Can an antibody tell which tyrosine carries the sulfate?

Why sulfotyrosine is a harder target than phospho

Tyrosine sulfation regulates signal transduction, immune recognition and viral entry, and it is chemically close enough to phosphorylation that generic reagents struggle to tell them apart. It has also lacked good tools: before this work there was no antibody that could detect sulfated tyrosine on CCR5 in a sequence-specific way, which left basic questions about the sulfation status of CCR5 on real cells unanswerable.

The stakes are concrete. Sulfation of the four N-terminal tyrosines of CCR5 has been reported to increase HIV infection efficiency by roughly 50-fold, and the sulfate groups participate directly in the interface with the HIV glycoprotein gp120. An antibody that reads sulfation state is therefore a research tool with a clear question attached to it.

What the antibody does

• Binary discrimination. Nanomolar binding to the sulfated CCR5 N-terminal peptide; no binding to the unsulfated peptide within the tested range by SPR, and no exothermic signal at all by isothermal titration calorimetry.
• It works on the real receptor, on cells. BA8 detected full-length CCR5 on the surface of HEK293 cells co-expressing the sulfotransferase TPST2, with a half-maximal binding concentration of 3.64 nM — the same order as the peptide affinity. It detected no other sulfated proteins in the lysate.
• Site selectivity, not just modification selectivity. Sulfation at one specific tyrosine (position 7 in the numbering used) is indispensable; peptides lacking it do not bind at all. Losing the sulfate at position 8 costs about 100-fold in affinity, with the binding enthalpy falling from −28 to −15 kcal/mol. The sulfate at position 3 turns out not to be required.
• Binding rigidifies the antibody. Melting temperature rose from 70.8 °C unbound to 75.6 °C in complex with the sulfated peptide, with no shift at all on adding the unsulfated peptide — an orthogonal confirmation of specificity.

Crystal structure of the Fab–peptide complex solved at 1.8 Å, deposited as PDB 9J8A. Alanine scanning across the interface identified R34, S52 and Y57 of the heavy chain as hot spots; the structure shows the critical sulfotyrosine inserted deep between heavy and light chain.

What this demonstrates about the platform

Three things transfer from this campaign to other difficult post-translational modifications.

Selection can be run against cells, not only peptide. Panning here used both the sulfated immunogen peptide and cells overexpressing CCR5 together with the sulfotransferases. Clones were then required to bind both the peptide and sulfated CCR5 on the cell surface before advancing. That is the screening discipline that prevents the classic outcome of an antibody that works only on the immunogen.

Convergence is informative. After selection, the recovered sequences were nearly identical — the campaign converged on a single solution. That is a different result from the methylation campaign, which returned six mechanistically distinct clones, and it tells you something real about how narrow the structural solution space for this epitope is.

The antibody paid for its specificity. Sequence analysis showed BA8 carries an arginine at a framework position where more than half of rabbit antibodies have serine. That unnatural arginine is what grips the sulfate — and it costs thermal stability, since the R34A mutant is actually more stable unbound than wild type. Knowing that trade-off exists is what lets it be engineered deliberately rather than discovered late.

As in the companion studies, the library and vector are Abwiz Bio technology; the paper’s conflict-of-interest statement records that the authors used patented technology WizAmp (US 9,890,414), invented by S. C. J. O. and T. M., for antibody acquisition.

Frequently asked questions