scholarly journals The sclerostin-neutralizing antibody AbD09097 recognizes an epitope adjacent to sclerostin's binding site for the Wnt co-receptor LRP6

Open Biology ◽  
2016 ◽  
Vol 6 (8) ◽  
pp. 160120 ◽  
Author(s):  
V. Boschert ◽  
C. Frisch ◽  
J. W. Back ◽  
K. van Pee ◽  
S. E. Weidauer ◽  
...  
Keyword(s):  
Rational Design ◽  
Bone Growth ◽  
Interaction Analysis ◽  
Neutralizing Antibody ◽  
Affinity Maturation ◽  
Negative Regulator ◽  
Binding Motif ◽  
Biochemical Analyses ◽  
Set Up

The glycoprotein sclerostin has been identified as a negative regulator of bone growth. It exerts its function by interacting with the Wnt co-receptor LRP5/6, blocks the binding of Wnt factors and thereby inhibits Wnt signalling. Neutralizing anti-sclerostin antibodies are able to restore Wnt activity and enhance bone growth thereby presenting a new osteoanabolic therapy approach for diseases such as osteoporosis. We have generated various Fab antibodies against human and murine sclerostin using a phage display set-up. Biochemical analyses have identified one Fab developed against murine sclerostin, AbD09097 that efficiently neutralizes sclerostin's Wnt inhibitory activity. In vitro interaction analysis using sclerostin variants revealed that this neutralizing Fab binds to sclerostin's flexible second loop, which has been shown to harbour the LRP5/6 binding motif. Affinity maturation was then applied to AbD09097, providing a set of improved neutralizing Fab antibodies which particularly bind human sclerostin with enhanced affinity. Determining the crystal structure of AbD09097 provides first insights into how this antibody might recognize and neutralize sclerostin. Together with the structure–function relationship derived from affinity maturation these new data will foster the rational design of new and highly efficient anti-sclerostin antibodies for the therapy of bone loss diseases such as osteoporosis.

scholarly journals Epitope–Paratope Interaction of a Neutralizing Human Anti-Hepatitis B Virus PreS1 Antibody That Recognizes the Receptor-Binding Motif

Vaccines ◽  
2021 ◽  
Vol 9 (7) ◽  
pp. 754
Author(s):  
Jisu Hong ◽  
Youngjin Choi ◽  
Yoonjoo Choi ◽  
Jiwoo Lee ◽  
Hyo Jeong Hong
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Receptor Binding ◽  
Neutralizing Antibody ◽  
Hbv Infection ◽  
Binding Motif ◽  
Alanine Scanning Mutagenesis ◽  
B Virus ◽  
Complementarity Determining Regions

Hepatitis B virus (HBV) is a global health burden that causes acute and chronic hepatitis. To develop an HBV-neutralizing antibody that effectively prevents HBV infection, we previously generated a human anti-preS1 monoclonal antibody (1A8) that binds to genotypes A–D and validated its HBV-neutralizing activity in vitro. In the present study, we aimed to determine the fine epitope and paratope of 1A8 to understand the mechanism of HBV neutralization. We performed alanine-scanning mutagenesis on the preS1 (aa 19–34, genotype C) and the heavy (HCDR) and light (LCDR) chain complementarity-determining regions. The 1A8 recognized the three residues (Leu22, Gly23, and Phe25) within the highly conserved receptor-binding motif (NPLGFFP) of the preS1, while four CDR residues of 1A8 were critical in antigen binding. Structural analysis of the epitope–paratope interaction by molecular modeling revealed that Leu100 in the HCDR3, Ala50 in the HCDR2, and Tyr96 in the LCDR3 closely interacted with Leu22, Gly23, and Phe25 of the preS1. Additionally, we found that 1A8 also binds to the receptor-binding motif (NPLGFLP) of infrequently occurring HBV. The results suggest that 1A8 may broadly and effectively block HBV entry and thus have potential as a promising candidate for the prevention and treatment of HBV infection.

Phosphoinositide 3-kinase is activated by MUC1 but not responsible for MUC1-induced suppression of Toll-like receptor 5 signaling

2007 ◽  
Vol 293 (3) ◽  
pp. L686-L692 ◽  
Author(s):  
Kosuke Kato ◽  
Wenju Lu ◽  
Hirofumi Kai ◽  
K. Chul Kim
Keyword(s):  
Cross Talk ◽  
Negative Regulator ◽  
Regulatory Subunit ◽  
Binding Motif ◽  
Toll Like Receptor ◽  
Akt Phosphorylation ◽  
Phosphoinositide 3 Kinase ◽  
Toll Like Receptor 5

MUC1 is a membrane-tethered mucin-like glycoprotein expressed on the surface of various mucosal epithelial cells as well as hematopoietic cells. Recently, we showed that MUC1 suppresses flagellin-induced Toll-like receptor (TLR) 5 signaling both in vivo and in vitro through cross talk with TLR5. In this study, we determined whether phosphoinositide 3-kinase (PI3K), a negative regulator of TLR5 signaling, is involved in the cross talk between MUC1 and TLR5 using various genetically modified epithelial cell lines. Our results showed 1) activation of MUC1 induced recruitment of the PI3K regulatory subunit p85 to the MUC1 cytoplasmic tail (CT) as well as Akt phosphorylation, 2) MUC1-induced Akt phosphorylation required the presence of Tyr20 within the PI3K binding motif of the MUC1 CT, and 3) mutation of Tyr20 or pharmacological inhibition of PI3K activation failed to block MUC1-induced suppression of TLR5 signaling. We conclude that whereas PI3K is downstream of MUC1 activation and negatively regulates TLR5 signaling, it is not responsible for MUC1-induced suppression of TLR5 signaling.

scholarly journals Homology Modeling-Based in Silico Affinity Maturation Improves the Affinity of a Nanobody

2019 ◽  
Vol 20 (17) ◽  
pp. 4187 ◽  
Author(s):  
Xin Cheng ◽  
Jiewen Wang ◽  
Guangbo Kang ◽  
Min Hu ◽  
Bo Yuan ◽  
...  
Keyword(s):  
Homology Modeling ◽  
Rational Design ◽  
Structural Model ◽  
Three Dimensional ◽  
Phage Display Library ◽  
Affinity Maturation ◽  
New Strategy ◽  
Design Protocol ◽  
High Binding Affinity

Affinity maturation and rational design have a raised importance in the application of nanobody (VHH), and its unique structure guaranteed these processes quickly done in vitro. An anti-CD47 nanobody, Nb02, was screened via a synthetic phage display library with 278 nM of KD value. In this study, a new strategy based on homology modeling and Rational Mutation Hotspots Design Protocol (RMHDP) was presented for building a fast and efficient platform for nanobody affinity maturation. A three-dimensional analytical structural model of Nb02 was constructed and then docked with the antigen, the CD47 extracellular domain (CD47ext). Mutants with high binding affinity are predicted by the scoring of nanobody-antigen complexes based on molecular dynamics trajectories and simulation. Ultimately, an improved mutant with an 87.4-fold affinity (3.2 nM) and 7.36 °C higher thermal stability was obtained. These findings might contribute to computational affinity maturation of nanobodies via homology modeling using the recent advancements in computational power. The add-in of aromatic residues which formed aromatic-aromatic interaction plays a pivotal role in affinity and thermostability improvement. In a word, the methods used in this study might provide a reference for rapid and efficient in vitro affinity maturation of nanobodies.

scholarly journals CXXC5 mediates growth plate senescence and is a target for enhancement of longitudinal bone growth

2019 ◽  
Vol 2 (2) ◽  
pp. e201800254 ◽  
Author(s):  
Sehee Choi ◽  
Hyun-Yi Kim ◽  
Pu-Hyeon Cha ◽  
Seol Hwa Seo ◽  
Chulho Lee ◽  
...  
Keyword(s):  
Growth Plate ◽  
Molecular Mechanisms ◽  
Bone Growth ◽  
Negative Regulator ◽  
Screening Assay ◽  
Longitudinal Bone Growth ◽  
Delayed Senescence ◽  
Before And After ◽  
Delayed Growth

Longitudinal bone growth ceases with growth plate senescence during puberty. However, the molecular mechanisms of this phenomenon are largely unexplored. Here, we examined Wnt-responsive genes before and after growth plate senescence and found that CXXC finger protein 5 (CXXC5), a negative regulator of the Wnt/β-catenin pathway, was gradually elevated with reduction of Wnt/β-catenin signaling during senescent changes of rodent growth plate. Cxxc5−/− mice demonstrated delayed growth plate senescence and tibial elongation. As CXXC5 functions by interacting with dishevelled (DVL), we sought to identify small molecules capable of disrupting this interaction. In vitro screening assay monitoring CXXC5–DVL interaction revealed that several indirubin analogs were effective antagonists of this interaction. A functionally improved indirubin derivative, KY19382, elongated tibial length through delayed senescence and further activation of the growth plate in adolescent mice. Collectively, our findings reveal an important role for CXXC5 as a suppressor of longitudinal bone growth involving growth plate activity.

scholarly journals Structures of the activator ofK. pneumoniabiofilm formation, MrkH, indicates PilZ domains involved in c-di-GMP and DNA binding

2016 ◽  
Vol 113 (36) ◽  
pp. 10067-10072 ◽  
Author(s):  
Maria A. Schumacher ◽  
Wenjie Zeng
Keyword(s):  
Dna Binding ◽  
Rational Design ◽  
Domain Architecture ◽  
Klebsiella Pneumonia ◽  
Binding Motif ◽  
Genes Encoding ◽  
Biochemical Analyses ◽  
Biofilm Development ◽  
Domain Reorientation ◽  
Structure Stabilization

The pathogenesis ofKlebsiella pneumoniais linked to the bacteria’s ability to form biofilms. Mannose-resistantKlebsiella-like (Mrk) hemagglutinins are critical forK.pneumoniabiofilm development, and the expression of the genes encoding these proteins is activated by a 3′,5′-cyclic diguanylic acid (c-di-GMP)–regulated transcription factor, MrkH. To gain insight into MrkH function, we performed structural and biochemical analyses. Data revealed MrkH to be a monomer with a two-domain architecture consisting of a PilZ C-domain connected to an N domain that unexpectedly also harbors a PilZ-like fold. Comparison of apo- and c-di-GMP–bound MrkH structures reveals a large 138° interdomain rotation that is induced by binding an intercalated c-di-GMP dimer. c-di-GMP interacts with PilZ C-domain motifs 1 and 2 (RxxxR and D/NxSxxG) and a newly described c-di-GMP–binding motif in the MrkH N domain. Strikingly, these c-di-GMP–binding motifs also stabilize an open state conformation in apo MrkH via contacts from the PilZ motif 1 to residues in the C-domain motif 2 and the c-di-GMP–binding N-domain motif. Use of the same regions in apo structure stabilization and c-di-GMP interaction allows distinction between the states. Indeed, domain reorientation by c-di-GMP complexation with MrkH, which leads to a highly compacted structure, suggests a mechanism by which the protein is activated to bind DNA. To our knowledge, MrkH represents the first instance of specific DNA binding mediated by PilZ domains. The MrkH structures also pave the way for the rational design of inhibitors that targetK.pneumoniabiofilm formation.

scholarly journals Potent germline-like monoclonal antibodies: Rapid identification of promising candidates for antibody-based antiviral therapy

2021 ◽  
Author(s):  
Xiaoyi Zhu ◽  
Fei Yu ◽  
Yanling Wu ◽  
Tianlei Ying
Keyword(s):  
Monoclonal Antibodies ◽  
Rational Design ◽  
Large Scale ◽  
Molecular Mechanisms ◽  
Viral Infections ◽  
Somatic Hypermutation ◽  
Affinity Maturation ◽  
Human Mabs

Abstract Recent years, fully human monoclonal antibodies (mAbs) are making up an increasing share of the pharmaceutical market. However, to improve affinity and efficacy of antibodies, many somatic hypermutation could be introduced during affinity maturation, which cause several issues including safety and efficacy and limit their application in clinic. Here, we propose a special class of human mAbs with limited level of somatic mutations, referred to as germline-like mAbs. Remarkably, germline-like mAbs could have high affinity and potent neutralizing activity in vitro and in various animal models, despite lacking of extensive affinity maturation. Furthermore, the germline nature of these mAbs implies that they exhibit lower immunogenicity and can be elicited relatively fast in vivo compared with highly somatically mutated antibodies. In this review, we summarize germline-like mAbs with strong therapeutic and protection activity against various viruses that caused large-scale outbreaks in the last decade, including influenza virus H7N9, Zika virus (ZIKV), Dengue virus (DENV), Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We also illustrate underlying molecular mechanisms of these germline-like antibodies against viral infections from the structural and genetic perspective, thus providing insight into further development as therapeutic agents for treatment of infectious diseases and implication for rational design of effective vaccines.

scholarly journals Regulation of TMPRSS6 by BMP6 and iron in human cells and mice

Blood ◽  
2011 ◽  
Vol 118 (3) ◽  
pp. 747-756 ◽  
Author(s):  
Delphine Meynard ◽  
Valentina Vaja ◽  
Chia Chi Sun ◽  
Elena Corradini ◽  
Shanzhuo Chen ◽  
...  
Keyword(s):  
Neutralizing Antibody ◽  
Negative Regulator ◽  
Deficiency Anemia ◽  
Hepcidin Expression ◽  
Protein Levels ◽  
Morphogenetic Protein ◽  
Smad Signaling Pathway ◽  
Feedback Inhibitor ◽  
Inhibitor Of Dna Binding

Abstract Mutations in transmembrane protease, serine 6 (TMPRSS6), encoding matriptase-2, are responsible for the familial anemia disorder iron-refractory iron deficiency anemia (IRIDA). Patients with IRIDA have inappropriately elevated levels of the iron regulatory hormone hepcidin, suggesting that TMPRSS6 is involved in negatively regulating hepcidin expression. Hepcidin is positively regulated by iron via the bone morphogenetic protein (BMP)-SMAD signaling pathway. In this study, we investigated whether BMP6 and iron also regulate TMPRSS6 expression. Here we demonstrate that, in vitro, treatment with BMP6 stimulates TMPRSS6 expression at the mRNA and protein levels and leads to an increase in matriptase-2 activity. Moreover, we identify that inhibitor of DNA binding 1 is the key element of the BMP-SMAD pathway to regulate TMPRSS6 expression in response to BMP6 treatment. Finally, we show that, in mice, Tmprss6 mRNA expression is stimulated by chronic iron treatment or BMP6 injection and is blocked by injection of neutralizing antibody against BMP6. Our results indicate that BMP6 and iron not only induce hepcidin expression but also induce TMPRSS6, a negative regulator of hepcidin expression. Modulation of TMPRSS6 expression could serve as a negative feedback inhibitor to avoid excessive hepcidin increases by iron to help maintain tight homeostatic balance of systemic iron levels.

scholarly journals The Penta-EF-Hand ALG-2 Protein Interacts with the Cytosolic Domain of the SOCE Regulator SARAF and Interferes with Ubiquitination

2020 ◽  
Vol 21 (17) ◽  
pp. 6315
Author(s):  
Wei Zhang ◽  
Ayaka Muramatsu ◽  
Rina Matsuo ◽  
Naoki Teranishi ◽  
Yui Kahara ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Binding Activity ◽  
Negative Regulator ◽  
Hek293 Cells ◽  
Binding Motif ◽  
Protein Database ◽  
Ww Domains ◽  
Ef Hand ◽  
Cytosolic Domain

ALG-2 is a penta-EF-hand Ca2+-binding protein and interacts with a variety of proteins in mammalian cells. In order to find new ALG-2-binding partners, we searched a human protein database and retrieved sequences containing the previously identified ALG-2-binding motif type 2 (ABM-2). After selecting 12 high-scored sequences, we expressed partial or full-length GFP-fused proteins in HEK293 cells and performed a semi-quantitative in vitro binding assay. SARAF, a negative regulator of store-operated Ca2+ entry (SOCE), showed the strongest binding activity. Biochemical analysis of Strep-tagged and GFP-fused SARAF proteins revealed ubiquitination that proceeded during pulldown assays under certain buffer conditions. Overexpression of ALG-2 interfered with ubiquitination of wild-type SARAF but not ubiquitination of the F228S mutant that had impaired ALG-2-binding activity. The SARAF cytosolic domain (CytD) contains two PPXY motifs targeted by the WW domains of NEDD4 family E3 ubiquitin ligases. The PPXY motif proximal to the ABM-2 sequence was found to be more important for both in-cell ubiquitination and post-cell lysis ubiquitination. A ubiquitination-defective mutant of SARAF with Lys-to-Arg substitutions in the CytD showed a slower degradation rate by half-life analysis. ALG-2 promoted Ca2+-dependent CytD-to-CytD interactions of SARAF. The ALG-2 dimer may modulate the stability of SARAF by sterically blocking ubiquitination and by bridging SARAF molecules at the CytDs.

scholarly journals In vitro affinity maturation of broader and more-potent variants of the HIV-1–neutralizing antibody CAP256-VRC26.25

2021 ◽  
Vol 118 (29) ◽  
pp. e2106203118
Author(s):  
Yiming Yin ◽  
Brian D. Quinlan ◽  
Tianling Ou ◽  
Yan Guo ◽  
Wenhui He ◽  
...  
Keyword(s):  
B Cell ◽  
Posttranslational Modifications ◽  
Neutralizing Antibodies ◽  
Cytidine Deaminase ◽  
Neutralizing Antibody ◽  
Affinity Maturation ◽  
Homology Directed Repair ◽  
Activation Induced Cytidine Deaminase ◽  
Hiv 1

Three variable 2 (V2) loops of HIV-1 envelope glycoprotein (Env) trimer converge at the Env apex to form the epitope of an important classes of HIV-1 broadly neutralizing antibodies (bNAbs). These V2-glycan/apex antibodies are exceptionally potent but less broad (∼60 to 75%) than many other bNAbs. Their CDRH3 regions are typically long, acidic, and tyrosine sulfated. Tyrosine sulfation complicates efforts to improve these antibodies through techniques such as phage or yeast display. To improve the breadth of CAP256-VRC26.25 (VRC26.25), a very potent apex antibody, we adapted and extended a B cell display approach. Specifically, we used CRISPR/Cas12a to introduce VRC26.25 heavy- and light-chain genes into their respective loci in a B cell line, ensuring that each cell expresses a single VRC26.25 variant. We then diversified these loci through activation-induced cytidine deaminase–mediated hypermutation and homology-directed repair using randomized CDRH3 sequences as templates. Iterative sorting with soluble Env trimers and further randomization selected VRC26.25 variants with successively improving affinities. Three mutations in the CDRH3 region largely accounted for this improved affinity, and VRC26.25 modified with these mutations exhibited greater breadth and potency than the original antibody. Our data describe a broader and more-potent form of VRC26.25 as well as an approach useful for improving the breadth and potency of antibodies with functionally important posttranslational modifications.

Effect of 300 mT static and 50 Hz 0.1 mT extremely low frequency magnetic fields on Tuber borchii mycelium

2012 ◽  
Vol 58 (10) ◽  
pp. 1174-1182 ◽  
Author(s):  
Lucia Potenza ◽  
Roberta Saltarelli ◽  
Emanuela Polidori ◽  
Paola Ceccaroli ◽  
Antonella Amicucci ◽  
...  
Keyword(s):  
Gene Expression ◽  
Magnetic Field ◽  
Low Frequency ◽  
Hyphal Growth ◽  
In Vitro Cultivation ◽  
Tuber Borchii ◽  
Extremely Low Frequency ◽  
Biochemical Analyses ◽  
Set Up

The present work aimed to investigate whether exposure to static magnetic field (SMF) and extremely low frequency magnetic field (ELF-MF) can induce biomolecular changes on Tuber borchii hyphal growth. Tuber borchii mycelium was exposed for 1 h for 3 consecutive days to a SMF of 300 mT or an ELF-MF of 0.1 mT 50 Hz. Gene expression and biochemical analyses were performed. In mycelia exposed to ELF-MF, some genes involved in hyphal growth, investigated using quantitative real-time polymerase chain reaction, were upregulated, and the activity of many glycolytic enzymes was increased. On the contrary, no differences were observed in gene expression after exposure to SMF treatment, and only the activities of glucose 6-phosphate dehydrogenase and hexokinase increased. The data herein presented suggest that the electromagnetic field can act as an environmental factor in promoting hyphal growth and can be used for applicative purposes, such as the set up of new in vitro cultivation techniques.

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