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 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.

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.

Fig 2.

RP-HPLC analysis of CCR5 2–18 sulfation products. (A) Characterization of the in vitro sulfation reaction. Peptide CCR5 2–18 (0.1 mg/ml, ≈50 μM) was incubated with a mixture of TPST-1 and TPST-2 (20 μg/ml each) and in the presence of the sulfation cosubstrate PAPS (400 μM). After 30 h or 100 h at 16°C, 60 μl aliquots were analyzed by RP-HPLC. In negative-control experiments (100 h incubation time), either the TPST mixture (no TPST) or PAPS (no PAPS) was omitted. Peaks were labeled a–f in increasing order of hydrophilicity. (B) Comparison of TSPT-1 and TPST-2. CCR5 2–18 (0.1 mg/ml, ≈50 μM) was incubated for 100 h with TPST-1 (40 μg/ml) or TPST-2 (40 μg/ml) in the presence of PAPS (400 μM).

Fig 3.

MALDI-TOF MS of CCR5 2–18 sulfation products. Peak fractions a–f from RP-HPLC were analyzed in negative ion mode by using an ultra-thin layer sample preparation method (27). Because of the lability of tyrosine sulfo–ester bonds under MS conditions (28), spectra for highly sulfated peptide species were dominated by loss-of-sulfate ions. Multisulfated peptide species could only be detected as sodium adducts. Peaks were labeled according to the number of identified sulfates as shown in Table 1. Ions with additional sodium adducts were not labeled. In peak fraction b, a small amount of the ion [(CCR5 3–18 + SO₃) − H]⁻ (*) was detected, consistent with the loss of the initial Asp.

Fig 4.

Analysis of sulfation sites in CCR5 2–18 sulfation products. (A) Strategy for the generation of proteolytic fragments. Fragments CCR5 2–10 and CCR5 11–18 were produced by endoproteinase Asp-N cleavage. Fragment CCR5 4–10 was generated from fragment CCR5 2–10 by chymotrypsin cleavage. For clarity, the dipeptide fragment (DY), which was not further analyzed, is not shown. Fragments CCR5 2–15 and CCR5 2–14 were obtained by carboxypeptidase Y cleavage of CCR5 2–18 sulfation products. (B) RP-HPLC chromatograms of endoproteinase Asp-N cleavage products. Nonsulfated CCR5 2–18 (peak a) and tyrosine sulfation products (peaks b–f), cleaved with endoproteinase Asp-N, were analyzed by RP-HPLC and identified by MALDI-TOF MS as fragments CCR5 2–10 and CCR5 11–18 containing zero, one, or two sulfates (MS data not shown). In the RP-HPLC chromatograms, peaks corresponding to the same fragment are connected by dotted red lines. The stepwise decrease in the elution time for a given fragment indicates incorporation of one or two sulfates. (C) RP-HPLC chromatograms of chymotrypsin cleavage products. Asp-N-generated fragments CCR5 2–10 were further cleaved with chymotrypsin. Peaks identified as uncleaved starting material CCR5 2–10 are drawn with dotted black lines, whereas peaks corresponding to the cleavage product CCR5 4–10 are drawn with solid black lines. Peaks corresponding to the same peptide sequence are connected by dotted red lines.

Fig 5.

Time course of CCR5 2–18 sulfation catalyzed by TPST-1 and TPST-2. CCR5 2–18 (0.1 mg/ml) was incubated with TPST-1 (A) or TPST-2 (B) (180 μg/ml each) in the presence of PAPS (400 μM) at 16°C. At the indicated time points, 40-μl aliquots were analyzed by RP-HPLC, and relative amounts for the different peptide species (a–f) were calculated from the peak areas.

Fig 6.

Reaction scheme for the sequential sulfation of CCR5 2–18 by TPST-1 and TPST-2. CCR5 2–18 species that arise from the sulfation of nonsulfated CCR5 2–18 (none), with either TPST-1 (Left) or TPST-2 (Right), are represented by the sulfotyrosine positions. Major sulfation products are depicted as bold numbers and corresponding reaction pathways are represented by bold arrows. Minor sulfation products are in smaller fonts and the corresponding reaction pathways are depicted as dotted arrows.