Dissection of Complex Molecular Interactions of Neurofascin with Axonin-1, F11, and Tenascin-R, Which Promote Attachment and Neurite Formation of Tectal Cells

Neurofascin is a member of the L1 subgroup of the Ig superfamily that promotes axon outgrowth by interactions with neuronal NgCAM-related cell adhesion molecule (NrCAM). We used a combination of cellular binding assays and neurite outgrowth experiments to investigate mechanisms that might modulate the interactions of neurofascin. In addition to NrCAM, we here demonstrate that neurofascin also binds to the extracellular matrix glycoprotein tenascin-R (TN-R) and to the Ig superfamily members axonin-1 and F11. Isoforms of neurofascin that are generated by alternative splicing show different preferences in ligand binding. While interactions of neurofascin with F11 are only slightly modulated, binding to axonin-1 and TN-R is strongly regulated by alternatively spliced stretches located in the NH2-terminal half, and by the proline-alanine-threonine-rich segment. In vitro neurite outgrowth and cell attachment assays on a neurofascin-Fc substrate reveal a shift of cellular receptor usage from NrCAM to axonin-1, F11, and at least one additional protein in the presence of TN-R, presumably due to competition of the neurofascin– NrCAM interaction. Thereby, F11 binds to TN-R of the neurofascin/TN-R complex, but not to neurofascin, whereas axonin-1 is not able to bind directly to the neurofascin/TN-R complex as shown by competition binding assays. In conclusion, these investigations indicate that the molecular interactions of neurofascin are regulated at different levels, including alternative splicing and by the presence of interacting proteins.

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Characterization of functional domains of the tenascin-R (restrictin) polypeptide: cell attachment site, binding with F11, and enhancement of F11-mediated neurite outgrowth by tenascin-R.

The Journal of Cell Biology ◽

10.1083/jcb.130.2.473 ◽

1995 ◽

Vol 130 (2) ◽

pp. 473-484 ◽

Cited By ~ 46

Author(s):

U Nörenberg ◽

M Hubert ◽

T Brümmendorf ◽

A Tárnok ◽

F G Rathjen

Keyword(s):

Neurite Outgrowth ◽

Cell Attachment ◽

Attachment Site ◽

Direct Interaction ◽

Bacterial Cells ◽

Fibronectin Type Iii ◽

Attachment Region ◽

A Cell ◽

Function Relationship

The extracellular matrix glycoprotein tenascin-R (TN-R) is a multidomain protein implicated in neural cell adhesion. To analyze the structure-function relationship of the different domains of TN-R, several recombinant TN-R fragments were expressed in bacterial cells. Two distinct binding regions were localized on the TN-R polypeptide: a region binding the axon-associated immunoglobulin (Ig)-like F11 protein and a cell attachment site. The binding region of the glycosylphosphatidylinositol (GPI)-anchored F11 was allocated to the second and third fibronectin type III (FNIII)-like domain within TN-R. By using a mutant polypeptide of F11 containing only Ig-like domains, a direct interaction between the Ig-like domains of F11 and FNIII-like domains 2-3 of TN-R was demonstrated. The interaction of TN-R with F11 in in vitro cultures enhanced F11-mediated neurite outgrowth, suggesting that the combined action of F11 and TN-R might be of regulatory influence on axon extension. A cell attachment region was identified in the FNIII-like domain eight of TN-R by domain-specific antibodies and fusion constructs. This site is distinct from the F11 binding site within TN-R.

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Comparative Effectiveness of Calabadion and Sugammadex to Reverse Non-depolarizing Neuromuscular-blocking Agents

Anesthesiology ◽

10.1097/aln.0000000000000868 ◽

2015 ◽

Vol 123 (6) ◽

pp. 1337-1349 ◽

Cited By ~ 45

Author(s):

Friederike Haerter ◽

Jeroen Cedric Peter Simons ◽

Urs Foerster ◽

Ingrid Moreno Duarte ◽

Daniel Diaz-Gil ◽

...

Keyword(s):

Dose Response ◽

Urine Analysis ◽

Ex Vivo ◽

Neuromuscular Blocking ◽

Response Relationship ◽

Binding Assays ◽

Dose Response Relationship ◽

Competition Binding ◽

Relationship Of

Abstract Background The authors evaluated the comparative effectiveness of calabadion 2 to reverse non-depolarizing neuromuscular-blocking agents (NMBAs) by binding and inactivation. Methods The dose–response relationship of drugs to reverse vecuronium-, rocuronium-, and cisatracurium-induced neuromuscular block (NMB) was evaluated in vitro (competition binding assays and urine analysis), ex vivo (n = 34; phrenic nerve hemidiaphragm preparation), and in vivo (n = 108; quadriceps femoris muscle of the rat). Cumulative dose–response curves of calabadions, neostigmine, or sugammadex were created ex vivo at a steady-state deep NMB. In living rats, the authors studied the dose–response relationship of the test drugs to reverse deep block under physiologic conditions, and they measured the amount of calabadion 2 excreted in the urine. Results In vitro experiments showed that calabadion 2 binds rocuronium with 89 times the affinity of sugammadex (Ka = 3.4 × 109 M−1 and Ka = 3.8 × 107 M−1). The results of urine analysis (proton nuclear magnetic resonance), competition binding assays, and ex vivo study obtained in the absence of metabolic deactivation are in accordance with an 1:1 binding ratio of sugammadex and calabadion 2 toward rocuronium. In living rats, calabadion 2 dose-dependently and rapidly reversed all NMBAs tested. The molar potency of calabadion 2 to reverse vecuronium and rocuronium was higher compared with that of sugammadex. Calabadion 2 was eliminated renally and did not affect blood pressure or heart rate. Conclusions Calabadion 2 reverses NMB induced by benzylisoquinolines and steroidal NMBAs in rats more effectively, i.e., faster than sugammadex. Calabadion 2 is eliminated in the urine and well tolerated in rats.

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Cell surface galactosyltransferase mediates the initiation of neurite outgrowth from PC12 cells on laminin.

The Journal of Cell Biology ◽

10.1083/jcb.110.2.461 ◽

1990 ◽

Vol 110 (2) ◽

pp. 461-470 ◽

Cited By ~ 67

Author(s):

P C Begovac ◽

B D Shur

Keyword(s):

Cell Surface ◽

Neurite Outgrowth ◽

Enzymatic Reaction ◽

Mesenchymal Cell ◽

Laminin Receptor ◽

Extracellular Matrix Molecule ◽

Neurite Initiation ◽

Neurite Formation ◽

And Migration

Neurite outgrowth from PC12 pheochromocytoma cells, as well as from peripheral and central nervous system neurons in vitro, is mediated by the extracellular matrix molecule, laminin. We have recently shown that mesenchymal cell spreading and migration on laminin is mediated, in part, by the cell surface enzyme, beta 1,4 galactosyltransferase (GalTase). GalTase is localized on lamellipodia of migrating cells where it functions as a laminin receptor by binding to specific N-linked oligosaccharides in laminin (Runyan et al., 1988; Eckstein and Shur, 1989). In the present study, we examined whether GalTase functions similarly during neutrite outgrowth on laminin using biochemical and immunological analyses. PC12 neurite outgrowth was inhibited by reagents that perturb cell surface GalTase activity, including anti-GalTase IgG and Fab fragments, as well as the GalTase modifier protein alpha-lactalbumin. Control reagents had no effect on neurite outgrowth. Furthermore, blocking GalTase substrates on laminin matrices by earlier galactosyltion or enzymatic removal of GalTase substrates also inhibited neurite outgrowth. Conversely, neurite outgrowth was enhanced by the addition of UDP-galactose, which completes the GalTase enzymatic reaction, while inappropriate sugar nucleotides had no effect. The effects of all these treatments were dose and/or time dependent. Surface GalTase was shown to function during both neurite initiation and elongation, although the effects of GalTase perturbation were most striking during the initiation stages of neurite formation. Consistent with this, surface GalTase was localized by indirect immunofluorescence to the growth cone and developing neurite. Collectively, these results demonstrate that GalTase mediates the initiation of neurite outgrowth on laminin, and to a lesser extent, neurite elongation. Furthermore, this study demonstrates that process extension from both mesenchymal cells and neuronal cells is partly dependent upon specific oligosaccharide residues in laminin.

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Comparative biochemical and pharmacological investigations of various newly developed opioid receptor ligands

10.14232/phd.10631 ◽

2021 ◽

Author(s):

Edina Szűcs

Keyword(s):

Rat Brain ◽

G Protein ◽

Antinociceptive Effect ◽

Peptide Ligand ◽

Selectivity Ratio ◽

Binding Assays ◽

Brain Membrane ◽

Activity Measurements ◽

Competition Binding

Bentley analogues: In vitro competition binding experiments all derivatives showed low subnanomolar affinity to MOR. For DOR the ligands showed comparable binding affinities than the selective DOR agonist Ile5,6-deltorphin II peptide ligand except 8 (Ki > 3000 nM). In the KOR binding assays the analogues still displayed nanomolar affinities. In G-protein activity measurements compound 1f, 2a, 2b had antagonistic; 1e, 2c, 8 had partial agonistic and 2d, 4, 5, 7 had full agonistic effects. Ligands were examined in G-protein activation tests in rat brain membranes, the selectivity could not be observed as the receptor selective antagonists such as Cyp, NTI, nor-BNI and the selective agonists such as DAMGO, Ile5,6-deltorphine II, U-69,593 are not able to inhibit the effects of the extremely potent Bentley analogues. In vivo tests in osteoarthritis inflammatory model the thevinol derivatives showed a significant antiallodynic effect, while orvinol derivatives, except for 2c, did not display this effect. Oligopeptides: In competition binding assays the KYNA‐containing peptide, KA1 bound selectively to the MOR with a low Ki value and a high selectivity ratio, the other oligopeptides also showed selectivity to MOR, except K3, which bound to MOR and DOR with similar affinity. In the G-protein activition tests the EM-2 containing compounds, K2 and K3 stimulated G-protein with low efficacy, compound KA1, K4, K5 behaved as full agonists, while K6 had efficacy and potency higher than those of the reference compound DAMGO. In functional binding assays all oligopetides were inhibited by Cyp (MOR) and NTI (DOR) in rat brain membrane. In guinea pig brain membrane K4 and K6 stimulated G-protein, the efficacy of K4 was inhibited by nor-BNI, while the effect of K6 was not. K6 exhibited a strong antinociceptive effect in formalin test.

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Identification of A Novel Class of Benzofuran Oxoacetic Acid-Derived Ligands that Selectively Activate Cellular EPAC1

Cells ◽

10.3390/cells8111425 ◽

2019 ◽

Vol 8 (11) ◽

pp. 1425 ◽

Cited By ~ 5

Author(s):

Elizabeth M. Beck ◽

Euan Parnell ◽

Angela Cowley ◽

Alison Porter ◽

Jonathan Gillespie ◽

...

Keyword(s):

Cyclic Nucleotide ◽

High Throughput Screening ◽

Partial Agonist ◽

Fold Increase ◽

Binding Assays ◽

Camp Analogue ◽

Competition Binding ◽

Multiple Cell ◽

Gtpase Activation

Cyclic AMP promotes EPAC1 and EPAC2 activation through direct binding to a specific cyclic nucleotide-binding domain (CNBD) within each protein, leading to activation of Rap GTPases, which control multiple cell responses, including cell proliferation, adhesion, morphology, exocytosis, and gene expression. As a result, it has become apparent that directed activation of EPAC1 and EPAC2 with synthetic agonists may also be useful for the future treatment of diabetes and cardiovascular diseases. To identify new EPAC agonists we have developed a fluorescent-based, ultra-high-throughput screening (uHTS) assay that measures the displacement of binding of the fluorescent cAMP analogue, 8-NBD-cAMP to the EPAC1 CNBD. Triage of the output of an approximately 350,000 compound screens using this assay identified a benzofuran oxaloacetic acid EPAC1 binder (SY000) that displayed moderate potency using orthogonal assays (competition binding and microscale thermophoresis). We next generated a limited library of 91 analogues of SY000 and identified SY009, with modifications to the benzofuran ring associated with a 10-fold increase in potency towards EPAC1 over SY000 in binding assays. In vitro EPAC1 activity assays confirmed the agonist potential of these molecules in comparison with the known EPAC1 non-cyclic nucleotide (NCN) partial agonist, I942. Rap1 GTPase activation assays further demonstrated that SY009 selectively activates EPAC1 over EPAC2 in cells. SY009 therefore represents a novel class of NCN EPAC1 activators that selectively activate EPAC1 in cellulae.

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Chicken Acidic Leucine-rich EGF-like Domain Containing Brain Protein (CALEB), a Neural Member of the EGF Family of Differentiation Factors, Is Implicated in Neurite Formation

The Journal of Cell Biology ◽

10.1083/jcb.136.4.895 ◽

1997 ◽

Vol 136 (4) ◽

pp. 895-906 ◽

Cited By ~ 37

Author(s):

Stefan Schumacher ◽

Hansjürgen Volkmer ◽

Fritz Buck ◽

Albrecht Otto ◽

Attila Tárnok ◽

...

Keyword(s):

Nervous System ◽

Brain Protein ◽

Binding Assays ◽

Multidomain Protein ◽

Differentiation Factors ◽

Neurite Formation ◽

Embryonic Nervous System ◽

Egf Family ◽

Developing Nervous System

Chicken acidic leucine-rich EGF-like domain containing brain protein (CALEB) was identified by combining binding assays with immunological screens in the chicken nervous system as a novel member of the EGF family of differentiation factors. cDNA cloning indicates that CALEB is a multidomain protein that consists of an NH2-terminal glycosylation region, a leucine-proline–rich segment, an acidic box, a single EGF-like domain, a transmembrane, and a short cytoplasmic stretch. In the developing nervous system, CALEB is associated with glial and neuronal surfaces. CALEB is composed of a 140/130-kD doublet, an 80-kD band, and a chondroitinsulfate-containing 200-kD component. The latter two components are expressed in the embryonic nervous system and are downregulated in the adult nervous system. CALEB binds to the extracellular matrix glycoproteins tenascin-C and -R. In vitro antibody perturbation experiments reveal a participation of CALEB in neurite formation in a permissive environment.

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Antisense oligonucleotides targeting alternative splicing of Nrcam exon 10 suppress neurite outgrowth of ganglion sensory neurons in vitro

Neuroreport ◽

10.1097/wnr.0000000000001625 ◽

2021 ◽

Vol Publish Ahead of Print ◽

Author(s):

Lixia Tian ◽

Yu Chen ◽

Shuyang Chang ◽

Linping Xu ◽

Xiaoqiong Zhou ◽

...

Keyword(s):

Alternative Splicing ◽

Neurite Outgrowth ◽

Sensory Neurons ◽

Antisense Oligonucleotides ◽

Exon 10

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Insights into the biochemical and functional characterization of sortase E transpeptidase of Corynebacterium glutamicum

Biochemical Journal ◽

10.1042/bcj20190812 ◽

2019 ◽

Vol 476 (24) ◽

pp. 3835-3847 ◽

Cited By ~ 1

Author(s):

Aliyath Susmitha ◽

Kesavan Madhavan Nampoothiri ◽

Harsha Bajaj

Keyword(s):

Protein Engineering ◽

Cell Attachment ◽

Functional Characterization ◽

Protein Structures ◽

Structural Similarity ◽

Surface Proteins ◽

Sortase A ◽

Peptide Cleavage ◽

New Findings

Most Gram-positive bacteria contain a membrane-bound transpeptidase known as sortase which covalently incorporates the surface proteins on to the cell wall. The sortase-displayed protein structures are involved in cell attachment, nutrient uptake and aerial hyphae formation. Among the six classes of sortase (A–F), sortase A of S. aureus is the well-characterized housekeeping enzyme considered as an ideal drug target and a valuable biochemical reagent for protein engineering. Similar to SrtA, class E sortase in GC rich bacteria plays a housekeeping role which is not studied extensively. However, C. glutamicum ATCC 13032, an industrially important organism known for amino acid production, carries a single putative sortase (NCgl2838) gene but neither in vitro peptide cleavage activity nor biochemical characterizations have been investigated. Here, we identified that the gene is having a sortase activity and analyzed its structural similarity with Cd-SrtF. The purified enzyme showed a greater affinity toward LAXTG substrate with a calculated KM of 12 ± 1 µM, one of the highest affinities reported for this class of enzyme. Moreover, site-directed mutation studies were carried to ascertain the structure functional relationship of Cg-SrtE and all these are new findings which will enable us to perceive exciting protein engineering applications with this class of enzyme from a non-pathogenic microbe.

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The In Vitro Antibiofilm Activity of Water Leaf Extract of Papaya (Carica papaya L.) against Pseudomonas aeruginosa

Current Biochemistry ◽

10.29244/cb.2.3.150-163 ◽

2017 ◽

Vol 2 (3) ◽

pp. 150-163

Author(s):

Ekajayanti Kining ◽

Syamsul Falah ◽

Novik Nurhidayat

Keyword(s):

Pseudomonas Aeruginosa ◽

Contact Time ◽

Cell Attachment ◽

Water Extract ◽

Opportunistic Pathogen ◽

Bacterial Biofilm ◽

Antibiofilm Activity ◽

Bacterial Survival ◽

Optimum Conditions

Pseudomonas aeruginosa is one of opportunistic pathogen forming bacterial biofilm. The biofilm sustains the bacterial survival and infections. This study aimed to assess the activity of water extract of papaya leaves on inhibition of cells attachment, growth and degradation of the biofilm using crystal violet (CV) biofilm assay. Research results showed that water extract of papaya leaves contains alkaloids, tanins, flavonoids, and steroids/terpenoids and showed antibacterial activity and antibiofilm against P. aeruginosa. Addition of extract can inhibit the cell attachment and was able to degrade the biofilm of 40.92% and 48.058% respectively at optimum conditions: extract concentration of 25% (v/v), temperature 37.5 °C and contact time 45 minutes. With a concentration of 25% (v/v), temperature of 50 °C and the contact time of 3 days, extract of papaya leaves can inhibit the growth of biofilms of 39.837% v/v.

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Spastin Interacts with CRMP2 to Regulate Neurite Outgrowth by Controlling Microtubule Dynamics through Phosphorylation Modifications

CNS & Neurological Disorders - Drug Targets ◽

10.2174/1871527319666201026165855 ◽

2020 ◽

Vol 19 ◽

Author(s):

Sumei Li ◽

Jifeng Zhang ◽

Jiaqi Zhang ◽

Jiong Li ◽

Longfei Cheng ◽

...

Keyword(s):

Neurite Outgrowth ◽

Hippocampal Neurons ◽

Neuronal Development ◽

Phosphorylation Site ◽

Microtubule Dynamics ◽

Microtubule Assembly ◽

C Terminus ◽

Mediator Protein ◽

Branch Formation

Aims: Our work aims to revealing the underlying microtubule mechanism of neurites outgrowth during neuronal development, and also proposes a feasible intervention pathway for reconstructing neural network connections after nerve injury. Background: Microtubule polymerization and severing are the basis for the neurite outgrowth and branch formation. Collapsin response mediator protein 2 (CRMP2) regulates axonal growth and branching as a binding partner of the tubulin heterodimer to promote microtubule assembly. And spastin participates in the growth and regeneration of neurites by severing microtubules into small segments. However, how CRMP2 and spastin cooperate to regulate neurite outgrowth by controlling the microtubule dynamics needs to be elucidated. Objective: To explore whether neurite outgrowth was mediated by coordination of CRMP2 and spastin. Method: Hippocampal neurons were cultured in vitro in 24-well culture plates for 4 days before being used to perform the transfection. Calcium phosphate was used to transfect the CRMP2 and spastin constructs and their control into the neurons. An interaction between CRMP2 and spastin was examined by using pull down, CoIP and immunofluorescence colocalization assays. And immunostaining was also performed to determine the morphology of neurites. Result: We first demonstrated that CRMP2 interacted with spastin to promote the neurite outgrowth and branch formation. Furthermore, our results identified that phosphorylation modification failed to alter the binding affinities of CRMP2 for spastin, but inhibited their binding to microtubules. CRMP2 interacted with the MTBD domain of spastin via its C-terminus, and blocking the binding sites of them inhibited the outgrowth and branch formation of neurites. In addition, we confirmed one phosphorylation site S210 at spastin in hippocampal neurons and phosphorylation spastin at site S210 promoted the neurite outgrowth but not branch formation by remodeling microtubules. Conclusion: Taken together, our data demonstrated that the interaction of CRMP2 and spastin is required for neurite outgrowth and branch formation and their interaction is not regulated by their phosphorylation.

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