNPRF KRAGCIVCNT LINEKWGREE ITYDTPFKRE
101 KSFEIVIMVL KDKFQVAVNG KHTLLYGHRI GPEKIDTLGI YGKVNIHSIG
151 FSF
a) b)
c) d)
e)
1000 1200 1400 1600 1800 m/z
1174.18
60ADVAFHFNPR69 [60-69]
1000 1200 1400 1600 1800 m/z
1000 1200 1400 1600 1800 m/z
1174.18
60ADVAFHFNPR69 [60-69]
73AGCIVCNTLINEKWGR88
900 1100 1300 1500 1700 1900 m/z
60ADVAFHFNPR69
1778.32 1174.03[60-69]
[73-88]
73AGCIVCNTLINEKWGR88
900 1100 1300 1500 1700 1900 m/z
900 1100 1300 1500 1700 1900 m/z
60ADVAFHFNPR69
800 1000 1400 1800 m/z
1174.10[60-69]
60ADVAFHFNPR69
800 1000 1400 1800 m/z
800 1000 1400 1800 m/z
1174.10[60-69]
73AGCIVCNTLINEKWGR88
60ADVAFHFNPR69
[73-88]
[60-69]
1000 1200 1400 1600 1800 m/z
1174.12
1000 1200 1400 1600 1800 m/z
1174.12
1778.01
1 MMLSLNNLQN IIYNPVIPFV GTIPDQLDPG TLIVIRGHVP SDADRFQVDL
51 QNGSSMKPRA DVAFHFNPRF KRAGCIVCNT LINEKWGREE ITYDTPFKRE
101 KSFEIVIMVL KDKFQVAVNG KHTLLYGHRI GPEKIDTLGI YGKVNIHSIG
151 FSF
Figure 65. (a) MALDI-TOF mass spectrum of the elution fraction from the tryptic extraction of hGal-8N on a lactose column, showing the signal of monoprotonated peptide [60-69]; (b) MALDI-TOF mass spectrum of the elution fraction from the 12 hours tryptic excision of hGal-8N on a lactose column, showing the signal of monoprotonated peptide [60-69]; (c) MALDI-TOF mass spectrum of the elution fraction from the 3 hours tryptic excision of hGal-8N on a lactose column, containing the ion signals of peptides [60-69] and [73-88];
(d) MALDI-TOF mass spectrum of the elution fraction from the clostripain extraction of hGal-8N on a lactose column, containing the ion signals of peptides [60-69] and [73-88]; (e) Sequence of hGal-8N, in which the identified peptides are highlighted in red.
For hGal-8C, both proteolytic excision and extraction using trypsin provided two lactose-binding peptides in the elution fractions, hGal-8[225-233] (peptide 14) and hGal-8[242-254] (peptide 15) (Figure 66a, b). An extraction experiment using clostripain led to the identification of two lactose-binding peptides, hGal-8[223-233]
(peptide 14a) and hGal-8[242-254] (peptide 15) (Figure 66c).
a) b)
c) d)
1049.86
1611.60
600 800 1000 1200 1400 1600 1800 m/z [242-254]
600 800 1000 1200 1400 1600 1800 m/z 1611.96
1049.01
[242-254]
[225-233]
700 900 1100 1300 1500 1700 m/z
1264.41
600 800 1000 1200 1400 1600 1800 m/z [242-254]
600 800 1000 1200 1400 1600 1800 m/z 600 800 1000 1200 1400 1600 1800 m/z
[242-254]
600 800 1000 1200 1400 1600 1800 m/z 1611.96
600 800 1000 1200 1400 1600 1800 m/z 600 800 1000 1200 1400 1600 1800 m/z
1611.96
1049.01
[242-254]
[225-233]
700 900 1100 1300 1500 1700 m/z
1264.41
700 900 1100 1300 1500 1700 m/z
1264.41
Figure 66. (a) MALDI-TOF mass spectrum of the elution fraction from the 12 hours tryptic excision of hGal-8C on a lactose column, showing the signals of monoprotonated peptides [225-233]
and [242-254]; (b) MALDI-TOF mass spectrum of the elution fraction from the trypsin extraction of hGal-8C on a lactose column, showing the signals of the same peptides [225-233] and [242-254]; (c) MALDI-TOF mass spectrum of the elution fraction from the clostripain extraction of hGal-8C on a lactose column, containing the signals of peptides [223-233] and [242-254]; (d) Sequence of hGal-8C, in which the identified peptides are highlighted in red.
For hGal-8S the tryptic excision-MS identified in the elution fraction the lactose-binding peptides hGal-8[60-69], hGal-8[225-233] and hGal-8[242-254]
(peptides 12, 14 and 15). A proteolytic excision experiment employing a reduced digestion time, aiming to obtain a missed cleavage at Lys85 and consequently hGal-8[73-88] (peptide 13) in the elution fraction, as previously for hGal-8N, did not succeed in generating the hGal-8[73-88] fragment. However, proteolytic extraction with clostripain (Figure 67) enabled the identification of hGal-8[73-88], as well as of peptides hGal-8[60-69], hGal-8[60-72], hGal-8[223-233] and hGal-8[242-254]
(peptides 12, 12a, 14a and 15).
a) b)
c) 1 MMLSLNNLQN IIYNPVIPFV GTIPDQLDPG TLIVIRGHVP SDADRFQVDL
51 QNGSSMKPRA DVAFHFNPRF KRAGCIVCNT LINEKWGREE ITYDTPFKRE
101 KSFEIVIMVL KDKFQVAVNG KHTLLYGHRI GPEKIDTLGI YGKVNIHSIG
151 FSFSSDLQST QASSLELTEI SRENVPKSGT PQLRLPFAAR LNTPMGPGRT
201 VVVKGEVNAN AKSFNVDLLA GKSKDIALHL NPRLNIKAFV RNSFLQESWG
251 EEERNITSFP FSPGMYFEMI IYCDVREFKV AVNGVHSLEY KHRFKELSSI
301 DTLEINGDIH LLEVRSW
1100 1300 1500 1700 1900 m/z
1174.32
60ADVAFHFNPR69 242NSFLQESWGEEER254
225DIALHLNPR233
600 800 1000 1200 1400 1600 1800 m/z [60-69]
c) 1 MMLSLNNLQN IIYNPVIPFV GTIPDQLDPG TLIVIRGHVP SDADRFQVDL
51 QNGSSMKPRA DVAFHFNPRF KRAGCIVCNT LINEKWGREE ITYDTPFKRE
101 KSFEIVIMVL KDKFQVAVNG KHTLLYGHRI GPEKIDTLGI YGKVNIHSIG
151 FSFSSDLQST QASSLELTEI SRENVPKSGT PQLRLPFAAR LNTPMGPGRT
201 VVVKGEVNAN AKSFNVDLLA GKSKDIALHL NPRLNIKAFV RNSFLQESWG
251 EEERNITSFP FSPGMYFEMI IYCDVREFKV AVNGVHSLEY KHRFKELSSI
301 DTLEINGDIH LLEVRSW
1100 1300 1500 1700 1900 m/z
1174.32
1100 1300 1500 1700 1900 m/z
1100 1300 1500 1700 1900 m/z
1174.32
60ADVAFHFNPR69 242NSFLQESWGEEER254
225DIALHLNPR233
600 800 1000 1200 1400 1600 1800 m/z [60-69]
[242-254]
[225-233]1174.35
1611.73
1049.37
60ADVAFHFNPR69 242NSFLQESWGEEER254
225DIALHLNPR233
600 800 1000 1200 1400 1600 1800 m/z [60-69]
[242-254]
[225-233]1174.35
1611.73
1049.37
Figure 67. (a) MALDI-TOF mass spectrum of the elution fraction from the 3 hours tryptic excision of hGal-8S on a lactose column, showing the signals of monoprotonated peptides [225-233], [60-69], and [242-254]; (b) MALDI-TOF mass spectrum of the elution fraction from the clostripain extraction of hGal-8S on a lactose column, containing the signals of peptides [60-69], [223-233], [242-254] and [73-88]; (c) Sequence of hGal-8S, in which the identified peptides are highlighted in red.
Peptide hGal-8[60-69] from the N-terminal CRD contains the same galactose-binding motif as hGal-8[225-233] from the C-terminal CRD, therefore they might be expected to compete for the interaction with lactose. However, both peptides were recovered in the elution fractions for hGal-8S (which contains both N- and C- domains). This indicated comparable affinities for the two peptides, close enough so that one peptide cannot completely displace the other. The same observation was made for peptides hGal-8[223-233] and hGal-8[242-254].
A comparison of the mass spectrometric results with available X-ray data for the N- terminal CRD of human galectin-8 is shown in Figure 68. Mass spectrometry identified lactose binding peptides hGal-8[60-69] and hGal-8[73-88] (peptides 12 and 13). The X-ray crystal structure [183, 184] showed that His65, Asn67 and Arg69 contained in peptide 12 form hydrogen bonds with the 4-OH group of β-galactose.
Arg69 also interacts with the galactose O-5. Peptide 13 contains Trp86, which ensures β-galactose specificity through stacking interactions with the galactose ring. Trp86 is one of only three amino acids (Trp-Gly-Arg) that were included in peptide 13 due to
the missed cleavage at Lys85. This elongation was sufficient to confer affinity to peptide 13, thus highlighting the importance of Trp86 in carbohydrate binding.
According to the X-ray data, Asn79 from peptide 13 interacts with the 6-OH group of galactose. The X-ray structure also shows the 6-OH group of galactose forming a direct hydrogen bond with Glu89 and participating in water-mediated hydrogen bonds with Gln47 and Arg59. However, the mass spectrometric data did not identify any peptides containing these amino acids.
Figure 68. X-ray crystal structure of the complex between the N-domain of human galectin-8 and lactose (from PDB entry 3VKL). The MS-identified lactose-binding peptides [60-69] and [73-88] are highlighted in red (a), while the lactose binding site identified by X-ray crystallography is shown in green (b). Amino acids in direct contact with the carbohydrate are labeled in bold. The N- and C-terminal ends of the protein chain are marked with "N"
and "C". Lactose is represented as stick model.
Both excision and extraction methods identified two lactose-binding peptides from the C-domain of galectin-8, namely hGal-8[225-233] and hGal-8[242-254]
(peptides 14 and 15). Similarly to the peptides identified for the N-domain, the C-domain peptides 14 and 15 harbor key ligand-contacting amino acids, in agreement with the X-ray crystal data (Figure 69). X-Ray crystallography of the complex between galectin-8 and lactose [183] showed that His229, Asn231, Arg233 from peptide 14 form hydrogen bonds with the galactose subunit. Peptide 15 forms hydrogen bonds with galactose through Asn242 and Glu252. Trp249 from peptide 15 is involved in stacking interactions with the galactose C4-C5-C6. The glucose moiety
forms hydrogen bonds with peptides 14 and 15 through Arg233 and Glu252, respectively.
Figure 69. X-ray crystal structure of the C-domain of human galectin-8 and lactose complex (from PDB entry 3VKL). The MS-identified lactose-binding peptides [225-233] and [242-254] are highlighted in red (a), while the lactose binding site identified by X-ray crystallography is shown in green (b). Amino acids which are in direct contact with the carbohydrate are labeled in bold. The N- and C-terminal ends are marked with "N" and "C". Lactose is represented as stick model.
2.4.7 Comparison of X-ray crystallography and mass spectrometry data