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

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 desorption͞ionization-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. 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-1␣, MIP-1␤, 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 (⌬32) 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. 1; residues 2-18) was identified as a major determinant of HIV-1 coreceptor function (11,12,(14)(15)(16)(17). Sequence similarities with proteins known to be modified by tyrosine O-sulfation, a posttranslational modification mediated by tyrosylprotein sulfotransfera...

Sulfated tyrosines 27 and 29 in the N-terminus of human CXCR3 participate in binding native IP-10

“…For example, the tyrosines of CCR5 at positions 3-15 are sulfated in a stepwise manner; tyrosines 14 and 15 are sulfated first, followed by tyrosines 10 and 3 [35] . In the present study, to verify that the reduced binding of IP-10 by cells expressing FDF reflects the loss of sulfate and not a change in receptor conformation, we attempted to increase or decrease the extent of sulfation by co-transfecting cells with the expression plasmids for CXCR3 and TPSTs, or with CXCR3 and shRNAs targeting TPSTs.…”

“…First of all, the previous authors report that the molecular weight of human CXCR3 is about 70 kDa, whereas our study shows that the molecular weight of human mature CXCR3 is about 45 kDa, consistent with the predicted amino acid composition. Secondly, tyrosines 27 and 29 are separated by one acidic amino acid, suggesting that both of these two tyrosines should be sulfated [12,35,37] . Thirdly, the previous authors were unable to exclude other factors that may have affected the binding of CXCR3 to IP-10, such as N-linked glycosylation.…”

Toward a framework for sulfoproteomics: Synthesis and characterization of sulfotyrosine‐containing peptides

“…In vitro sulfation studies with model peptides demonstrate a considerable degree of overlap in substrate specificity between TPST-1 and TPST-2. 8,11 However, gene disruption studies in mice suggest that in vivo both isoenzymes have distinct physiological functions. 12,13 Due to the cellular localization of the TPST enzymes, Tyr sulfation can only occur in proteins that transit the transGolgi network and therefore is limited to secretory and transmembrane proteins.…”

Female Infertility in grt Mice is Caused by Thyroid Hormone Deficiency, not by Insufficient TPST2 Activity in the Reproductive Organs

“…This may account for the decreased rates of copulation and parturition in both T-grt/grt and Tgrt/+ mice. The fact that there were no differences in the mean ratio of parturitions per copulation and first litter size among T-grt/grt, T-grt/+ and grt/+ mice suggests that the side effect of thyroid powder feeding disturb folliculogene- TPST has two isoforms with distinct acceptor preferences [31,37]. The sulfated tyrosine residues have recently been identified in LHR and FSHR, and they are predicted to play an important role in the interaction with their natural ligands [4].…”

“…In mammals, two isozymes, TPST1 and 2, catalyze the tyrosine sulfation of proteins. Although TPST1 and 2 in mammals have 65~67% identical amino acid sequences [28], they seem to have different acceptor preferences [31,37]. Evidence indicates that the posttranslational modification by tyrosine sulfation regulates many important protein-protein interactions and modulates binding affinity and specificity [8,10,11,18,24,26,29,[33][34][35][36].…”