SARS-CoV-2 Omicron subvariant spike N405 unlikely to rapidly deamidate.

The RGD motif on the SARS-CoV-2 spike protein has been suggested to interact with RGD-binding integrins αVß3 and α5ß1 to enhance viral cell entry and alter downstream signaling cascades. The D405N mutation on the Omicron subvariant spike proteins, resulting in an RGN motif, has recently been shown to inhibit binding to integrin αVß3. Deamidation of asparagines in protein ligand RGN motifs has been demonstrated to generate RGD and RGisoD motifs that permit binding to RGD-binding integrins. Two asparagines, N481 and N501, on the Wild-type spike receptor-binding domain have been previously shown to have deamidation half-lives of 16.5 and 123 days, respectively, which may occur during the viral life cycle. Deamidation of Omicron subvariant N405 may recover the ability to interact with RGD-binding integrins. Thus, herein, all-atom molecular dynamics simulations of the Wild-type and Omicron subvariant spike protein receptor-binding domains were conducted to investigate the potential for asparagines, the Omicron subvariant N405 in particular, to assume the optimized geometry for deamidation to occur. In summary, the Omicron subvariant N405 was primarily found to be stabilized in a state unfavourable for deamidation after hydrogen bonding with downstream E406. Nevertheless, a small number of RGD or RGisoD motifs on the Omicron subvariant spike proteins may restore the ability to interact with RGD-binding integrins. The simulations also provided structural clarification regarding the deamidation rates of Wild-type N481 and N501 and highlighted the utility of tertiary structure dynamics information in predicting asparagine deamidation. Further work is needed to characterize the effects of deamidation on spike-integrin interactions.

The effects of inhibition and siRNA knockdown of collagen-binding integrins on human umbilical vein endothelial cell migration and tube formation.

Blood vessels in the body are lined with endothelial cells which have vital roles in numerous physiological and pathological processes. Collagens are major constituents of the extracellular matrix, and many adherent cells express several collagen-binding adhesion receptors. Here, we study the endothelium-collagen interactions mediated by the collagen-binding integrins, α1ß1, α2ß1, α10ß1 and α11ß1 expressed in human umbilical vein endothelial cells (HUVECs). Using qPCR, we found expression of the α10 transcript of the chondrocyte integrin, α10ß1, along with the more abundant α2, and low-level expression of α1. The α11 transcript was not detected. Inhibition or siRNA knockdown of the α2-subunit resulted in impaired HUVEC adhesion, spreading and migration on collagen-coated surfaces, whereas inhibition or siRNA knockdown of α1 had no effect on these processes. In tube formation assays, inhibition of either α1 or α2 subunits impaired the network complexity, whereas siRNA knockdown of these integrins had no such effect. Knockdown of α10 had no effect on cell spreading, migration or tube formation in these conditions. Overall, our results indicate that the collagen-binding integrins, α1ß1 and α2ß1 play a central role in endothelial cell motility and self-organisation.

In silico analysis of mutations near S1/S2 cleavage site in SARS-CoV-2 spike protein reveals increased propensity of glycosylation in Omicron strain.

Cleavage of the severe respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein has been demonstrated to contribute to viral-cell fusion and syncytia formation. Studies have shown that variants of concern (VOC) and variants of interest (VOI) show differing membrane fusion capacity. Mutations near cleavage motifs, such as the S1/S2 and S2' sites, may alter interactions with host proteases and, thus, the potential for fusion. The biochemical basis for the differences in interactions with host proteases for the VOC/VOI spike proteins has not yet been explored. Using sequence and structure-based bioinformatics, mutations near the VOC/VOI spike protein cleavage sites were inspected for their structural effects. All mutations found at the S1/S2 sites were predicted to increase affinity to the furin protease but not TMPRSS2. Mutations at the spike residue P681 in several strains, such P681R in the Delta strain, resulted in the disruption of a proline-directed kinase phosphorylation motif at the S1/S2 site, which may lessen the impact of phosphorylation for these variants. However, the unique N679K mutation in the Omicron strain was found to increase the propensity for O-linked glycosylation at the S1/S2 cleavage site, which may prevent recognition by proteases. Such glycosylation in the Omicron strain may hinder entry at the cell surface and, thus, decrease syncytia formation and induce cell entry through the endocytic pathway as has been shown in previous studies. Further experimental work is needed to confirm the effect of mutations and posttranslational modifications on SARS-CoV-2 spike protein cleavage sites.

Tyrosine-sulfated dermatopontin shares multiple binding sites and recognition determinants on triple-helical collagens with proteins implicated in cell adhesion and collagen folding, fibrillogenesis, cross-linking, and degradation.

Dermatopontin (DPT), a small extracellular matrix protein that stimulates collagen fibrillogenesis, contains sulfotyrosine residues but neither its level of sulfation nor its binding sites on fibrillar collagens are known. Here, we discovered that DPT is present in a relatively high mass concentration (~ 0.02%) in porcine corneal stroma, from which we purified five DPT charge variants (A-E) containing up to six sulfations. The major variant (C), containing four sulfotyrosine residues, was used to locate binding sites for DPT on triple-helical collagens II and III using the Collagen Toolkits. DPT-binding loci included the triple helix crosslinking sites and collagenase cleavage site. We find that strong DPT-binding sites on triple-helical collagen comprise an arginine-rich, positively-charged sequence that also contains hydrophobic residues. This collagen-binding signature of DPT is similar to that of the chaperone HSP47. Thus, we propose that DPT assumes the role of HSP47 as a collagen chaperone during and after the secretion. Peptide II-44, harbouring the conserved collagenase cleavage site, shows the strongest DPT-binding of the Collagen Toolkit II peptides. Substituting any of the three arginine residues (R) with alanine in the sequence GLAGQRGIVGLOGQRGER of II-44 resulted in almost complete loss of DPT binding. Since osteogenesis imperfecta, spondyloepiphyseal dysplasia, and spondyloepimetaphyseal dysplasia congenita are associated with missense mutations that substitute the corresponding arginine residues in collagens alpha-1(I) and alpha-1(II), we suggest that disrupted DPT binding to fibrillar collagens may contribute to these connective tissue disorders. In conclusion, the present work provides a cornerstone for further elucidation of the role of DPT.

Can the SARS-CoV-2 Spike Protein Bind Integrins Independent of the RGD Sequence?

The RGD motif in the Severe Acute Syndrome Coronavirus 2 (SARS-CoV-2) spike protein has been predicted to bind RGD-recognizing integrins. Recent studies have shown that the spike protein does, indeed, interact with αVß3 and α5ß1 integrins, both of which bind to RGD-containing ligands. However, computational studies have suggested that binding between the spike RGD motif and integrins is not favourable, even when unfolding occurs after conformational changes induced by binding to the canonical host entry receptor, angiotensin-converting enzyme 2 (ACE2). Furthermore, non-RGD-binding integrins, such as αx, have been suggested to interact with the SARS-CoV-2 spike protein. Other viral pathogens, such as rotaviruses, have been recorded to bind integrins in an RGD-independent manner to initiate host cell entry. Thus, in order to consider the potential for the SARS-CoV-2 spike protein to bind integrins independent of the RGD sequence, we investigate several factors related to the involvement of integrins in SARS-CoV-2 infection. First, we review changes in integrin expression during SARS-CoV-2 infection to identify which integrins might be of interest. Then, all known non-RGD integrin-binding motifs are collected and mapped to the spike protein receptor-binding domain and analyzed for their 3D availability. Several integrin-binding motifs are shown to exhibit high sequence similarity with solvent accessible regions of the spike receptor-binding domain. Comparisons of these motifs with other betacoronavirus spike proteins, such as SARS-CoV and RaTG13, reveal that some have recently evolved while others are more conserved throughout phylogenetically similar betacoronaviruses. Interestingly, all of the potential integrin-binding motifs, including the RGD sequence, are conserved in one of the known pangolin coronavirus strains. Of note, the most recently recorded mutations in the spike protein receptor-binding domain were found outside of the putative integrin-binding sequences, although several mutations formed inside and close to one motif, in particular, may potentially enhance binding. These data suggest that the SARS-CoV-2 spike protein may interact with integrins independent of the RGD sequence and may help further explain how SARS-CoV-2 and other viruses can evolve to bind to integrins.

Selectivity of the collagen-binding integrin inhibitors, TC-I-15 and obtustatin.

Integrins are a family of 24 adhesion receptors which are both widely-expressed and important in many pathophysiological cellular processes, from embryonic development to cancer metastasis. Hence, integrin inhibitors are valuable research tools which may have promising therapeutic uses. Here, we focus on the four collagen-binding integrins α1ß1, α2ß1, α10ß1 and α11ß1. TC-I-15 is a small molecule inhibitor of α2ß1 that inhibits platelet adhesion to collagen and thrombus deposition, and obtustatin is an α1ß1-specific disintegrin that inhibits angiogenesis. Both inhibitors were applied in cellular adhesion studies, using synthetic collagen peptide coatings with selective affinity for the different collagen-binding integrins and testing the adhesion of C2C12 cells transfected with each. Obtustatin was found to be specific for α1ß1, as described, whereas TC-I-15 is shown to be non-specific, since it inhibits both α1ß1 and α11ß1 as well as α2ß1. TC-I-15 was 100-fold more potent against α2ß1 binding to a lower-affinity collagen peptide, suggestive of a competitive mechanism. These results caution against the use of integrin inhibitors in a therapeutic or research setting without testing for cross-reactivity.

Identification of HSP47 Binding Site on Native Collagen and Its Implications for the Development of HSP47 Inhibitors.

HSP47 (heat shock protein 47) is a collagen-specific molecular chaperone that is essential for procollagen folding and function. Previous studies have shown that HSP47 binding requires a critical Arg residue at the Y position of the (Gly-Xaa-Yaa) repeats of collagen; however, the exact binding sites of HSP47 on native collagens are not fully defined. To address this, we mapped the HSP47 binding sites on collagens through an ELISA binding assay using collagen toolkits, synthetic collagen peptides covering the entire amino acid sequences of collagen types II and III assembled in triple-helical conformation. Our results showed that HSP47 binds to only a few of the GXR motifs in collagen, with most of the HSP47 binding sites identified located near the N-terminal part of the triple-helical region. Molecular modelling and binding energy calculation indicated that residues flanking the key Arg in the collagen sequence also play an important role in defining the high-affinity HSP47 binding site of collagen. Based on this binding mode of HSP47 to collagen, virtual screening targeting both the Arg binding site and its neighboring area on the HSP47 surface, and a subsequent bioassay, we identified two novel compounds with blocking activity towards HSP47 binding of collagen. Overall, our study revealed the native HSP47 binding sites on collagen and provided novel information for the design of small-molecule inhibitors of HSP47.

Multimerin 1 supports platelet function in vivo and binds to specific GPAGPOGPX motifs in fibrillar collagens that enhance platelet adhesion.

BACKGROUND: Multimerin 1 (human: MMRN1, mouse: Mmrn1) is a homopolymeric, adhesive, platelet and endothelial protein that binds to von Willebrand factor and enhances platelet adhesion to fibrillar collagen ex vivo. OBJECTIVES: To examine the impact of Mmrn1 deficiency on platelet adhesive function, and the molecular motifs in fibrillar collagen that bind MMRN1 to enhance platelet adhesion. METHODS: Mmrn1-deficient mice were generated and assessed for altered platelet adhesive function. Collagen Toolkit peptides, and other triple-helical collagen peptides, were used to identify multimerin 1 binding motifs and their contribution to platelet adhesion. RESULTS: MMRN1 bound to conserved GPAGPOGPX sequences in collagens I, II, and III (including GPAGPOGPI, GPAGPOGPV, and GPAGPOGPQ) that enhanced activated human platelet adhesion to collagen synergistically with other triple-helical collagen peptides (P < .05). Mmrn1-/- and Mmrn1+/- mice were viable and fertile, with complete and partial platelet Mmrn1 deficiency, respectively. Relative to wild-type mice, Mmrn1-/- and Mmrn1+/- mice did not have overt bleeding, increased median bleeding times, or increased wound blood loss (P ≥ .07); however, they both showed significantly impaired platelet adhesion and thrombus formation in the ferric chloride injury model (P ≤ .0003). Mmrn1-/- platelets had impaired adhesion to GPAGPOGPX peptides and fibrillar collagen (P ≤ .03) and formed smaller aggregates than wild-type platelets when captured onto collagen, triple-helical collagen mimetic peptides, von Willebrand factor, or fibrinogen (P ≤ .008), despite preserved, low shear, and high shear aggregation responses. CONCLUSIONS: Multimerin 1 supports platelet adhesion and thrombus formation and binds to highly conserved, GPAGPOGPX motifs in fibrillar collagens that synergistically enhance platelet adhesion.

Tunable bioactivity and mechanics of collagen-based tissue engineering constructs: A comparison of EDC-NHS, genipin and TG2 crosslinkers.

Due to its ubiquity and versatility in the human body, collagen is an ideal base material for tissue-engineering constructs. Chemical crosslinking treatments allow precise control of the biochemical and mechanical properties through macromolecular modifications to the structure of collagen. In this work, three key facets regarding the collagen crosslinking process are explored. Firstly, a comparison is drawn between the carbodiimide-succinimide (EDC-NHS) system and two emerging crosslinkers utilising alternate chemistries: genipin and tissue transglutaminase (TG2). By characterising the chemical changes upon treatment, the effect of EDC-NHS, genipin and TG2 crosslinking mechanisms on the chemical structure of collagen, and thus the mechanical properties conferred to the substrate is explored. Secondly, the relative importance of mechanical and biochemical cues on cellular phenomena are investigated, including cell viability, integrin-specific attachment, spreading and proliferation. Here, we observe that for human dermal fibroblasts, long-term, stable proliferation is preconditioned by the availability of suitable binding sites, irrespective of the substrate modulus post-crosslinking. Finally, as seen in the graphical abstract we show that by choosing the appropriate crosslinker chemistries, a materials selection map can be drawn for collagen films, encompassing both a range of tensile modulus and fibroblast proliferation which can be modified independently. Thus, in addition to a range of parameters that can be modified in collagen constructs, we demonstrate a route to obtaining tunable bioactivity and mechanics in collagen constructs is uncovered, that is exclusively driven by the crosslinking process.

Identification of the cis­molecular neighbours of the immune checkpoint protein B7­H4 in the breast cancer cell­line SK­BR­3 by proteomic proximity labelling.

The immune checkpoint protein B7­H4 plays an important role in the positive as well as the negative regulation of immune T­cell responses. When expressed on cancer cells, B7­H4 inhibits T­cell activity, and numerous types of cancer cells use upregulation of B7­H4 as a survival strategy. Thus, B7­H4 is a potential target for anticancer drug therapy. Unfortunately, the cell biology of this molecule has yet to be fully elucidated. Even basic properties, such as the nature of B7­H4 interactors, are controversial. In particular, the cis­interactors of B7­H4 on cancer cell plasma membranes have not been investigated to date. The present study used a proteomic proximity­labelling assay to investigate the molecular neighbours of B7­H4 on the surface of the human breast cancer cells SK­BR­3. By comparison to a comprehensive proteome analysis of SK­BR­3 cells, the proximity method detected a relatively small number of low abundance plasma membrane proteins highly enriched for proteins known to modulate cell adhesion and immune recognition. It may be inferred that these molecules contribute to the immunosuppressive behaviour that is characteristic of B7­H4 on cancer cells.

Chain alignment of collagen I deciphered using computationally designed heterotrimers.

The most abundant member of the collagen protein family, collagen I (also known as type I collagen; COL1), is composed of one unique (chain B) and two similar (chain A) polypeptides that self-assemble with one amino acid offset into a heterotrimeric triple helix. Given the offset, chain B can occupy either the leading (BAA), middle (ABA) or trailing (AAB) position of the triple helix, yielding three isomeric biomacromolecules with different protein recognition properties. Despite five decades of intensive research, there is no consensus on the position of chain B in COL1. Here, three triple-helical heterotrimers that each contain a putative von Willebrand factor (VWF) and discoidin domain receptor (DDR) recognition sequence from COL1 were designed with chain B permutated in all three positions. AAB demonstrated a strong preference for both VWF and DDR, and also induced higher levels of cellular DDR phosphorylation. Thus, we resolve this long-standing mystery and show that COL1 adopts an AAB register.

Impact of UV- and carbodiimide-based crosslinking on the integrin-binding properties of collagen-based materials.

Collagen constructs are widely used for tissue engineering. These are frequently chemically crosslinked, using EDC, to improve their stability and tailor their physical properties. Although generally biocompatible, chemical crosslinking can modify crucial amino acid side chains, such as glutamic acid, that are involved in integrin-mediated cell adhesion. Instead UV crosslinking modifies aromatic side chains. Here we elucidate the impact that EDC, in combination with UV, exerts on the activity of integrin-binding motifs. By employing a model cell line that exclusively utilises integrin α2ß1, we found that whilst EDC crosslinking modulated cell binding, from cation-dependent to cation-independent, UV-mediated crosslinking preserved native-like cell binding, proliferation and surface colonisation. Similar results were observed using a purified recombinant I-domain from integrin α1. Conversely, binding of the I-domain from integrin α2 was sensitive to UV, particularly at low EDC concentrations. Therefore, from this in vitro study, it appears that UV can be used to augment EDC whist retaining a specific subset of integrin-binding motifs in the native collagen molecule. These findings, delineating the EDC- and UV-susceptibility of cell-binding motifs, permit controlled cell adhesion to collagen-based materials through specific integrin ligation in vitro. However, in vivo, further consideration of the potential response to UV wavelength and dose is required in the light of literature reports that UV initiated collagen scission may lead to an adverse inflammatory response. STATEMENT OF SIGNIFICANCE: Recently, there has been rapid growth in the use of extracellular matrix-derived molecules, and in particular collagen, to fabricate biomaterials that replicate the cellular micro-environment. Often chemical or physical crosslinkers are required to enhance the biophysical properties of these materials. Despite extensive use of these crosslinkers, the cell-biological consequences have not been ascertained. To address this, we have investigated the integrin-binding properties of collagen after chemically crosslinking with EDC and physically crosslinking with UV-irradiation. We have established that whilst EDC crosslinking abates all of the integrin binding sites in collagen, UV selectively inhibits interaction with integrin-α2 but not -α1. By providing a mechanistic model for this behaviour, we have, for the first time, defined a series of crosslinking parameters to systematically control the interaction of collagen-based materials with defined cellular receptors.

MMP-13 binds to platelet receptors αIIbß3 and GPVI and impairs aggregation and thrombus formation.

BACKGROUND: Acute thrombotic syndromes lead to atherosclerotic plaque rupture with subsequent thrombus formation, myocardial infarction and stroke. Following rupture, flowing blood is exposed to plaque components, including collagen, which triggers platelet activation and aggregation. However, plaque rupture releases other components into the surrounding vessel which have the potential to influence platelet function and thrombus formation. OBJECTIVES: Here we sought to elucidate whether matrix metalloproteinase-13 (MMP-13), a collagenolytic metalloproteinase up-regulated in atherothrombotic and inflammatory conditions, affects platelet aggregation and thrombus formation. RESULTS: We demonstrate that MMP-13 is able to bind to platelet receptors alphaIIbbeta3 (αIIbß3) and platelet glycoprotein (GP)VI. The interactions between MMP-13, GPVI and αIIbß3 are sufficient to significantly inhibit washed platelet aggregation and decrease thrombus formation on fibrillar collagen. CONCLUSIONS: Our data demonstrate a role for MMP-13 in the inhibition of both platelet aggregation and thrombus formation in whole flowing blood, and may provide new avenues of research into the mechanisms underlying the subtle role of MMP-13 in atherothrombotic pathologies.

Fundamental insight into the effect of carbodiimide crosslinking on cellular recognition of collagen-based scaffolds.

Research on the development of collagen constructs is extremely important in the field of tissue engineering. Collagen scaffolds for numerous tissue engineering applications are frequently crosslinked with 1-ethyl-3-(3-dimethylaminopropyl-carbodiimide hydrochloride (EDC) in the presence of N-hydroxy-succinimide (NHS). Despite producing scaffolds with good biocompatibility and low cellular toxicity the influence of EDC/NHS crosslinking on the cell interactive properties of collagen has been overlooked. Here we have extensively studied the interaction of model cell lines with collagen I-based materials after crosslinking with different ratios of EDC in relation to the number of carboxylic acid residues on collagen. Divalent cation-dependent cell adhesion, via integrins α1ß1, α2ß1, α10ß1 and α11ß1, were sensitive to EDC crosslinking. With increasing EDC concentration, this was replaced with cation-independent adhesion. These results were replicated using purified recombinant I domains derived from integrin α1 and α2 subunits. Integrin α2ß1-mediated cell spreading, apoptosis and proliferation were all heavily influenced by EDC crosslinking of collagen. Data from this rigorous study provides an exciting new insight that EDC/NHS crosslinking is utilising the same carboxylic side chain chemistry that is vital for native-like integrin-mediated cell interactions. Due to the ubiquitous usage of EDC/NHS crosslinked collagen for biomaterials fabrication this data is essential to have a full understanding in order to ensure optimized collagen-based material performance. STATEMENT OF SIGNIFICANCE: Carbodiimide stabilised collagen is employed extensively for the fabrication of biologically active materials. Despite this common usage, the effect of carbodiimide crosslinking on cell-collagen interactions is unclear. Here we have found that carbodiimide crosslinking of collagen inhibits native-like, whilst increasing non-native like, cellular interactions. We propose a mechanistic model in which carbodiimide modifies the carboxylic acid groups on collagen that are essential for cell binding. As such we feel that this research provides a crucial, long awaited, insight into the bioactivity of carbodiimide crosslinked collagen. Through the ubiquitous use of collagen as a cellular substrate we feel that this is fundamental to a wide range of research activity with high impact across a broad range of disciplines.

Unique charge-dependent constraint on collagen recognition by integrin α10ß1.

The collagen-binding integrins recognise collagen through their inserted (I) domain, where co-ordination of a Mg2+ ion in the metal ion-dependent site is reorganised by ligation by a collagen glutamate residue found in specific collagen hexapeptide motifs. Here we show that GROGER, found in the N-terminal domain of collagens I and III, is only weakly recognised by α10ß1, an important collagen receptor on chondrocytes, contrasting with the other collagen-binding integrins. Alignment of I domain sequence and molecular modelling revealed a clash between a unique arginine residue (R215) in α10ß1 and the positively-charged GROGER. Replacement of R215 with glutamine restored binding. Substituting arginine at the equivalent locus (Q214) in integrins α1 and α2 I domains impaired their binding to GROGER. Collagen II, abundant in cartilage, lacks GROGER. GRSGET is uniquely expressed in the C-terminus of collagen II, but this motif is similarly not recognised by α10ß1. These data suggest an evolutionary imperative to maintain accessibility of the terminal domains of collagen II in tissues such as cartilage, perhaps during endochondral ossification, where α10ß1 is the main collagen-binding integrin.

Intrinsic local destabilization of the C-terminus predisposes integrin α1 I domain to a conformational switch induced by collagen binding.

Integrin-collagen interactions play a critical role in a myriad of cellular functions that include immune response, and cell development and differentiation, yet their mechanism of binding is poorly understood. There is increasing evidence that conformational flexibility assumes a central role in the molecular mechanisms of protein-protein interactions and here we employ NMR hydrogen-deuterium exchange (HDX) experiments to explore the impact of slower timescale dynamic events. To gain insight into the mechanisms underlying collagen-induced conformational switches, we have undertaken a comparative study between the wild type integrin α1 I and a gain-of-function E317A mutant. NMR HDX results suggest a relationship between regions exhibiting a reduced local stability in the unbound I domain and those that undergo significant conformational changes upon binding. Specifically, the αC and α7 helices within the C-terminus are at the center of such major perturbations and present reduced local stabilities in the unbound state relative to other structural elements. Complementary isothermal titration calorimetry experiments have been performed to derive complete thermodynamic binding profiles for association of the collagen-like triple-helical peptide with wild type α1 I and E317A mutant. The differential energetics observed for E317A are consistent with the HDX experiments and support a model in which intrinsically destabilized regions predispose conformational rearrangement in the integrin I domain. This study highlights the importance of exploring different timescales to delineate allosteric and binding events.

The synthesis and coupling of photoreactive collagen-based peptides to restore integrin reactivity to an inert substrate, chemically-crosslinked collagen.

Collagen is frequently advocated as a scaffold for use in regenerative medicine. Increasing the mechanical stability of a collagen scaffold is widely achieved by cross-linking using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS). However, this treatment consumes the carboxylate-containing amino acid sidechains that are crucial for recognition by the cell-surface integrins, abolishing cell adhesion. Here, we restore cell reactivity to a cross-linked type I collagen film by covalently linking synthetic triple-helical peptides (THPs), mimicking the structure of collagen. These THPs are ligands containing an active cell-recognition motif, GFOGER, a high-affinity binding site for the collagen-binding integrins. We end-stapled peptide strands containing GFOGER by coupling a short diglutamate-containing peptide to their N-terminus, improving the thermal stability of the resulting THP. A photoreactive Diazirine group was grafted onto the end-stapled THP to allow covalent linkage to the collagen film upon UV activation. Such GFOGER-derivatized collagen films showed restored affinity for the ligand-binding I domain of integrin α2ß1, and increased integrin-dependent cell attachment and spreading of HT1080 and Rugli cell lines, expressing integrins α2ß1 and α1ß1, respectively. The method we describe has wide application, beyond collagen films or scaffolds, since the photoreactive diazirine will react with many organic carbon skeletons.

Optimisation of UV irradiation as a binding site conserving method for crosslinking collagen-based scaffolds.

Short wavelength (λ = 254 nm) UV irradiation was evaluated over a range of intensities (0.06 to 0.96 J/cm(2)) as a means of cross-linking collagen- and gelatin-based scaffolds, to tailor their material characteristics whilst retaining biological functionality. Zero-link carbodiimide treatments are commonly applied to collagen-based materials, forming cross-links from carboxylate anions (for example the acidic E of GFOGER) that are an essential part of integrin binding sites on collagen. Cross-linking these amino acids therefore disrupts the bioactivity of collagen. In contrast, UV irradiation forms bonds from less important aromatic tyrosine and phenylalanine residues. We therefore hypothesised that UV cross-linking would not compromise collagen cell reactivity. Here, highly porous (~99 %) isotropic, collagen-based scaffolds were produced via ice-templating. A series of scaffolds (pore diameters ranging from 130-260 µm) with ascending stability in water was made from gelatin, two different sources of collagen I, or blends of these materials. Glucose, known to aid UV crosslinking of collagen, was added to some lower-stability formulations. These scaffolds were exposed to different doses of UV irradiation, and the scaffold morphology, dissolution stability in water, resistance to compression and cell reactivity was assessed. Stabilisation in aqueous media varied with both the nature of the collagen-based material employed and the UV intensity. Scaffolds made from the most stable materials showed the greatest stability after irradiation, although the levels of cross-linking in all cases were relatively low. Scaffolds made from pure collagen from the two different sources showed different optimum levels of irradiation, suggesting altered balance between stabilisation from cross-linking and destabilisation from denaturation. The introduction of glucose into the scaffold enhanced the efficacy of UV cross-linking. Finally, as hypothesized, cell attachment, spreading and proliferation on collagen materials were unaffected by UV cross-linking. UV irradiation may therefore be used to provide relatively low level cross-linking of collagen without loss of biological functionality.

The recognition of collagen and triple-helical toolkit peptides by MMP-13: sequence specificity for binding and cleavage.

Remodeling of collagen by matrix metalloproteinases (MMPs) is crucial to tissue homeostasis and repair. MMP-13 is a collagenase with a substrate preference for collagen II over collagens I and III. It recognizes a specific, well-known site in the tropocollagen molecule where its binding locally perturbs the triple helix, allowing the catalytic domain of the active enzyme to cleave the collagen α chains sequentially, at Gly(775)-Leu(776) in collagen II. However, the specific residues upon which collagen recognition depends within and surrounding this locus have not been systematically mapped. Using our triple-helical peptide Collagen Toolkit libraries in solid-phase binding assays, we found that MMP-13 shows little affinity for Collagen Toolkit III, but binds selectively to two triple-helical peptides of Toolkit II. We have identified the residues required for the adhesion of both proMMP-13 and MMP-13 to one of these, Toolkit peptide II-44, which contains the canonical collagenase cleavage site. MMP-13 was unable to bind to a linear peptide of the same sequence as II-44. We also discovered a second binding site near the N terminus of collagen II (starting at helix residue 127) in Toolkit peptide II-8. The pattern of binding of the free hemopexin domain of MMP-13 was similar to that of the full-length enzyme, but the free catalytic subunit bound none of our peptides. The susceptibility of Toolkit peptides to proteolysis in solution was independent of the very specific recognition of immobilized peptides by MMP-13; the enzyme proved able to cleave a range of dissolved collagen peptides.

New insights into the DT40 B cell receptor cluster using a proteomic proximity labeling assay.

In the vertebrate immune system, each B-lymphocyte expresses a surface IgM-class B cell receptor (BCR). When cross-linked by antigen or anti-IgM antibody, the BCR accumulates with other proteins into distinct surface clusters that activate cell signaling, division, or apoptosis. However, the molecular composition of these clusters is not well defined. Here we describe a quantitative assay we call selective proteomic proximity labeling using tyramide (SPPLAT). It allows proteins in the immediate vicinity of a target to be selectively biotinylated, and hence isolated for mass spectrometry analysis. Using the chicken B cell line DT40 as a model, we use SPPLAT to provide the first proteomic analysis of any BCR cluster using proximity labeling. We detect known components of the BCR cluster, including integrins, together with proteins not previously thought to be BCR-associated. In particular, we identify the chicken B-lymphocyte allotypic marker chB6. We show that chB6 moves to within about 30-40 nm of the BCR following BCR cross-linking, and we show that cross-linking chB6 activates cell binding to integrin substrates laminin and gelatin. Our work provides new insights into the nature and composition of the BCR cluster, and confirms SPPLAT as a useful research tool in molecular and cellular proteomics.