Proc Natl Acad Sci U S A 99(17): 11031-11036

Tyrosine sulfation of CCR5 N-terminal peptide by tyrosylprotein sulfotransferases 1 and 2 follows a discrete pattern and temporal sequence

^{Laboratory of Molecular Biology and Biochemistry,}^{Laboratory for Mass Spectrometry and Gaseous Ion Chemistry,}^{Howard Hughes Medical Institute, The Rockefeller University, New York, NY 10021}

^{To whom reprint requests should be addressed at: Box 284, The Rockefeller University, 1230 York Avenue, New York, NY 10021. E-mail:}ude.rellefekcor.liam@ramkas.

Communicated by Bruce Merrifield, The Rockefeller University, New York, NY

Received 2002 Apr 24; Accepted 2002 Jun 26.

Abstract

The CC-chemokine receptor 5 (CCR5) is the major coreceptor for the entry of macrophage-tropic (R5) HIV-1 strains into target cells. Posttranslational sulfation of tyrosine residues in the N-terminal tail of CCR5 is critical for high affinity interaction of the receptor with the HIV-1 envelope glycoprotein gp120 in complex with CD4. Here, we focused on defining precisely the sulfation pattern of the N terminus of CCR5 by using recombinant human tyrosylprotein sulfotransferases TPST-1 and TPST-2 to modify a synthetic peptide that corresponds to amino acids 2–18 of the receptor (CCR5 2–18). Analysis of the reaction products was made with a combination of reversed-phase HPLC, proteolytic cleavage, and matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry (MALDI-TOF MS). We found that CCR5 2–18 is sulfated by both TPST isoenzymes leading to a final product with four sulfotyrosine residues. Sulfates were added stepwise to the peptide producing specific intermediates with one, two, or three sulfotyrosines. The pattern of sulfation in these intermediates suggests that Tyr-14 and Tyr-15 are sulfated first, followed by Tyr-10, and finally Tyr-3. These results represent a detailed analysis of the multiple sulfation reaction of a peptide substrate by TPSTs and provide a structural basis for understanding the role of tyrosine sulfation of CCR5 in HIV-1 coreceptor and chemokine receptor function.

Abstract

The CC-chemokine receptor 5 (CCR5) is a member of the protein superfamily of G protein-coupled receptors (GPCRs) (1–3). High-affinity binding of the CC-chemokines MIP-1α, MIP-1β, RANTES (1–3), or MCP-2 (4) to CCR5 induces signaling through G proteins of the G_i subfamily (1) and leads to chemotactic responses in CCR5-expressing leukocytes (5).

In addition to their physiological function in chemokine signaling, some chemokine receptors are used as coreceptors by HIV-1. Entry of HIV-1 into target cells is mediated by the sequential interaction of the envelope glycoprotein gp120 with CD4 and a chemokine receptor on the cell membrane (6). CCR5 and CXCR4 are the primary HIV-1 coreceptors in vivo (7, 8). CCR5, in particular, is the principal coreceptor for macrophage-tropic HIV-1 strains (R5 isolates) that are commonly transmitted between individuals (6, 9). A naturally occurring CCR5 mutant (Δ32) with a deletion in the second extracellular loop results in impaired membrane expression of the receptor and leads to resistance to HIV-1 infection in homozygous individuals (6).

Interaction with both types of CCR5 ligands, CC-chemokines and the HIV-1 gp120-CD4 complex, involves the N-terminal domain, as well as other extracellular regions of the receptor (10–13). Within the N-terminal domain, a region rich in tyrosine residues and acidic amino acid residues (Fig. (Fig.1;1; residues 2–18) was identified as a major determinant of HIV-1 coreceptor function (11, 12, 14–17). Sequence similarities with proteins known to be modified by tyrosine O-sulfation, a posttranslational modification mediated by tyrosylprotein sulfotransferases (TPSTs) in the trans-Golgi network (18), led to the recent discovery of tyrosine sulfation within this region of CCR5 (19).

An external file that holds a picture, illustration, etc.
Object name is pq1723808001.jpg

Fig 1.

Schematic representation of human CCR5 with potential tyrosine sulfation sites. The seven transmembrane helices are shown as cylinders. Connecting extracellular and cytosolic loops as well as N- and C-terminal domains are depicted as black lines. Amino acid residues in the N-terminal sequence corresponding to peptide CCR5 2–18 are represented in single-letter codes. Met-1, which is not present in CCR5 2–18, is marked by a dashed circle. The potentially sulfated tyrosine residues at positions 3, 10, 14, and 15 are highlighted in red, and the acidic amino acid residues Asp-2, Asp-11, and Glu-18 are shown in green.

Although the role of tyrosine sulfation in protein function is not generally well understood, in the case of CCR5 it has been shown that tyrosine sulfation is crucial for efficient gp120–CD4 binding and HIV-1 coreceptor function (19–21). Sulfotyrosines were also reported to contribute to the binding of the CC-chemokines MIP-1α and MIP-1β to CCR5 (19, 21).

The specific contribution of the four potential tyrosine sulfation sites in CCR5 to the HIV-1 coreceptor function was investigated in different studies by using either a mutagenesis approach (15, 19) or binding and competition experiments with synthetic sulfotyrosine peptides (20, 21). While these studies agree on the importance of sulfotyrosines at positions 10 and 14, the role of Tyr-3 and Tyr-15 remains in question.

Here we present the pattern observed by enzymatic in vitro sulfation of a peptide corresponding to amino acids 2–18 of CCR5 (CCR5 2–18) by using recombinant human TPSTs. We analyzed intermediates and the final product of the sulfation reaction by using a combination of reversed-phase (RP)-HPLC, proteolytic cleavage, and matrix-assisted laser desorption/ionization–time-of-flight MS (MALDI-TOF MS). By using this in vitro approach, we found that CCR5 2–18 is sulfated by the two known human TPSTs, TPST-1 (22) and TPST-2 (23, 24), resulting in a final product in which all four tyrosine residues are sulfated. We also found that sulfates are added stepwise to the peptide and that Tyr-14 and Tyr-15 are sulfated first, followed by Tyr-10, and finally Tyr-3. These results represent a detailed analysis of the multiple sulfation reaction of a peptide substrate by TPSTs and provide a basis for understanding the role of posttranslational tyrosine sulfation of CCR5 in HIV-1 coreceptor function and chemokine signaling.

n.d., not determined.

Acknowledgments

We thank Dr. K. L. Moore for the gift of TPST expression vectors, and Dr. C. G. Unson for helpful discussions. This work was supported by a grant from the National Institutes of Health (RR00862 from the National Center for Research Resources). C.S. is an Associate and T.P.S. is an Associate Investigator of the Howard Hughes Medical Institute.

Acknowledgments

Abbreviations

CCR5, CC-chemokine receptor 5
MALDI-TOF MS, matrix-assisted laser desorption/ionization–time-of-flight MS
PAPS, 3′-phosphoadenosine 5′-phosphosulfate
RP, reversed-phase
TPST, tyrosylprotein sulfotransferase

Abbreviations

References

1. Combadiere C., Ahuja, S. K., Tiffany, H. L. & Murphy, P. M. (1996) J. Leukocyte Biol.60, 147-152. [[PubMed]
2. Samson M., Labbe, O., Mollereau, C., Vassart, G. & Parmentier, M. (1996) Biochemistry35, 3362-3367. [[PubMed]
3. Raport C. J., Gosling, J., Schweickart, V. L., Gray, P. W. & Charo, I. F. (1996) J. Biol. Chem.271, 17161-17166. [[PubMed]
4. Gong W., Howard, O. M., Turpin, J. A., Grimm, M. C., Ueda, H., Gray, P. W., Raport, C. J., Oppenheim, J. J. & Wang, J. M. (1998) J. Biol. Chem.273, 4289-4292. [[PubMed]
5. Murphy P. M., Baggiolini, M., Charo, I. F., Hebert, C. A., Horuk, R., Matsushima, K., Miller, L. H., Oppenheim, J. J. & Power, C. A. (2000) Pharmacol. Rev.52, 145-176. [[PubMed]
6. Berger E. A., Murphy, P. M. & Farber, J. M. (1999) Annu. Rev. Immunol.17, 657-700. [[PubMed]
7. Zhang Y., Lou, B., Lal, R. B., Gettie, A., Marx, P. A. & Moore, J. P. (2000) J. Virol.74, 6893-6910.
8. Zhang Y. J. & Moore, J. P. (1999) J. Virol.73, 3443-3448.
9. Dragic T(2001) J. Gen. Virol.82, 1807-1814. [[PubMed][Google Scholar]
10. Rucker J., Samson, M., Doranz, B. J., Libert, F., Berson, J. F., Yi, Y., Smyth, R. J., Collman, R. G., Broder, C. C., Vassart, G., et al. (1996) Cell87, 437-446. [[PubMed]
11. Ross T. M., Bieniasz, P. D. & Cullen, B. R. (1998) J. Virol.72, 1918-1924.
12. Blanpain C., Doranz, B. J., Vakili, J., Rucker, J., Govaerts, C., Baik, S. S., Lorthioir, O., Migeotte, I., Libert, F., Baleux, F., et al. (1999) J. Biol. Chem.274, 34719-34727. [[PubMed]
13. Howard O. M., Shirakawa, A. K., Turpin, J. A., Maynard, A., Tobin, G. J., Carrington, M., Oppenheim, J. J. & Dean, M. (1999) J. Biol. Chem.274, 16228-16234. [[PubMed]
14. Dragic T., Trkola, A., Lin, S. W., Nagashima, K. A., Kajumo, F., Zhao, L., Olson, W. C., Wu, L., Mackay, C. R., Allaway, G. P., et al. (1998) J. Virol.72, 279-285.
15. Rabut G. E., Konner, J. A., Kajumo, F., Moore, J. P. & Dragic, T. (1998) J. Virol.72, 3464-3468.
16. Farzan M., Choe, H., Vaca, L., Martin, K., Sun, Y., Desjardins, E., Ruffing, N., Wu, L., Wyatt, R., Gerard, N., et al. (1998) J. Virol.72, 1160-1164.
17. Doranz B. J., Lu, Z. H., Rucker, J., Zhang, T. Y., Sharron, M., Cen, Y. H., Wang, Z. X., Guo, H. H., Du, J. G., Accavitti, M. A., et al. (1997) J. Virol.71, 6305-6314.
18. Baeuerle P. A. & Huttner, W. B. (1987) J. Cell Biol.105, 2655-2664.
19. Farzan M., Mirzabekov, T., Kolchinsky, P., Wyatt, R., Cayabyab, M., Gerard, N. P., Gerard, C., Sodroski, J. & Choe, H. (1999) Cell96, 667-676. [[PubMed]
20. Cormier E. G., Persuh, M., Thompson, D. A., Lin, S. W., Sakmar, T. P., Olson, W. C. & Dragic, T. (2000) Proc. Natl. Acad. Sci. USA97, 5762-5767.
21. Farzan M., Vasilieva, N., Schnitzler, C. E., Chung, S., Robinson, J., Gerard, N. P., Gerard, C., Choe, H. & Sodroski, J. (2000) J. Biol. Chem.275, 33516-33521. [[PubMed]
22. Ouyang Y. B., Lane, W. S. & Moore, K. L. (1998) Proc. Natl. Acad. Sci. USA95, 2896-2901.
23. Ouyang Y. B. & Moore, K. L. (1998) J. Biol. Chem.273, 24770-24774. [[PubMed]
24. Beisswanger R., Corbeil, D., Vannier, C., Thiele, C., Dohrmann, U., Kellner, R., Ashman, K., Niehrs, C. & Huttner, W. B. (1998) Proc. Natl. Acad. Sci. USA95, 11134-11139.
25. Merrifield R. B. (1963) J. Am. Chem. Soc.85, 2149-2154. [PubMed]
26. Carpino L. A. & Han, G. Y. (1972) J. Org. Chem.37, 3404-3409. [PubMed]
27. Cadene M. & Chait, B. T. (2000) Anal. Chem.72, 5655-5658. [[PubMed]
28. Gibson B. W. & Cohen, P. (1990) Methods Enzymol.193, 480-501. [[PubMed]
29. Bundgaard J. R., Vuust, J. & Rehfeld, J. F. (1997) J. Biol. Chem.272, 21700-21705. [[PubMed]
30. Huttner W. B. & Baeuerle, P. A. (1988) Mod. Cell Biol.6, 97-140. [PubMed]
31. Kehoe J. W. & Bertozzi, C. R. (2000) Chem. Biol.7, R57-R61. [[PubMed]
32. Baeuerle P. A. & Huttner, W. B. (1985) J. Biol. Chem.260, 6434-6439. [[PubMed]
33. Cormier E. G., Tran, D. N., Yukhayeva, L., Olson, W. C. & Dragic, T. (2001) J. Virol.75, 5541-5549.