Abstract Solid‐state NMR‐based structure determination of membrane proteins and large protein complexes faces the challenge of limited spectral resolution when the proteins are uniformly 13 C‐labeled. A strategy to meet this challenge is chemical ligation combined with site‐specific or segmental labeling. While chemical ligation has been adopted in NMR studies of water‐soluble proteins, it has not been demonstrated for membrane proteins. Here we show chemical ligation of the influenza M2 protein, which contains a transmembrane (TM) domain and two extra‐membrane domains. The cytoplasmic domain, which contains an amphipathic helix (AH) and a cytoplasmic tail, is important for regulating virus assembly, virus budding, and the proton channel activity. A recent study of uniformly 13 C‐labeled full‐length M2 by spectral simulation suggested that the cytoplasmic tail is unstructured. To further test this hypothesis, we conducted native chemical ligation of the TM segment and part of the cytoplasmic domain. Solid‐phase peptide synthesis of the two segments allowed several residues to be labeled in each segment. The post‐AH cytoplasmic residues exhibit random‐coil chemical shifts, low bond order parameters, and a surface‐bound location, thus indicating that this domain is a dynamic random coil on the membrane surface. Interestingly, the protein spectra are similar between a model membrane and a virus‐mimetic membrane, indicating that the structure and dynamics of the post‐AH segment is insensitive to the lipid composition. This chemical ligation approach is generally applicable to medium‐sized membrane proteins to provide site‐specific structural constraints, which complement the information obtained from uniformly 13 C, 15 N‐labeled proteins.
The M2 protein of influenza A viruses forms a tetrameric proton channel that is targeted by the amantadine class of antiviral drugs. A S31N mutation in the transmembrane (TM) domain of the protein has caused widespread amantadine resistance in most of the currently circulating flu viruses. Recently, a new family of compounds based on amantadine- and aryl-substituted isoxazole were discovered to inhibit the S31N channel activity and reduce replication of S31N-harboring viruses. We now use solid-state NMR spectroscopy to investigate the effects of one of these isoxazole compounds, WJ352, on the conformation of the S31N TM segment and the dynamics of the proton-selective residue, His37. Chemical shift perturbations show that WJ352 changes the conformational equilibrium of multiple TM residues, with the maximal perturbation occurring at the crucial Asn31. (13)C-(2)H distance measurements and (1)H-(1)H NOE cross peaks indicate that the adamantane moiety of the drug is bound in the spacious pore between Asn31 and Gly34 while the phenyl tail is located near Val27. Thus, the polar amine points to the channel exterior rather than to His37, in contrast to amantadine and rimantadine in the wild-type channel, suggesting that the drug is significantly stabilized by hydrophobic interactions between the adamantane and the TM peptide. (15)N and (13)C chemical shifts indicate that at low pH, His37 undergoes fast exchange among the τ tautomer, the π tautomer, and the cationic state due to proton transfer with water. The exchange rate is higher than the wild-type channel, consistent with the larger single-channel conductance of the mutant. Drug binding at acidic pH largely suppresses this exchange, reverting the histidines to a similar charge distribution as that of the high-pH closed state.
Article15 February 2021Open Access Transparent process CA10 regulates neurexin heparan sulfate addition via a direct binding in the secretory pathway Laia Montoliu-Gaya Laia Montoliu-Gaya orcid.org/0000-0001-7684-6318 Department of Laboratory Medicine, Institute for Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden Wallenberg Centre for Molecular and Translational Medicine, University of Gothenburg, Gothenburg, Sweden Search for more papers by this author Daniel Tietze Daniel Tietze orcid.org/0000-0002-9251-1902 Wallenberg Centre for Molecular and Translational Medicine, University of Gothenburg, Gothenburg, Sweden Department of Chemistry and Molecular Biology, Faculty of Science, University of Gothenburg, Gothenburg, Sweden Search for more papers by this author Debora Kaminski Debora Kaminski Department of Laboratory Medicine, Institute for Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden Wallenberg Centre for Molecular and Translational Medicine, University of Gothenburg, Gothenburg, Sweden Department of Clinical Chemistry, Sahlgrenska University Hospital, Gothenburg, Sweden Search for more papers by this author Ekaterina Mirgorodskaya Ekaterina Mirgorodskaya Proteomics Core Facility, University of Gothenburg, Gothenburg, Sweden Search for more papers by this author Alesia A Tietze Alesia A Tietze Wallenberg Centre for Molecular and Translational Medicine, University of Gothenburg, Gothenburg, Sweden Department of Chemistry and Molecular Biology, Faculty of Science, University of Gothenburg, Gothenburg, Sweden Search for more papers by this author Fredrik H Sterky Corresponding Author Fredrik H Sterky [email protected] orcid.org/0000-0001-8881-0523 Department of Laboratory Medicine, Institute for Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden Wallenberg Centre for Molecular and Translational Medicine, University of Gothenburg, Gothenburg, Sweden Department of Clinical Chemistry, Sahlgrenska University Hospital, Gothenburg, Sweden Search for more papers by this author Laia Montoliu-Gaya Laia Montoliu-Gaya orcid.org/0000-0001-7684-6318 Department of Laboratory Medicine, Institute for Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden Wallenberg Centre for Molecular and Translational Medicine, University of Gothenburg, Gothenburg, Sweden Search for more papers by this author Daniel Tietze Daniel Tietze orcid.org/0000-0002-9251-1902 Wallenberg Centre for Molecular and Translational Medicine, University of Gothenburg, Gothenburg, Sweden Department of Chemistry and Molecular Biology, Faculty of Science, University of Gothenburg, Gothenburg, Sweden Search for more papers by this author Debora Kaminski Debora Kaminski Department of Laboratory Medicine, Institute for Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden Wallenberg Centre for Molecular and Translational Medicine, University of Gothenburg, Gothenburg, Sweden Department of Clinical Chemistry, Sahlgrenska University Hospital, Gothenburg, Sweden Search for more papers by this author Ekaterina Mirgorodskaya Ekaterina Mirgorodskaya Proteomics Core Facility, University of Gothenburg, Gothenburg, Sweden Search for more papers by this author Alesia A Tietze Alesia A Tietze Wallenberg Centre for Molecular and Translational Medicine, University of Gothenburg, Gothenburg, Sweden Department of Chemistry and Molecular Biology, Faculty of Science, University of Gothenburg, Gothenburg, Sweden Search for more papers by this author Fredrik H Sterky Corresponding Author Fredrik H Sterky [email protected] orcid.org/0000-0001-8881-0523 Department of Laboratory Medicine, Institute for Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden Wallenberg Centre for Molecular and Translational Medicine, University of Gothenburg, Gothenburg, Sweden Department of Clinical Chemistry, Sahlgrenska University Hospital, Gothenburg, Sweden Search for more papers by this author Author Information Laia Montoliu-Gaya1,2, Daniel Tietze2,3, Debora Kaminski1,2,4, Ekaterina Mirgorodskaya5, Alesia A Tietze2,3 and Fredrik H Sterky *,1,2,4 1Department of Laboratory Medicine, Institute for Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden 2Wallenberg Centre for Molecular and Translational Medicine, University of Gothenburg, Gothenburg, Sweden 3Department of Chemistry and Molecular Biology, Faculty of Science, University of Gothenburg, Gothenburg, Sweden 4Department of Clinical Chemistry, Sahlgrenska University Hospital, Gothenburg, Sweden 5Proteomics Core Facility, University of Gothenburg, Gothenburg, Sweden *Corresponding author. Tel: +46 31 7865357; E-mail: [email protected] EMBO Reports (2021)22:e51349https://doi.org/10.15252/embr.202051349 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Neurexins are presynaptic adhesion molecules that shape the molecular composition of synapses. Diversification of neurexins in numerous isoforms is believed to confer synapse-specific properties by engaging with distinct ligands. For example, a subset of neurexin molecules carry a heparan sulfate (HS) glycosaminoglycan that controls ligand binding, but how this post-translational modification is controlled is not known. Here, we observe that CA10, a ligand to neurexin in the secretory pathway, regulates neurexin-HS formation. CA10 is exclusively found on non-HS neurexin and CA10 expressed in neurons is sufficient to suppress HS addition and attenuate ligand binding and synapse formation induced by ligands known to recruit HS. This effect is mediated by a direct interaction in the secretory pathway that blocks the primary step of HS biosynthesis: xylosylation of the serine residue. NMR reveals that CA10 engages residues on either side of the serine that can be HS-modified, suggesting that CA10 sterically blocks xylosyltransferase access in Golgi. These results suggest a mechanism for the regulation of HS on neurexins and exemplify a new mechanism to regulate site-specific glycosylations. Synopsis CA10 binds to neurexins in the secretory pathway and blocks the addition of heparan sulfate to neurexins, which affects their ligand-binding properties. These findings illustrate a new mechanism to regulate site-specific glycosylations and a means to further diversify neurexin isoforms. Binding of the secreted protein CA10 to neurexins in the secretory pathway blocks the addition of heparan sulfate (HS) to neurexins. A direct interaction between CA10 and neurexin residues near the glycosylated serine prevents the primary step of HS biosynthesis: xylosylation of the serine residue. Introduction The flow of information through the circuits of our brain depends on synaptic connections that relay signals between interconnected neurons. While all synapses share a set of common features—such as a presynaptic vesicle release machinery juxtaposed to appropriate receptors on the postsynaptic membrane—structural and functional diversity allows for differential information processing and plasticity at the level of individual synapses (Grant & O'Dell, 2001; Sheng & Kim, 2011; Südhof, 2013). The properties of a synapse depend on the composition and organization of its molecular building blocks. Synaptic cell adhesion molecules and their trans-synaptic interactions are believed to be major determinants of this molecular architecture by coordinating the recruitment and assembly of components on either side of the synaptic cleft (de Wit & Ghosh, 2016; Rudenko, 2017; Sudhof, 2018). Neurexins are a major class of presynaptic cell adhesion molecules (Reissner et al, 2013; Sudhof, 2017; Rudenko, 2019), which are genetically linked to multiple psychiatric and neuropsychiatric diseases, including schizophrenia, intellectual disability and Tourette's syndrome (Schaaf et al, 2012; Rees et al, 2014; Huang et al, 2017; Kasem et al, 2018). Neurexins are widely expressed in the nervous system by three genes (NRXN1-3) that each expresses both larger ɑ- and shorter β-neurexins (Ushkaryov et al, 1992; Ushkaryov et al, 1994; Reissner et al, 2013), with NRXN1 also encoding as a small γ-neurexin isoform (Yan et al, 2015; Sterky et al, 2017). α-Neurexins carry on their extracellular part six LNS (laminin-NRXN-sex hormone binding globulin) and three EGF (epidermal growth factor-like) domains, while β-neurexins contain only a single LNS domain and Nrxn1γ lacks folded extracellular domains. However, all isoforms encompass a glycosylated "stalk" region containing a conserved cysteine-loop (Cys-loop) (Gokce & Sudhof, 2013), followed by a transmembrane domain and a relatively short intracellular sequence ending with a PDZ-binding motif (Hata et al, 1996). Extensive research has identified more than 20 structurally diverse ligands that may participate in neurexin complexes by direct interactions (reviewed in Sudhof, 2017; Rudenko, 2019). The carbonic anhydrase (CA)-related protein CA10 constitutes a special type of neurexin ligand by binding robustly and stoichiometrically only when expressed in the same presynaptic neuron (i.e., in cis), suggesting that the interaction forms in the secretory pathway. Indeed, a chaperone-like function is suggested by the finding that overexpressed CA10 could increase neurexin surface levels (Sterky et al, 2017). CA10 and its homologue, CA11, are secreted proteins that each contain an enzymatically inactive CA domain (Lovejoy et al, 1998; Okamoto et al, 2001), similar to the extracellular CA-like domains of the tyrosine phosphatase receptors R-PTPγ/PTPRG and R-PTPζ/PTPRZ1 (Krueger & Saito, 1992; Barnea et al, 1993). However, the CA-like domain of CA10/11 is followed by a unique ~ 25 residues long C-terminal "tail" that contains a single conserved cysteine. This cysteine can form an intermolecular disulfide between CA10 and the N-terminal of the two cysteines in the neurexin Cys-loop (Sterky et al, 2017). As CA10 is expressed in subsets of neurons, with highest expression found in cerebellum (Aspatwar et al, 2010), it may serve to regulate neurexin complexes in specific cell types. CA10 has been found to be important for normal development in Zebrafish (Aspatwar et al, 2015) and has also been shown to suppress glioma growth by an unknown mechanism (Tao et al, 2019). However, the exact roles of CA10 during normal development and as part of neurexin complexes remain unknown. Molecular diversity of neurexin isoforms contributes to functional specialization of specific synapses. For example, alternative splicing at six conserved sites (SS1-6), generates hundreds—possibly a thousand—of unique neurexin transcripts (Treutlein et al, 2014; Schreiner et al, 2014). Alternative splicing at a single site (splice site 4; SS4) is sufficient to regulate postsynaptic receptor responses in an isoform-dependent manner (Aoto et al, 2013; Dai et al, 2019). Further diversification arises from glycosylation. Recent work has shown that a substantial fraction of neurexins (70–80% in mouse brains) carry a heparan sulfate (HS) glycosaminoglycan (GAG) chain (Zhang et al, 2018). The HS-modified serine is conserved among neurexins and present in ɑ-, β-, and γ-neurexin isoforms, which all can carry this post-translational modification (Zhang et al, 2018; Roppongi et al, 2020). The HS chain has been shown to cooperate in the protein–protein interactions between neurexins and postsynaptic neuroligins (Nlgns) and leucine-rich repeat transmembrane neuronal protein 2 (LRRTM2) to enhance and/or stabilize these interactions (Zhang et al, 2018). The HS chain can also recruit additional HS-binding proteins to neurexin complexes, for example postsynaptic LRRTM4 (Roppongi et al, 2020). Whether it may also recruit secreted proteins that influence synapse formation (Yuzaki, 2018), for example, growth factors and signaling molecules known to bind other HSPGs (Esko & Selleck, 2002; Xie & Li, 2019), remains unknown. While more remains to be learned about this modification, it is clearly important for at least some of the synaptic functions of neurexins. For example, mice that lack HS on Nrxn1 show structural and functional impairments of hippocampal mossy fiber-CA3 synapses (Zhang et al, 2018). Biosynthesis of HS begins with the conjugation of a xylose residue to a serine on the core protein by O-xylosyltransferase activity, accounted for in vertebrates by one of two xylosyltransferases (XYLT1/2) (Esko & Selleck, 2002; Briggs & Hohenester, 2018). Additional glycosyltransferases in turn attach two galactose and one glucuronic acid sugars to form the tetrasaccharide core structure that is shared between all GAGs and is the starting point for further chain elongation (Kreuger & Kjellén, 2012). In this process, alternating glucuronic acid (GlcA) and N-acetylglucosamine (GlcNAc) residues are added, resulting in a 40–100 residues linear polysaccharide chain. Different enzymatic modifications, including epimerization, deacetylation and sulfation give rise to the mature chain and functionally specialized segments. For example, highly sulfated regions preferentially engage with specific interacting proteins (Xu & Esko, 2014). How the neurexin HS chain may be modified and how this relates to its function at the synapse is not known. Also unknown is whether the neurexin HS chain is ubiquitous to all neurexins or regulated to be expressed only in select cell types or at specific synapses. In this work, we observe that the addition of HS to neurexin can be regulated by CA10. We find that CA10 can block the neurexin HS addition by directly binding to neurexin before HS en route in the secretory pathway. Resulting non-HS neurexin showed reduced binding to LRRTM2 and capacity for Nlgn1-mediated synapse formation, demonstrating that CA10 can modify synaptic properties of neurexins. Localized protein–protein interactions within the secretory pathway, such as that between CA10 and neurexin, exemplifies a cell-biological mechanism able to directly control substrate-specific glycosylation without affecting global proteoglycan biosynthesis. Results Neurexin HS addition depends on residues within its Cys-loop To learn more about neurexin and its heparan sulfate (HS), we analyzed the sequence context required for this post-translational modification. We used a set of secreted, Fc-tagged neurexin-1β variants with different mutations in residues surrounding the HS-modified serine residue (Fig 1A). The proteins were expressed in HEK293 cells and purified from the media using protein A beads. The samples were subjected to on-bead digestion with heparinases and analyzed by immunoblotting under reducing conditions. Heparinase treatment did not result in detectable size shifts, consistent with the previous observations that only a minor fraction of neurexin expressed in HEK293 cells contain HS (Zhang et al, 2018). Instead, we relied on a monoclonal antibody which detects the HS "stub" that remains after heparinase digestion [3G10 epitope; (David et al, 1992)] and that allowed us to detect species that carry HS against the background of non-HS species (Fig 1B). Using this assay, we found that wild-type neurexin-1 contained HS as expected, but not a negative control in which the modified serine was mutated (Fig 1B and C). The serine is located just N-terminal of a Cys-loop (Gokce & Sudhof, 2013) that is predicted to form between two conserved cysteines flanking a stretch of negatively charged residues. Deletion of the entire Cys-loop (ΔCysL) or the acidic residues within it (CysL>G, CysL>R) blocked HS addition. However, mutating both cysteines (CysL C>A) to prevent the loop to form had no effect on HS addition, suggesting a requirement for residues within the loop rather than the loop itself. Moreover, we found that the leucine–valine residues (DILV) N-terminal of the HS-modified serine could be mutated to glycines (GIGG), but not to positively charged arginines (DIRR) (Fig 1B and C). We observed a similar tolerance for glycines N-terminal of the serine when testing variants of neurexin-3β (Fig EV1-EV5). Figure 1. Analysis of Nrxn1 sequence determinants required for HS addition Sequences of secreted Fc-tagged Nrxn1β variants analyzed. Mutated residues are shown in red, the HS-modified serine in green, and cysteines forming the Cys-loop in blue. Right column ('HS?') summarizes the results from (B, C). Asterisks, colons, and periods indicate fully, strongly, or weakly conserved residues, respectively. Representative immunoblot of Nrxn1β-Fc variants harvested from HEK293 cell media and subjected to heparinase treatment ('Heps'). Samples were analyzed by reducing SDS–PAGE and immunoblotting with antibodies against the Fc tag and the 3G10 epitope (to detect the HS stub which reveal HS after heparinase digestion). Quantification of 3G10 (reflecting HS) signal normalized to that of Fc (Nrxn1). Bar graph shows means ± SEM of 3 independent experiments. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Analysis of Nrxn3 sequences required for its HS addition (related to Fig 1) Sequences of secreted Fc-tagged Nrxn3β variants used. Mutated residues are shown in red, the HS-modified serine in green, and cysteines forming the Cys-loop in blue. Asterisks, colons, and periods indicate fully, strongly, or weakly conserved residues, respectively. Right column ('HS?') summarizes the results from (B, C). Representative immunoblot of Nrxn3β-Fc variants harvested from HEK293 cell media and subjected to heparinase treatment ("Heps"). Samples were analyzed by reducing SDS–PAGE and immunoblotting with antibodies against the Fc tag (Nrxn) and the 3G10 epitope (for the HS stub). Quantification of 3G10 (reflecting HS) signal normalized to that of Fc (Nrxn). Bar graph shows means ± SEM of 3 independent experiments. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Heparinase digestion and detection of the HS stub is not inhibited by CA10 and prevention of HS addition by CA10 is specific for neurexins (related to Fig 2) Fc-tagged Nrxn1β was captured on protein A beads and digested with heparinases ("Heps") with or without recombinant V5-tagged CA10 (3.5 μM final concentration). Samples were analyzed by SDS–PAGE under reducing conditions, followed by immunoblotting against the Fc tag and the HS stub (3G10). Fc-tagged Nrxn1β expressed in HEK293 cells, alone or together with CA10-V5, were captured on protein A beads and treated with heparinases ("Heps") in the presence of indicated concentrations of DTT (to dissociate the Nrxn1β-CA10 complex). Samples were analyzed by SDS–PAGE under non-reducing conditions, followed by immunoblotting against the Fc tag and the HS stub (3G10). DTT at a concentration of 20 mM fully dissociated the CA10- Nrxn1β complex without inhibiting heparinase activities. HA-tagged GPC1 (left) or HA-Nrxn1α, processed in parallel as a control (right), were expressed alone or together with V5-tagged CA10 in HEK293 cells, immunoprecipitated for HA and subjected to heparinase treatment ("Heps") to reveal HS-carrying isoforms (similar to the experiment outlined in Fig 2A). Samples were analyzed by immunoblotting with antibodies against HA, the 3G10 epitope (for the HS stub) and V5. Non-HS GPC1 is marked by an arrowhead, while HS-containing GPC1 is not visible prior to heparinase treatment (Wen et al, 2014). Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Characterization of the Nrxn1-CA10 antiserum (related to Fig 3) Soluble Nrxn1γ -Fc, CA10-V5 and both were expressed in HEK293 cells. Media was analyzed by non-reducing SDS–PAGE and blotted with "Hadlai" antiserum raised against recombinant covalent Nrxn1γ-CA10 complex or pre-immune serum from the same rabbit. The antiserum recognizes CA10 and the Nrxn1-CA10 complex, but not Nrxn1γ alone. Download figure Download PowerPoint Click here to expand this figure. Figure EV4. Analytical characterization of recombinant CA10 and the Nrxn1 stalk peptide (related to Fig 4) Analytical HPLC chromatogram of the purified Nrxn1 stalk peptide. ESI mass spectrum of the purified Nrxn1 stalk peptide. The calculated average mass is 3,237.35 [M + H]+. Coomassie-stained gel of recombinant HIS-FLAG-tagged CA10, separated under reducing conditions. Download figure Download PowerPoint Click here to expand this figure. Figure EV5. MS/MS spectra of peptides from recombinant Nrxn1 expressed with or without CA10 (related to Fig 5) A, B. SDS–PAGE gel stained for (A) total protein and (B) immunoblot of the replicate experiments used for MS/MS analysis. C. Fragment spectrum of the unmodified DDILVASAECPSDDE peptide, precursor at m/z 818.338 ([M + 2H]2+). D. Fragment spectrum of the DDILVASAECPSDDE+ HexNAc, precursor at m/z 919.878 ([M + 2H]2+). E. Fragment spectrum of the DDILVASAECPSDDE+ HexNAcHex, precursor at m/z 1,000.904 ([M + 2H]2+). F. Fragment spectrum of the DDILVASAECPSDDE+ XylGalGalGlcA-H2O, precursor at m/z 1,125.423 ([M + 2H]2+). G. Table with theoretical MH and retention times (RT) of each glycopeptide, and % total occupancy of each replicate in the control and CA10 samples (values represented in Fig 5D). Data information: All fragment spectra (C–F) display high similarity with the same major peptide fragments being observed, confirming the peptide identities. The O-glycosylated peptides further displayed oxonium ions associated with mucin-type O-GalNAc glycosylation. Download figure Download PowerPoint The conserved leucine–valine residues in positions −3 and −2 relative to the HS-modified serine, as well as the cysteine in position +3, are both required for the binding of CA10 to neurexins (Sterky et al, 2017). This observation, together with the finding that exogenous CA10 expression dramatically shifts neurexin isoform distribution in neurons (Sterky et al, 2017), prompted us to investigate whether CA10 may play a role in regulating the addition of HS to neurexin. CA10 blocks addition of HS to neurexin To assess whether CA10 had an effect on neurexin HS, we expressed hemagglutinin (HA)-tagged Nrxn1α alone or in combination with V5-tagged CA10 in HEK293 cells. Deletion of the modified serine (S > A) was used as a negative control. We then immunoprecipitated neurexin from cell lysates and detected species carrying HS using the monoclonal antibody 3G10, after treatment with heparinases (Fig 2A). We readily detected HS-carrying neurexin in cells that expressed wildtype Nrxn1α alone, but not the (S > A) mutant (Fig 2B). However, no HS-carrying neurexin could be detected in cells that also expressed CA10, suggesting that CA10 completely blocked the formation of HS-carrying species. To further corroborate that CA10-bound neurexin did not carry HS, we immunoprecipitated CA10 by means of its V5-tag and analyzed the co-immunoprecipitated Nrxn1α (Fig 2C). As expected, no HS-carrying Nrxn1α was detected in the complex (Fig 2D). Figure 2. CA10 prevents HS addition to neurexin A–E. HEK293 cells: (A) Schematic outline of the experiment shown in (B). HA-Nrxn1α or HA-Nrxn1α (S > A), a mutant of the modified serine were expressed alone or in combination with V5-tagged CA10 in HEK293 cells. Neurexins were immunoprecipitated for HA and treated with heparinases ('Heps'). (B) Representative immunoblots (of three independent experiments) with antibodies against HA, the 3G10 epitope (for the HS stub) and V5. (C) Schematic outline of the experiment shown in (D). HA-Nrxn1α was expressed alone or in combination with CA10-V5 in HEK293 cells, lysates were immunoprecipitated for HA (for Nrxn1α) or V5 (for CA10). (D) Representative immunoblots (of three independent experiments) analyzed as described for (B). (E) Schematic illustration of how endogenous neurexin HS was analyzed. HS-modified α-neurexin migrates at higher apparent molecular weight (HMW) than non-modified low molecular weight (LMW; white arrowheads) species (left). Following treatment with heparinases (right), both HS-modified and non-modified α-neurexin migrates as LMW species, but HS-modified α-neurexin can be detected by the 3G10 monoclonal. F–H. Primary cortical neurons: Cortical neurons were infected with control lentivirus (empty vector) or lentivirus expressing FLAG-tagged CA10, and cell lysates subjected to immunoprecipitation for neurexin and heparinase treatment. (F) Samples were analyzed by immunoblotting using antibodies against neurexins, 3G10 (for the HS stub) and FLAG (for CA10). Stars indicate non-neurexin bands (see Results). (G) Quantification of low molecular weight neurexin (LMW; white arrowhead) in relation to total neurexin amounts [upper band (line) + LMW]. Data shown as mean ± SEM (n = 3 independent experiments); *P < 0.05 by 2-way ANOVA and Holm–Sidak tests. (H) Quantification of the 3G10 signal normalized to the signal of the corresponding neurexin band. Data shown as mean ± SEM of biological replicates. *P < 0.05 by 2-way ANOVA and Holm–Sidak tests (n = 3). I–K. Mouse brains: Neurexins and CA10 were immunoprecipitated from total mouse brain lysates and subject to heparinase treatment. (I) Samples were analyzed by immunoblotting using antibodies against neurexins, the 3G10 epitope (for the HS stub) and CA10. Stars indicate non-neurexin bands (see Results). (J) Quantification of low molecular weight neurexin (LMW; white arrowhead) in relation to total neurexin amounts [upper band (line) + LMW]. Data shown as mean ± SEM (n = 3 independent experiments); *P < 0.05 by 2-way ANOVA and Holm–Sidak tests. (K) Quantification of the 3G10 signal normalized to the signal of the corresponding neurexin band. Data shown as mean ± SEM of 3 biological replicates. *P < 0.05 by 2-way ANOVA and Holm–Sidak tests. Download figure Download PowerPoint Because the abovementioned experiments rely on trimming of HS by heparinases to reveal the neo-epitope recognized by the 3G10 monoclonal, we performed several control experiments to exclude that CA10 may inhibit heparinase activity. First, we expressed secreted Nrxn1β in HEK293 cells, as described above, and added recombinant CA10 prior to the heparinase digestion step. Supplemented CA10 had no noticeable effect on the detection of HS- carrying species (Fig EV2A). As CA10 and neurexins can form an intermolecular disulfide bond when co-expressed (Sterky et al, 2017), we also considered the possibility that covalently bound CA10 may prevent access to the HS chain by heparinases. To test this, we co-expressed Fc-tagged Nrxn1β and V5-tagged CA10, then partially dissociated the complexes with increasing amounts of DTT, treated samples with heparinases and analyzed by SDS–PAGE under non-reducing conditions. The Nrxn1β-CA10 complexes were completely dissociated by 20 mM DTT. At the same time, concentrations of up 50 mM had no noticeable effect on heparinase activities, as the 3G10 epitope could be detected when neurexin was expressed alone (Fig EV2B). No HS could be detected on neurexin dissociated from complexes with CA10. Thus, CA10 blocks the biosynthesis of the HS chain on neurexins. Moreover, we tested whether CA10 also blocks biosynthesis of another HSPG, glypican-1 (GPC1). We expressed HA-tagged GPC1 alone or together with CA10 in HEK293 cells and analyzed the HS of GPC1 as described for Nrxn1α (Fig 2A). In contrast to Nrxn1α, CA10 did not affect the levels of GPC1 that carry HS (Fig EV2C). To test whether CA10 could block HS added on endogenous neurexin, we studied mixed neuron/glia cultures from mouse cortex. Cells were harvested after 14 days in vitro and neurexin was immunoprecipitated from the lysates, heparinase-treated and detected using a pan-neurexin antibody. Endogenous α-neurexins, which dominate in mouse brains (Anderson et al, 2015; Sterky et al, 2017), migrate on SDS–PAGE gels as both a distinct lower band and more diffuse bands of ~ 30 kDa higher apparent molecular mass (Zhang et al, 2018). Digestion of the HS GAG chain with heparinases compressed most (but not all) of the apparently larger isoforms to the lower band (open arrowheads; Fig 2E and F). The relative amounts of the lower isoforms increased from ~ 25 to ~ 70%, indicating that ~ 50% of α-neurexins in our cultures carry the HS chain (Fig 2F). Exogenous expression of FLAG-tagged CA10 by lentiviral transduction resulted, as shown previously (Sterky et al, 2017), in a redistribution of α-neurexin isoforms that mimicked treatment with heparinases. In this case, the distribution of isoforms did not shift further upon heparinase treatment (Fig 2F and G). Moreover, the 3G10 monoclonal showed no reactivity toward neurexin from CA10-expressing cells, indicating that CA10 fully blocked formation of HS-carrying neurexin in neurons (Fig 2F and H). However, consistent with what has been observed by others (Zhang et al, 2018), two 3G10-reactive bands that did not correspond to neurexin isoforms consistently appeared following immunoprecipitation and heparinase treatment, possibly reflecting a background of other abundant HSPGs. The above results suggest that CA10 may regulate neurexin-HS formation. If so, endogenous CA10 would exclusively associate with non-HS neurexin. To test this, we subjected mouse brain lysates to immunoprecipitations using either a pan-neurexin antibody or an antiserum raised against the CA10-Nrxn1γ complex (Fig EV3). The resulting fractions, representing the total pool of neurexin as well as neurexin bound to CA10, were subject to heparinase treatm
The neat ionic liquid (IL) [C2mim][OAc] is not just capable of dissolving thiol- and disulfide-containing compounds, but is able to chemically react with them without addition of any catalytic reagent. Through the analysis of four small organic molecules and a cysteine-containing peptide we could postulate a general reaction mechanism. Here, the imidazolium-carbenes preferentially react with the disulfide bond, but not thiol group. Moreover, the imidazole moiety was found to abstract the sulfur atom from the cysteine residue, providing an alternative way to transform Cys residues, which were artificially inserted into a peptide sequence in order to perform native chemical ligation (NCL) of two peptide fragments. Finally, the chemical reaction of [C2mim][OAc] with a cysteine-containing biomolecules can be tuned or even suppressed through the addition of at least 30% of water to the reaction mixture.
During recent years several peptide-based Ni superoxide dismutase (NiSOD) models have been developed. These NiSOD models show an important structural difference compared to the native NiSOD enzyme, which could cause a completely different mechanism of superoxide dismutation. In the native enzyme the peptide bond between Leu4 and Pro5 is cis-configured, while the NiSOD models exhibit a trans-configured peptide bond between these two residues. To shed light on how the configuration of this single peptide bond influences the activity of the NiSOD model peptides, a new cis-prolyl bond surrogate was developed. As surrogate we chose a leucine/alanine-based disubstituted 1,2,3-triazole, which was incorporated into the NiSOD model peptide replacing residues Leu4 and Pro5. The yielded 1,5-disubstituted triazole nickel peptide exhibited high SOD activity, which was approximately the same activity as its parent trans-configured analogue. Hence, the conformation of the prolyl peptide bond apparently has of minor importance for the catalytic activity of the metallopeptides as postulated in literature. Furthermore, it is shown that the triazole metallopeptide is forming a stable cyanide adduct as a substrate analogue model complex.
Flower power: Potent protease inhibitors containing triazolyl mimics of cis and trans backbone amides were engineered based on the structure of the sunflower trypsin inhibitor 1. The biologically relevant cis-Pro motif was successfully replaced with a non-prolyl unit. High-resolution crystal structures of 1,4- and 1,5-disubstituted 1,2,3-triazolyl peptidomimetics can serve in the design of tailor-made Bowman–Birk inhibitors. Detailed facts of importance to specialist readers are published as "Supporting Information". Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
For the first time, the existence of a substrate adduct of a nickel superoxide dismutase (NiSOD) model, based on the first nine residues from the N terminus of the active form of Streptomyces coelicolor NiSOD, has been proven and the adduct has been isolated. This adduct is based on the cyanide anion (CN(-)), as a substrate analogue of the superoxide anion (O(2)(*-)), and the nickel metallopeptide H-HCDLPCGVY-NH(2)-Ni. Spectroscopic studies, including IR, UV/Vis, and liquid- and solid-state NMR spectroscopy, show a single nickel-bound cyanide anion, which is embedded in the metallopeptide structure. This complex sheds new light on the question of whether the mode of action of the NiSOD enzyme is an inner- or outer-sphere mechanism. Whereas discussion was previously biased in favor of an outer-sphere electron-transfer mechanism due to the fact that binding of cyanide or azide moieties to the nickel active site had never been observed, our results are a clear indication in favor of the inner-sphere electron-transfer mechanism for the disproportionation of the O(2)(*-) ion, whereby the substrate is attached to the Ni atom in the active site of the NiSOD.
