Biology subjects

Birchenough, H. L.

Publications and source records attributed to Birchenough, H. L..

4 recordsLinked to original sources

bioRxiv2024

The structural organisation of pentraxin-3 and its interactions with heavy chains of inter-alpha-inhibitor regulate crosslinking of the hyaluronan matrix

Pentraxin-3 (PTX3) is an octameric protein, comprised of eight identical protomers, that has diverse functions in reproductive biology, innate immunity and cancer. PTX3 interacts with the large polysaccharide hyaluronan (HA) to which heavy chains (HCs) of the inter--inhibitor (II) family of proteoglycans are covalently attached, playing a key role in the (non-covalent) crosslinking of HC*HA complexes. These interactions stabilise the cumulus matrix, essential for ovulation and fertilisation in mammals, and are also implicated in the formation of pathogenic matrices in the context of viral lung infections. To better understand the physiological and pathological roles of PTX3 we have analysed how its quaternary structure underpins HA crosslinking via its interactions with HCs. A combination of X-ray crystallography, cryo-electron microscopy (cryo-EM) and AlphaFold predictive modelling revealed that the C-terminal pentraxin domains of the PTX3 octamer are arranged in a central cube, with two long extensions on either side, each formed from four protomers assembled into tetrameric coiled-coil regions, essentially as described by (Noone et al., 2022; doi:10.1073/pnas.2208144119). From crystallography and cryo-EM data, we identified a network of inter-protomer salt bridges that facilitate the assembly of the octamer. Small angle X-ray scattering (SAXS) validated our model for the octameric protein, including the analysis of two PTX3 constructs: a tetrameric Half-PTX3 and a construct missing the 24 N-terminal residues ({Delta}1-24-PTX3). SAXS determined a length of [~]520 [A] for PTX3 and, combined with 3D variability analysis of cryo-EM data, defined the flexibility of the N-terminal extensions. Biophysical analyses revealed that the prototypical heavy chain HC1 does not interact with PTX3 at pH 7.4, consistent with our previous studies showing that, at this pH, PTX3 only associates with HC*HA complexes if they are formed in its presence. However, PTX3 binds to HC1 at acidic pH, and can also be incorporated into pre-formed HC*HA complexes under these conditions. This provides a novel mechanism for the regulation of PTX3-mediated HA crosslinking (e.g., during inflammation), likely mediated by a pH-dependent conformational change in HC1. The PTX3 octamer was found to associate simultaneously with up to eight HC1 molecules and, thus, has the potential to form a major crosslinking node within HC*HA matrices, i.e., where the physical and biochemical properties of resulting matrices could be tuned by the HC/PTX3 composition.

biochemistry↗

bioRxiv2024

An avidity-driven mechanism of extracellular BMP regulation by Twisted gastrulation

During dorsoventral patterning of bilaterian embryos, the conserved regulator Twisted gastrulation (Tsg) modulates BMP signalling by binding Chordin/Short gastrulation (Sog). Here we elucidate the mechanism by which Tsg interacts with Sog/Chordin to promote formation of the inhibitory Tsg-Sog/Chordin-BMP complex and regulate BMP signalling extracellularly. We identify and validate in vitro a hydrophobic interface in the Tsg C-terminal domain that binds Chordin. Mutation of this epitope in Drosophila Tsg (TsgL100A) results in an unexpectedly mild perturbation to embryonic BMP gradient formation. We show that a protosome-specific Tsg C-terminal extension also binds Sog, and the presence of this second binding site allows partial rescue of Sog interaction with TsgL100A in the presence of BMP. Consistent with this, a truncated Tsg protein lacking both Sog binding regions is unable to support BMP gradient formation in vivo. As our data show that disruption of either Sog binding site in Tsg, but not both, can be overcome by Tsg-BMP and Sog-BMP interactions, we present a new avidity-driven mechanism of BMP gradient formation that will be relevant to a broad range of developmental contexts. Summary statementIdentification and mutational analysis of the binding epitopes that mediate Twisted gastrulation interaction with Short gastrulation/Chordin reveals an avidity-based model of embryonic BMP gradient formation.

developmental biology↗

bioRxiv2023

CXCL17 binds efficaciously to glycosaminoglycans with the potential to modulate chemokine signalling

CXCL17 is a mucosally secreted protein, and the most recently identified human chemokine, an assignment based on protein fold prediction and chemotactic activity for leukocytes. However, these credentials have been the subject of much recent discussion and no experimental evidence has been presented regarding the definitive structure of CXCL17. In this study, we evaluated the structural and chemoattractant credentials of CXCL17 to better characterise this molecule, and gain deeper insights into its functional role as a glycosaminoglycan (GAG) binding protein. In the absence of structural information, in silico modelling techniques assessed the likelihood of CXCL17 adopting a chemokine-fold. Recombinant CXCL17 was synthesized in mammalian and prokaryotic systems. Modified Boyden chamber and real-time chemotaxis assays assessed the ability of CXCL17 to promote chemotaxis of murine splenocytes, human neutrophils and CXCR1-transfectants. The efficacy of CXCL17 binding to GAGs was quantified with solid-phase assays and bio-layer interferometry techniques. All modelling efforts failed to support classification of CXCL17 as a chemokine based on its predicted conformation. Recombinant CXCL17 was observed to dimerize as a function of concentration, a characteristic of several chemokines. Contrary to a previous report, CXCL17 was not chemotactic for murine splenocytes, although it was a low-potency chemoattractant for human neutrophils at micromolar concentrations, several orders of magnitude higher than those required for CXCL8. As anticipated due to its highly basic nature, CXCL17 bound to GAGs robustly, with key C-terminal motifs implicated in this process. While inactive via CXCR1, CXCL17 was found to inhibit CXCR1-mediated chemotaxis of transfectants to CXCL8 in a dose-dependent manner. In summary, despite finding little evidence for chemokine-like structure and function, CXCL17 readily bound GAGs, and could modulate chemotactic responses to another chemokine in vitro. We postulate that such modulation is a consequence of superior GAG-binding, and that C-terminal fragments of CXCL17 may serve as prototypic inhibitors of chemokine function.

immunology↗

bioRxiv2022

Chemokines form complex signals during inflammation and disease that can be decoded by extracellular matrix proteoglycans

Chemokine driven leukocyte recruitment is a key component of the immune response and is central to a wide range of diseases. However, there has yet to be a clinically successful therapeutic approach that targets the chemokine system during inflammatory disease; possibly due to the supposed redundancy of the chemokine system. A range of recent studies have demonstrated that the chemokine system is in fact based on specificity of function. Here we have generated a resource to analyse chemokine gene (ligand and receptor) expression across different species, tissues and diseases; revealing complex expression patterns whereby multiple chemokine ligands that mediate recruitment of the same leukocyte type are expressed in the same context, e.g. the CXCR3 ligands CXCL9, 10 and 11. We use biophysical approaches to show that CXCL9, 10 and 11 have very different interactions with extracellular matrix glycosaminoglycans (GAGs) which is exacerbated by specific GAG sulphation. Finally, in vivo approaches demonstrate that GAG-binding is critical for CXCL9 driven recruitment of specific T cell subsets (e.g. CD4+) but not others (e.g. CD8+), independent of CXCR3 expression. Our data demonstrate that chemokine expression is complex and that multiple ligands are likely needed for robust leukocyte recruitment across tissues and diseases. We also demonstrate that ECM GAGs facilitate decoding of these complex chemokine signals so that they are either primarily presented on GAG-coated cell surfaces or remain more soluble. Our findings represent a new mechanistic understanding of chemokine mediated immune cell recruitment and identify novel avenues to target specific chemokines during inflammatory disease.

immunology↗