tyrosine sulfation lecture(edited)

VBMS 7180 ReceptorologyPresented by: Chris Ramhold Ph.D. Student

16-March-15

TYROSINE SULFATION OF THE AMINO TERMINUS

OF CCR5 FACILITATES HIV-1 ENTRY

• 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

• 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.

• 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.

• Aromatic- Benzene• Reactive hydroxyl group• Can be phosphorylated or

sulfated

QUICK REVIEW- TYROSINE

• 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

• 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.

• 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

• 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.

• 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

• 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

• 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

• Research group published paper regarding N-terminus of CCR5 receptor

• Gradual increase in focus

• Need to identify targets for HIV therapy

THE PAPER

• 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.

• 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 CD4Label

IP

• 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)

• 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

• 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

• 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

• 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)


FACS- Fluorescence Activated Cell Sorting

• Confirmation of results– Mutated tyrosine to phenylalanine– Similar results (data not shown)– Decreased recognition specific to loss of sulfate


• 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

• 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



MIP= Macrophage inflammatory protein-1β a.k.a. CCL4

• 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



sYU2- HIV-1 env gp120, strain YU2

• 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

• 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

• 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

• 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

• 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).

• 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

• 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



• 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



• 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

• 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

• 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

• 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?

• 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 TERMINUSOF 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.

• 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

tyrosine sulfation lecture(edited)

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