Tyrosine sulfation is a post-translational modification of proteins and peptides. Many secreted and trans-membrane proteins contain sulfated tyrosine residues, including chemokine receptors. The enzymes tyrosylprotein sulfotransferases or TPSTs (EC 2.8.2.20) catalyze the transfer of a sulfonate group from the donor compound 3’-phosphoadenosine 5’-phosphosulfate or PAPS to the hydroxyl group of a luminally oriented peptidyltyrosine residue to form a tyrosine O4-sulfate ester and 3’,5’-ADP. The reaction is illustrated in figure 1 below.
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Figure 1: The tyrosylprotein sulfotransferase reaction. The enzymes tyrosylprotein sulfotransferases or TPSTs (EC 2.8.2.20) catalyze the transfer of a sulfonate group from the donor compound 3’-phosphoadenosine 5’-phosphosulfate or PAPS to the hydroxyl group of a luminally oriented peptidyltyrosine residue to form a tyrosine O4-sulfate ester and 3’,5’-ADP.
The addition of a sulfate group (SO3) to a tyrosine peptide results in a mass shift of ~80 Da to a higher mass. This mass shift is nearly isobaric with that of a phosphate group due to phosphorylation (monoisotopic masses: SO3 79.9568; HPO3 79.9663). The very small difference of this mass difference results in an analytical challenge for the detection of this group when conjugated to tyrosine residues in proteins or peptides when using most mass spectrometers.
However, the use of alkaline phosphatase allows the removal of phosphorylated peptides, leaving only sulfonated peptides for the analysis. Also, anti-sulfotyrosine antibodies allow for the selective enrichment of sulfotyrosine peptides thereby enabling the identification of these type of peptides.
Synthetic sulfated tyrosine peptides containing sulfotyrosine can be utilized as substrates or model peptides for the study of sulfation kinetics and protein-peptide or protein-protein interactions. Optimized peptide synthesis strategies can be used to generate sulfated peptides. Simpson et al. in 2009 reported a peptide synthesis strategy for the synthesis of sulfotyrosine derivatives. The research group utilized the neopentyl protecting group for sulfate monoesters to achieve a high-yield synthesis of tyrosine-sulfated peptides.
Receptor tyrosine sulfation enhances chemokine affinity in a site-specific manner thereby influencing chemokine interactions. A review article published by Ludeman and Stone in 2013 describes how sulfation of the same receptor peptide at different positions can have a different effect on chemokine binding affinity. Variations in peptide or protein sulfation may alter affinities of chemokines for their receptors resulting in a regulation of cellular responses to chemokines. These findings support the possibility that differential receptor sulfation can modulate chemokine selectivity and chemokine oligomerization.
Table 1 contains a list of substrates and sulfated peptides, and table 2 contains info gained from biophysical studies of chemokine binding by receptor sulfopeptides.
Table 1: Tyrosine substrate peptides or tyrosine sulfated peptides.
(Source: Ludeman and Stone; 2014).
|
Peptide
|
Substrate
|
MW
|
|
|
HPO3
|
79.9663
|
|
|
SO3
|
79.9568
|
|
C4P5y3
|
EDFEDYEFD
|
1,208.18
|
|
C4P5y3
|
EDFEDY(SO3H)EFD
|
1,289.14
|
|
Human complement C4
|
EDYEDYEYD
|
1,223.41
|
|
Human complement C4
|
EDYEDY(SO3H)EYD
|
1,304.38
|
|
Selectin P ligand, CRA b
|
YEYLDYDF
|
1,127.18
|
|
|
YEY(SO3H)LDYDF
|
1,208.14
|
|
|
LDYDF
|
671.71
|
|
|
LDY(SO3H)DF
|
752.66
|
|
|
LDY(PO3)DF
|
752.68
|
|
Selectin P ligand, isoform CRA b
|
QATEXEXLDXDFLPETEPP
|
|
|
Psgl-1 peptide
|
QATEXEXLDXDFLPETEPPRPMMDDDDK
|
|
|
|
QATEYEYLDYDFLPETEPP
|
2,320.53
|
|
|
QATEY(SO3H)EYLDYDFLPETEPP
|
2,401.5
|
|
|
QATEYEY(SO3H)LDYDFLPETEPP
|
2,401.5
|
|
|
QATEYEYLDY(SO3H)DFLPETEPP
|
2,401.5
|
|
|
QATEY(SO3H)EY(SO3H)LDYDFLPETEPP
|
2,382.46
|
|
|
QATEY(SO3H)EY(SO3H)LDY(SO3H)DFLPETEPP
|
2,482.44
|
|
|
QATEYEYLDYDFLPETEPPRPMMDDDDK
|
3,424.78
|
|
|
QATEY(SO3H)EYLDYDFLPETEPPRPMMDDDDK
|
3,505.74
|
|
|
QATEYEY(SO3H)LDYDFLPETEPPRPMMDDDDK
|
3,505.74
|
|
|
QATEYEYLDY(SO3H)DFLPETEPPRPMMDDDDK
|
3,505.74
|
|
|
QATEY(SO3H)EY(SO3H)LDYDFLPETEPPRPMMDDDDK
|
3,586.69
|
|
|
QATEY(SO3H)EY(SO3H)LDY(SO3H)DFLPETEPPRPMMDDDDK
|
3,667.65
|
|
|
|
3,586.69
|
|
Fibrinogen beta peptide, mouse
|
ENENVINEYSSILEDQR
|
2,052.23
|
|
|
ENENVINEY(SO3H)SSILEDQR
|
2,133.19
|
|
|
|
|
|
Cholecytokinin (26-33)
|
DYMGWMDF-NH2
|
1,063.25
|
|
Cholecytokinin (26-33)
|
DY(SO3H)MGWMDF-NH2
|
1,141.3
|
|
Leu-Enkephalin
|
YGGFL
|
554.68
|
|
Leu-Enkephalin
|
Y(SO3H)GGFL
|
634.2
|
|
Hirudin
|
DFEEIPEEYLQ
|
1,409.7
|
|
Hirudin
|
DFEEIPEEY(SO3H)LQ
|
1,489.6
|
|
|
|
|
|
Model peptide
|
KESDYLKNT
|
1,096.25
|
|
Model peptide
|
KESDY(SO3H)LKNT
|
1,177.2
|
Table 2: Chemokine receptors known to be sulfated and their cognate chemokines.
(Source: Ludeman and Stone; 2014).
|
Receptor
|
Chemokine ligands
|
Receptor N-terminal amino acid sequence
|
Key findings
|
|
CCR2
|
CCL2/MCP-1
CCL7/MCP-3
CCL8/MCP-2
CCL11/eotaxin-1
CCL13/MCP-4
CCL16/HCC-4/LEC
|
1MLSTSRSRFIRNTNESGEEVTTFFDYDYGAPC32
|
Y26 is sulfated
Y26A mutant has reduced receptor binding/activation.
Mutation of D25 reduces sulfation.
(Preobrazhensky et al.,
2000; Tan et al., 2013)
|
|
CCR5
|
CCL3/MIP-1α
CCL4/MIP-1β CCL5/RANTES
CCL8/MCP-2
CCL11/eotaxin-1
CCL14/HCC-1
CCL16/HCC-4/LEC
|
1MDYQVSSPIYDINYYTSEPC20
|
CCR5 is Tyr-sulfated.
Sulfated Tyr residues contribute to binding
of CCL3, CCL4 and HIV-1 surface
proteins.
(Farzan et al., 1999)
|
|
CCR8
|
CCL1/I-309
CCL4/MIP-1β
CCL16/ HCC-4/LEC
CCL17/TARC
|
1MDYTLDLSVTTVTDYYYPDIFSSPC25
|
N-terminal Tyr residues are sulfated.
Sulfated Tyr residues contribute to binding
of I-309.
(Gutierrez et al., 2004)
|
|
CXCR3
|
CXCL9/Mig
CXCL10/IP-10
CXCL11/I-TAC
|
1MVLEVSDHQVLNDAEVAALLENFSSSYDYGENESDSC37
|
Y27 and Y29 or CXCR3 are sulfated.
Mutation of Y27 or Y29 reduces binding and activation by CXCL9-11.
(Colvin et al., 2006; Gao
et al., 2009)
|
|
CXCR4
|
CXCL12/SDF-1
|
1MEGISIYTSDNYTEEMGSGDYDSMKEPC28
|
N-terminal Tyr residues are sulfated.
Mutation of N-terminal Tyr residues reduces CXCL12 binding.
(Farzan et al., 2002a)
|
|
CX3CR1
|
CX3CL1/fractalkine
|
1MDQFPESVTENFEYDDLAEACYIGDIV27
|
Mutation of N-terminal Tyr residues or sulfatase treatment reduces fractalkine
binding affinity.
(Fong et al., 2002)
|
|
DARC
|
Many CC and CXC chemokines
|
1MGNCLHRAELSPSTENSSQLDFEDVWNSSYGVNDSFP
DGDYDANLEAAAPCHSCNLLDDS60
|
Y30 and Y41 are sulfated.
Mutation of Y30 and Y41 reduces binding
to different chemokines.
Mutation of Y41 reduces binding to
Plasmodium vivax Duffy binding protein.
(Choe et al., 2005)
|
Receptor Chemokine ligands1 Receptor N-terminal amino acid sequence2 Key findings References
1Chemokine ligands are those listed in Szpakowska et al. (2012). 2Potentially sulfated Tyr residues are shown in bold; acidic residues are underlined.
Table 2: Biophysical studies of chemokine binding by receptor sulfopeptides.
(Ludeman and Stone; 2014).
|
Receptor
|
Chemokine(s) studied
|
Sulfopeptide
|
Key findings
|
|
CCR2
|
CCL2/MCP-1
(wild type; obligate
monomer P8A; obligate
dimer T10C)
|
18EEVTTFFDYDYGAP31
(all four sulfation states)
|
All three forms of CCL2 bind CCR2
sulfopeptides.
Sulfation of single Tyr residues enhances affinity by 4- to 30-fold.
Sulfation of both Tyr residues enhances affinity additively for monomer and cooperatively for dimer.
Sulfopeptides destabilize dimeric CCL2 in favour of active monomer.
Sulfopeptides bind to N-loop/β3 site.
(Tan et al., 2013)
|
|
CCR2
|
CCL7/MCP-3
|
21TTFFDYDYGA30
(non-sulfated, monosulfated and disulfated)
|
Single sulfation enhances CCL7 affinity ∼4-fold.
Double sulfation enhances CCL7 affinity ∼36-fold.
Binding affinities can be measured by
electrospray mass spectrometry.
(Jen and Leary, 2010)
|
|
CCR3
|
CCL11/eotaxin-1
CCL24/eotaxin-2
CCL26/eotaxin-3
|
8VETFGTTSYYDDVGLL23
(all four sulfation states)
|
Single sulfation enhances binding to
CCL11, 24 and 26 ∼3- to ∼30-fold.
Sulfation of Y16 and Y17 gives different
affinity enhancements.
Double sulfation enhances affinity
additively for CCL11 and CCL24,
cooperatively for CCL26.
Sulfopeptides bind to N-loop/β3 site.
(Simpson et al., 2009;
Zhu et al., 2011)
|
|
CCR5
|
CCL5/RANTES
|
1NleDYQVSSPIYDINYYTSEPSQKINV25
Nle = norleucine
(non-sulfated; Y10/Y14-disulfated)
|
Sulfation of Y10 and Y14 enhances affinity for CCL5 by >100-fold.
Sulfopeptide binds to N-loop/β3 site.
(Duma et al., 2007)
|
|
CXCR4
|
CXCL12/SDF-1
|
GS1MEGISIYTSDNYTEEMGSGDYDSMKEPAFREENANFNK38
(non-sulfated; Y21-sulfated; Y7/Y12/Y21-trisulfated)
|
Single and triple sulfation enhances affinity
∼3- and ∼30-fold respectively.
Sulfopeptides stabilize dimeric CXCL12.
NMR structures of complexes determined
and sTyr binding sites identified.
(Veldkamp et al., 2006;
2008; Seibert et al.,
2008)
|
1Potentially sulfated Tyr residues are shown in bold; acidic residues are underlined.
Reference
http://www.ncbi.nlm.nih.gov/books/NBK56012/ Neuroproteomics
Justin P Ludeman and Martin J Stone;The structural role of receptor tyrosine sulfation in chemokine recognition. British Journal of Pharmacology (2014) 171 1167–1179.
Levi S. Simpson, John Z. Zhu, Theodore S. Widlanski, and Martin J. Stone; Regulation of Chemokine Recognition by Site-Specific Tyrosine Sulfation of Receptor Peptides. Chem Biol. 2009 February 27; 16(2): 153–161. doi:10.1016/j.chembiol.2008.12.007.