1. VBMS 7180 Receptorology
Presented by: Chris Ramhold Ph.D. Student
16-March-15
TYROSINE SULFATION OF THE AMINO TERMINUS
OF CCR5 FACILITATES HIV-1 ENTRY

2. • Setting the stage
• Post-translational modification
– Tyrosine Sulfation
• Chemokine receptors
– What are chemokine receptors?
• Characteristics and families
– How do chemokine receptors work?
– CCR5
• HIV
– Classification
• Primary research article
OVERVIEW

3. • Previously studied N-
terminus region of
CCR5
• Site directed
mutagenesis
PRIOR RESEARCH
Farzan, Michael, et al. "A tyrosine-rich region in the N terminus of CCR5 is important for human immunodeficiency virus type 1 entry and mediates an association between gp120 and CCR5." Journal of virology 72.2
(1998): 1160-1164.

4. • N-terminus important
for internalization
• Found something
interesting
• Half of the mutants
were originally
tyrosine
PRIOR RESEARCH
Farzan, Michael, et al. "A tyrosine-rich region in the N terminus of CCR5 is important for human immunodeficiency virus type 1 entry and mediates an association between gp120 and CCR5." Journal of virology 72.2
(1998): 1160-1164.

5. • Aromatic- Benzene
• Reactive hydroxyl group
• Can be phosphorylated or
sulfated
QUICK REVIEW- TYROSINE

6. • Modifications
occurring after
translation
• Responsible for
proteome diversity
• Most often includes
covalent
attachment of
functional groups
POSTTRANSLATIONAL MODIFICATION
http://asfaculty.syr.edu/pages/chem/_images/hougland_fig1.jpg

7. • The transfer of a sulfate to
the hydroxyl group of the
tyrosine residue
• Enzymatic assistance by
tyrosylprotein
sulfotransferase (TPST)
• Occurs in the golgi
• Donor molecule 3’-
phosphoadenosine-
5’phosphosulfate (PAPS)
TYROSINE SULFATION
Kanan, Yogita, and Muayyad R. Al-Ubaidi. "Tyrosine OSulfation: An Overview." JSM 1.1 (2013): 1003.

8. • Receptors for cytokines
that induce chemotaxis
• 7TM structure
– Member of GPCR
• Four classes
– C, CC, CXC, and CX3C
CHEMOKINE RECEPTORS
http://www.biolegend.com/media_assets/chemokine_receptors/CK_structure.jpg

9. • Chemokine binds N-terminus of receptor
– Binding enhanced by tyrosine sulfation
• Complex interacts with cytoplasmic face of receptor to
activate signaling
CHEMOKINE RECEPTOR BINDING MODEL
Ludeman, Justin P., and Martin J. Stone. "The structural role of receptor tyrosine sulfation in chemokine recognition." British journal of pharmacology 171.5 (2014): 1167-1179.

10. • Expressed on T-cells,
macrophages, dendritic
cells, eosinophils and
microglia
• Most common receptor for
HIV-1 internalization
• Most common target of HIV
drugs
– CCR5 receptor antagonists
– Maraviroc (Pfizer)
– Block CCR5 binding site
CHEMOKINE RECEPTOR TYPE 5
http://idshowcase.lshtm.ac.uk/id501/ID501/S1S4/ID501_S1S4_030_010.html

11. • Human immunodeficiency virus
(HIV)
– Lentivirus, group of retroviruses
• Cause of AIDS (acquired
immunodeficiency syndrome)
• Viral RNA is reverse transcribed
and integrated into host DNA
• Infects immune cells
– CD4+ T cells
– Macrophages
– Dendritic cells
HIV
http://s.hswstatic.com/gif/aids-hiv-anatomy.gif

12. • HIV-1
– High virulence
– High infectivity
– Global
• HIV-2
– Moderate virulence
– Low infectivity
– Localized to West
Africa
HIV CLASSIFICATIONS
http://treasuresoftheinternet.org/health/aids/us_census/aids_maps/Map3c.gif

13. • Research group
published paper
regarding N-terminus
of CCR5 receptor
• Gradual increase in
focus
• Need to identify
targets for HIV therapy
THE PAPER

14. • N-terminal CCR5 residues
important
• Tyrosines specifically
• Sulfate plays several roles in
HIV-1 internalization
– Virus-cell interactions
– Inhibit internalization by
binding gp120*
– Inhibit viral replication
TYROSINE SULFATION OF THE AMINO TERMINUS OF
CCR5 FACILITATES HIV-1 ENTRY
Baleux, Françoise, et al. "A synthetic CD4–heparan sulfate glycoconjugate inhibits CCR5 and CXCR4 HIV-1 attachment and entry." Nature chemical biology 5.10 (2009): 743-748.

15. • Sulfation common in
chemokines and HIV-1 env
glycoproteins
• Is CCR5 sulfated?
• Red- Negative controls
• Blue- Stably transfected
CD4/CCR5
• Sulfate is specifically
incorporated in CCR5
POSTTRANSLATIONAL MODIFICATION BY SULFATE
[35S] C [35S] M [35S] Sulfate
CCR5 CCR5CD4 CD4
Label
IP

16. • Sulfation occurs at either O-, N-linked glycosylation sites,
or on tyrosines
• Endoglycosidase F (endo F) & Tunicamycin treatment
ineffective against CCR5
– Cleavage and blockage of N-linked glycosylation
– Not N-linked glycosylation
CCR5 SITE OF SULFATION (N-LINKED GLYCOSYLATION)

17. • Red- Negative controls
– No expression of CD4 or CCR5
• IP with CCR5 antibody
• 1, 4, 5, & 8- Enzyme buffer
• 2 & 6 - Neuraminidase (green)
• 3 & 7 – Four O-glycosidases
(yellow)
• [35S] Sulfate still present after
treatments
– Sulfation must occur on tyrosines
CCR5 SITE OF SULFATION (O-LINKED GLYCOSYLATION)
[35S] Sulfate [35S] C & M

18. • Thin layer chromatography
• Labeled CCR5 from previous
experiment removed from gel
• Digested, pH neutralized, and
run next to unlabeled tyrosine
and serine sulfates (1 & 3)
• 2 & 4 show presence of
labeled Tyrosine sulfate
• Presence of 35S due to Tyrosine
sulfation
CCR5 MODIFICATIONS (TYROSINE SULFATION)
Ninhydrin X-ray film

19. • Negative control cells treated with
sulfate (white)
• Treatment with chlorate- inhibit
sulfation (gray)
• Treatment with sulfate- allow
sulfation (thick line)
• 2D7- Antibody specific to 2nd
extracellular loop
• No difference between treatments
• Sulfation does not interfere with
binding- no alteration in 2nd
extracellular loop
ROLE OF TYROSINE SULFATION
FACS- Fluorescence Activated
Cell Sorting

20. • Negative control cells treated with
sulfate (white)
• Treatment with chlorate- inhibit
sulfation (gray)
• Treatment with sulfate- allow sulfation
(thick line)
• 5C7- Antibody specific to N terminus
• Decreased binding of 5C7 when
sulfation is inhibited
• N-terminus recognition altered
• No effect on CCR5 expression levels
(AUC)
ROLE OF TYROSINE SULFATION
FACS- Fluorescence Activated
Cell Sorting

21. • Confirmation of results
– Mutated tyrosine to phenylalanine
– Similar results (data not shown)
– Decreased recognition specific to loss of sulfate
ROLE OF TYROSINE SULFATION

22. • Chlorate treatment (circles/red)
• Sulfate treatment (squares/blue)
• 125I-labeled MIP-1a incubated
with CCR5 transfected cells
• Increasing amounts of MIP-1a
competitor added
• Loss of sulfate leads to
decreased binding of native
ligand MIP-1a
• Sulfate-treated cells with 2D7
antibody (diamond)
• Negative control cells (triangle)
ROLE OF TYROSINE SULFATION (CCR5 LIGANDS)
MIP= Macrophage inflammatory
protein-1a a.k.a. CCL3

23. • Chlorate treatment (circles/red)
• Sulfate treatment (squares/blue)
• 125I-labeled MIP-1β incubated
with CCR5 transfected cells
• Increasing amounts of MIP-1β
competitor added
• Loss of sulfate leads to
decreased binding of native
ligand MIP-1β
• Sulfate-treated cells with 2D7
antibody (diamond)
• Negative control cells (triangle)
ROLE OF TYROSINE SULFATION (CCR5 LIGANDS)
MIP= Macrophage inflammatory
protein-1β a.k.a. CCL4

24. • Chlorate treatment (circles/red)
• Sulfate treatment (squares/blue)
• 125I-labeled gp120 complexed
with soluble CD4 incubated with
CCR5 transfected cells
• Typically binds with high affinity
• Loss of sulfate leads to decreased
binding efficiency
• Sulfate-treated cells with 2D7
antibody (diamond)
• Negative control cells (triangle)
ROLE OF TYROSINE SULFATION (CCR5 LIGANDS)
sYU2- HIV-1 env gp120, strain YU2

25. • Which of the four tyrosines are sulfated?
• CCR5 construct used
• Nine amino acid tag
– Motif from rhodopsin
– Identified with anti-rhodopsin antibody 1D4
• New cell line used
– HeLa cells
– Six transfection lines created
• Mock transfection- Empty vector
• All four tyrosines replaced with phenylalaline
• Four constructs where three of four tyrosines are replaced with
phenylalaline
• Assay for sulfation
IDENTIFICATION OF SULFATED TYROSINES

26. • HeLa transfected cell lysates
• IP with 1D4 antibody
• WT does incorporate sulfate
• FFFF construct fails to
incorporate sulfate
• Only YFFF could incorporate
sulfate
– Faint bands on other 3 constructs
after extended exposure (not
shown)
– FYYY (next 2 slides) showed sulfation
greater than 3 constructs previously
mentioned
IDENTIFICATION OF SULFATED TYROSINES

27. • HeLa transfected cell lysates
• IP with 1D4 antibody
• WT does incorporate sulfate
• Sulfation greater in three adjacent
tyrosine construct
• Due to the absence of phenylalanine?
• Due to the presence of neighboring
tyrosines?
• Replace phenylalaline with AA that
mimics a sulfated tyrosine
SULFATION DEPENDENT ON NEIGHBORING RESIDUES

28. • HeLa transfected cell lysates
• IP with 1D4 antibody
• WT does incorporate sulfate
• FFFF construct fails to
incorporate sulfate
• New panel of mutants using
aspartic acid vs phenylalanine
• Sulfation of single tyrosines
• Negative charge may assist in
sulfation of neighboring tyrosines
SULFATION OF CCR5 TYROSINES DEPENDENT ON N-
TERMINAL RESIDUES

29. • Do sulfotyrosines in CCR5 play
a role in HIV-1 internalization?
• Generate pseudotype viruses
– Virus containing CAT reporter
gene
– Lacking env gene
• Host cell expresses env gene
(pseudotyped gene)
• Generate PV, capable of only
one round of infection
• Use envelope glycoproteins
ADA, YU2, and 89.6 ( from
strains that use CCR5 for entry)
SULFATION OF CCR5 TYROSINES DEPENDENT ON N-
TERMINAL RESIDUES
Tani, Hideki, Shigeru Morikawa, and Yoshiharu Matsuura. "Development and applications of VSV vectors based on cell tropism." Frontiers in microbiology 2 (2011).

30. • YU2 pseudotyped HIV-1
• WT CCR5 (black squares/
blue)
• FFFF CCR5 construct
(circles/ red)
• Negative control (triangle)
• Lack of tyrosine residues
resulted in decrease of
viral entry
HIV-1 ENTRY EFFICIENCY MODULATED BY SULFATED
TYROSINES
• % entry relative to wild type CCR5

31. • ADA pseudotyped HIV-1
• WT CCR5 (black squares/
blue)
• FFFF CCR5 construct
(circles/ red)
• Negative control (triangle)
• Lack of tyrosine residues
resulted in greater
decrease of viral entry
relative to YU2
pseudotyped HIV-1
HIV-1 ENTRY EFFICIENCY MODULATED BY SULFATED
TYROSINES
• % entry relative to wild type CCR5

32. • 89.6 pseudotyped HIV-1
• WT CCR5 (black squares/
blue)
• FFFF CCR5 construct
(circles/ red)
• Negative control (triangle)
• Lack of tyrosine residues
resulted in greatest
decrease of viral entry
relative to all tested
pseudotypes of HIV-1
HIV-1 ENTRY EFFICIENCY MODULATED BY SULFATED
TYROSINES
• % entry relative to wild type CCR5

33. • YU2 pseudotyped HIV-1
• WT CCR5 (YYYY)
standard
• Test entry mediated by
sulfate group vs phenyl
ring
• Presence of sulfated
tyrosine leads to
significant viral entry
HIV-1 YU2 ENTRY ENHANCED BY SULFATION OF N-
TERMINAL TYROSINE RESIDUES

34. • ADA pseudotyped HIV-1
• WT CCR5 (YYYY)
standard
• Test entry mediated by
sulfate group vs phenyl
ring
• Presence of sulfated
tyrosine leads to
significant viral entry
HIV-1 ADA ENTRY ENHANCED BY SULFATION OF N-
TERMINAL TYROSINE RESIDUES

35. • AA sequences of N-
termini of related
receptors
• All but two are known
HIV-1/SIV coreceptors*
• Tyrosines and adjacent
AAs are bolded
DO THESE FINDINGS TRANSLATE TO OTHER RELATED
RECEPTORS?
Receptors incorporating [35S] sulfate

36. • CXCR4 tested in human embryonic kidney
cells expressing SV40 large T antigen (HEK
293T)
• CXCR4 contained c-terminal tag
recognized by Ab 1D4
• Receptor labeled with [35S] sulfate
• Band indicates presence of [35S] sulfate in
CXCR4 expressing cells
DO THESE FINDINGS TRANSLATE TO OTHER RELATED
RECEPTORS?

37. • Confirmed CCR5 is sulfated
• Site of sulfation on tyrosines
– Confirmed tyrosine sulfation
• Tyrosine sulfation occurs on N-terminus
– Affects binding but not receptor expression
– Blockage of sulfation leads to decreased binding ability of native ligands
• Tyrosine sulfation enhanced by negative charge of proximal AAs
• Sulfated tyrosines are important in HIV-1 entry
• Sulfation may play a role in other coreceptors and the
internalization of HIV-1
TYROSINE SULFATION OF THE AMINO TERMINUS
OF CCR5 FACILITATES HIV-1 ENTRY

38. • Baleux, Françoise, et al. "A synthetic CD4–heparan sulfate glycoconjugate inhibits CCR5
and CXCR4 HIV-1 attachment and entry." Nature chemical biology 5.10 (2009): 743-748.
• Farzan, Michael, et al. "A tyrosine-rich region in the N terminus of CCR5 is important for
human immunodeficiency virus type 1 entry and mediates an association between
gp120 and CCR5." Journal of virology 72.2 (1998): 1160-1164.
• Farzan, Michael, et al. "Tyrosine sulfation of the amino terminus of CCR5 facilitates HIV-1
entry." Cell 96.5 (1999): 667-676.
• Kanan, Yogita, and Muayyad R. Al-Ubaidi. "Tyrosine OSulfation: An Overview." JSM 1.1
(2013): 1003.
• Tani, Hideki, Shigeru Morikawa, and Yoshiharu Matsuura. "Development and
applications of VSV vectors based on cell tropism." Frontiers in microbiology 2 (2011).
• http://asfaculty.syr.edu/pages/chem/_images/hougland_fig1.jpg
• http://www.biolegend.com/media_assets/chemokine_receptors/CK_structure.jpg
• http://s.hswstatic.com/gif/aids-hiv-anatomy.gif
• http://treasuresoftheinternet.org/health/aids/us_census/aids_maps/Map3c.gif
• http://idshowcase.lshtm.ac.uk/id501/ID501/S1S4/ID501_S1S4_030_010.html
REFERENCES