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Glyco-sulfo hotspots in the chemokine receptor system

Glycosylation and sulfation – N-terminal PTMs on chemokine receptors

The interaction of chemokine receptors with their cognate chemokine ligands is generally described by the two-step/two-site model - the first step characterized by the interaction between the extracellular domains of the receptor and the structural core domain of the chemokine (CRS1); and the second step featured by the interaction between the N-terminus of the chemokine and the extracellular loops and transmembrane domains of the receptor (CRS2), which will trigger conformational changes that ultimately lead to receptor activation (Scholten D et al. 2012, Kufavera I et al. 2017). Within the CRS1 ineraction mode, the N-terminal region of chemokine receptors is indispensable for chemokine binding, where the negatively charged residues, as a consequence of the negatively charged aminoacids and post-translational modifications (PTMs), contribute to the high affinity binding to the positively charged groove on the chemokine. N-terminal PTMs include sulfation and glycosylation which contribute to the overall negative charge of the N-terminus fine-tuning chemokine binding.

Chemokine receptors have either reported or predicted sites of N-acetyl galactosamine (GalNAc)-type O-glycosylation in their N-termini as well as sulfation, both PTMs which co-localize in the Trans-Golgi network (Mehta AY et al. 2020). In silico analyses done in this study with the NetOGlyc 4.0 prediction algorithm (Steentoft C et al. 2013) for O-glycosylation and the sulfinator tool for tyrosine sulfation sites (Monigatti F et al. 2002), suggest that CC chemokine receptors have general patterns of O-glycosylation and tyrosine sulfation in their N-terminal region which seem to be widely conserved between human and murine sequences.

GalNAc-type or mucin-type O-glycosylation is initiated by the transfer of Gal-NAc to a serine or threonine residue by polypeptide GalNAc-transferases (GalNAc-Ts) which is further elongated with the linkage of other monosaccharides (Schjoldager KT et al. 2020), and usually capped by a single sialic acid, although, in rare cases glycans can carry polysialylation (PolySia) (Mindler K et al. 2021). PolySia has been described for CCR7 where it specifically affects the recognition of CCL21 but not CCL19, interfering with dendritic cell trafficking (Kiermaier E et al. 2016). Other examples of O-glycosylation impact on chemokine receptors include the viral receptor US28 (Bagdonaite I et al. 2016) where O-glycosylation contributes to the differential binding to CC or CX3C chemokine (Casarosa P et al. 2005). CCR5 O-glycosylation, which is under investigation in this study, also plays a major role in promoting the interaction with CCL3 and CCL5, being also important for HIV infection (Bannert N et al. 2001).

Tyrosine sulfation consists in the transfer of a sulfate group from the adenosine 3’-phosphate 5’-phosphosulfate (PAPS) donor to the hydroxyl group of a tyrosine residue of the protein chain (Seibert C and Sakmar TP. 2008) by tyrosine sulfotransferase 1 or 2 (TPST1/2). This PTM has been shown to be heterogeneous [Li X et al. 2018; Scurci I et al. 2021) and to improve the affinity of chemokines through the charge interactions between the negative sulfate groups in the N-terminus and the positively charged chemokines (Ludeman JP and Stone MJ. 2014). CCR5 sulfation has been also reported to play a key role in CCL3 and CCL5 binding (Bannert N et al. 2001) as well as to exist in heterogeneous forms inside the cell (Scurci I et al. 2021). The atypical chemokine receptor 2 (ACKR2), US28 and sphingosine-1-phosphate receptor 1 (S1PR1) also carry tyrosine sulfation sites in their N-termini that mediate ligand binding and signaling (Bannert N et al. 2001; Casarosa P et al. 2005; Gao J et al. 2003; Fieger CB et al. 2005).

Probing the modulation of O-glycosylation and tyrosine sulfation on CCR5 and CCR1 function

In this study engineered cell lines, inhibitors and mutagenesis approaches were performed to evaluate the effects of O-glycosylation and tyrosine sulfation modulation on the chemokine receptors CCR1 and CCR5 pharmacology. Both PTMs were shown to contribute to the binding of CCL5 and CCL8 and to a minor extend CCL3. The interplay between these PTMs was also revealed where mutagenesis of tyrosine sulfation sites considerably impacted O-glycosylation. Removal of the terminal sialic acid was also shown to negatively impact signaling as previously appreciated (Bannert N et al. 2001). Interestingly, expression of PolySia was able to partially rescue the signaling upon reduction of sulfation, suggesting the fine-tuning role of O-glycosylation although the mechanism through which PolySia exerts its compensatory effect remains unclear. This effect could be driven by a specific carrier or it can potentially be related with the increase in the general negative charge of the cell surface glycosaminoglycans which have an established role in chemokine gradients and oligomerization (Deshauer C et al. 2015; Dyer DP et al. 2016). In this work authors set out to investigate GalNAc-Ts candidates involved in CCR5 O-glycosylation with CHO GalNAc-T knock-outs transfected with CCR5. From the five GalNAc-Ts, GalNAc-T1 was shown to be the most likely candidate for directly glycosylating CCR5 although T11 may also be involved.

Sulfo-glyco barcode - perspectives and added value in drug discovery

A growing body of evidence proposes that O-glycosylation and sulfation are important PTMs in chemokine receptor biology and pharmacology however the reported effects can vary depending on the receptor-ligand pairs and potentially cell line and tissue tested. The combined effects of both PTMs as well as the relevance of specific acceptor sites and glycan composition remains to be investigated in more depth. Dissecting the biological relevance of these PTMs requires relevant cells expressing the chemokine receptor endogenously together with relevant enzymes and co-receptor systems. In this study, although direct effects of O-glycosylation removal are ruled out it is possible that indirect effects may also contribute to the observed phenotype since many glycosyltransferases and the two TPSTs also carry O-glycosylation (King SL et al.2017). In addition, tyrosine sulfation is heterogenous between cell lines or even on the same cell (Scurci I et al. 2021).

It remains to be discovered how much the modulation of these PTMs is relevant for future drug design. There are few examples of the applicability of the sulfo-glyco barcode in drug discovery. For CCR5, it has been shown that N-terminal antibodies show different sulfo-sensitivities (Scurci I et al. 2021). It has been also reported that PSGL-1 glycosulfo peptide analogue GSnP-6 displays nanomolar affinity and promising potential for blocking PSGL-1/P-selectin interaction (Wong DJ et al. 2021). Moreover, sulfated mCCR2 peptides can outcompete available chemokines, decreasing retinal degradation in mice (Jung SA et al. 2021).

Further research will be needed to boost our understanding on the dynamics and biological relevance of these PTMs in the chemokine receptor system which may ultimately allow for new precise targeting of the immune system.

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