Mechanistic principles of an ultra-long bovine CDR reveal strategies for antibody design

AbstractAntibodies bind antigens via flexible loops called complementarity-determining regions (CDRs). These are usually 6-20 residues long. However, some bovine antibodies have ultra-long CDRs comprising more than 50 residues organized in a stalk and a disulfide-rich knob. The design features of this structural unit and its influence on antibody stability remained enigmatic. Here, we show that the stalk length is critical for the folding and stability of antibodies with an ultra-long CDR and that the disulfide bonds in the knob do not contribute to stability; they are important for organizing the antigen-binding knob structure. The bovine ultra-long CDR can be integrated into human antibody scaffolds. Furthermore, mini-domains from de novo design can be reformatted as ultra-long CDRs to create unique antibody-based proteins neutralizing SARS-CoV-2 and the Alpha variant of concern with high efficiency. Our findings reveal basic design principles of antibody structure and open new avenues for protein engineering.

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Bovine antibodies with ultra long H3 Complementarity Determining Regions

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314097447 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C255-C255

Author(s):

Robyn Stanfield ◽

Vaughn Smider ◽

Ian Wilson

Keyword(s):

Disulfide Bonds ◽

The Other ◽

Sequence Length ◽

Antibody Repertoire ◽

Human Antibody ◽

Structural Homology ◽

Disulfide Bonding ◽

Cysteine Rich Protein ◽

Complementarity Determining Regions ◽

Bovine Antibody

About 10% of the bovine antibody repertoire exhibit extremely long H3 complementarity determining regions (CDRs). These H3 CDRs are usually described as `loops' in the more familiar mouse and human antibody Fab structures, but the ultra long bovine H3 CDRs are actually small, cysteine-rich protein domains that vary in size from 44 to 64 amino acids. We have recently determined the structures for two bovine antibody Fab fragments, and will describe these, as well as compare them with two other previously determined bovine Fab structures (Wang et al., Cell, 2013). One new Fab has a relatively short H3 CDR region of 44 residues, with just one disulfide bond, while the other boasts one of the longest H3 CDRs, with 63 residues and four disulfide bonds. These H3 CDRs fold to form apparently rigid `stem' regions, that present the disulfide bonded `knob' domain far above the five other Fab CDR loops. Despite extreme diversity in sequence, length and disulfide bonding patterns, the CDRs share structural homology, both in their long stems and in the more variable knob regions.

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Adaption of human antibody λ and κ light chain architectures to CDR repertoires

Protein Engineering Design and Selection ◽

10.1093/protein/gzz012 ◽

2019 ◽

Vol 32 (3) ◽

pp. 109-127 ◽

Cited By ~ 1

Author(s):

Rob van der Kant ◽

Joschka Bauer ◽

Anne R Karow-Zwick ◽

Sebastian Kube ◽

Patrick Garidel ◽

...

Keyword(s):

Light Chain ◽

High Specificity ◽

Field Analysis ◽

Human Antibody ◽

Antigen Binding ◽

Variable Regions ◽

Wide Range ◽

Distinct Sequence ◽

Statistical Coupling ◽

Complementarity Determining Regions

Abstract Monoclonal antibodies bind with high specificity to a wide range of diverse antigens, primarily mediated by their hypervariable complementarity determining regions (CDRs). The defined antigen binding loops are supported by the structurally conserved β-sandwich framework of the light chain (LC) and heavy chain (HC) variable regions. The LC genes are encoded by two separate loci, subdividing the entity of antibodies into kappa (LCκ) and lambda (LCλ) isotypes that exhibit distinct sequence and conformational preferences. In this work, a diverse set of techniques were employed including machine learning, force field analysis, statistical coupling analysis and mutual information analysis of a non-redundant antibody structure collection. Thereby, it was revealed how subtle changes between the structures of LCκ and LCλ isotypes increase the diversity of antibodies, extending the predetermined restrictions of the general antibody fold and expanding the diversity of antigen binding. Interestingly, it was found that the characteristic framework scaffolds of κ and λ are stabilized by diverse amino acid clusters that determine the interplay between the respective fold and the embedded CDR loops. In conclusion, this work reveals how antibodies use the remarkable plasticity of the beta-sandwich Ig fold to incorporate a large diversity of CDR loops.

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General synthetic strategy for regioselective ultrafast formation of disulfide bonds in peptides and proteins

Nature Communications ◽

10.1038/s41467-021-21209-0 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Shay Laps ◽

Fatima Atamleh ◽

Guy Kamnesky ◽

Hao Sun ◽

Ashraf Brik

Keyword(s):

Drug Discovery ◽

Disulfide Bonds ◽

Unsolved Problem ◽

De Novo ◽

Therapeutic Potential ◽

Selective Activation ◽

One Pot ◽

Synthetic Strategy ◽

The One ◽

Game Changer

AbstractDespite six decades of efforts to synthesize peptides and proteins bearing multiple disulfide bonds, this synthetic challenge remains an unsolved problem in most targets (e.g., knotted mini proteins). Here we show a de novo general synthetic strategy for the ultrafast, high-yielding formation of two and three disulfide bonds in peptides and proteins. We develop an approach based on the combination of a small molecule, ultraviolet-light, and palladium for chemo- and regio-selective activation of cysteine, which enables the one-pot formation of multiple disulfide bonds in various peptides and proteins. We prepare bioactive targets of high therapeutic potential, including conotoxin, RANTES, EETI-II, and plectasin peptides and the linaclotide drug. We anticipate that this strategy will be a game-changer in preparing millions of inaccessible targets for drug discovery.

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Heterogeneous expression of human antibody lambda chains by concanavalin A-resistant hybridomas lead to changed antigen binding.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)62013-2 ◽

1994 ◽

Vol 269 (46) ◽

pp. 29061-29066

Author(s):

H Tachibana ◽

I Kido ◽

H Murakami

Keyword(s):

Concanavalin A ◽

Human Antibody ◽

Antigen Binding ◽

Heterogeneous Expression

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SLR-superscaffolder: a de novo scaffolding tool for synthetic long reads using a top-to-bottom scheme

BMC Bioinformatics ◽

10.1186/s12859-021-04081-z ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Lidong Guo ◽

Mengyang Xu ◽

Wenchao Wang ◽

Shengqiang Gu ◽

Xia Zhao ◽

...

Keyword(s):

High Efficiency ◽

De Novo ◽

Next Generation Sequencing Data ◽

Sequencing Data ◽

Draft Assembly ◽

Screening Algorithm ◽

Long Reads ◽

Hybrid Genome ◽

Genomics Research ◽

Negative Effect

Abstract Background Synthetic long reads (SLR) with long-range co-barcoding information are now widely applied in genomics research. Although several tools have been developed for each specific SLR technique, a robust standalone scaffolder with high efficiency is warranted for hybrid genome assembly. Results In this work, we developed a standalone scaffolding tool, SLR-superscaffolder, to link together contigs in draft assemblies using co-barcoding and paired-end read information. Our top-to-bottom scheme first builds a global scaffold graph based on Jaccard Similarity to determine the order and orientation of contigs, and then locally improves the scaffolds with the aid of paired-end information. We also exploited a screening algorithm to reduce the negative effect of misassembled contigs in the input assembly. We applied SLR-superscaffolder to a human single tube long fragment read sequencing dataset and increased the scaffold NG50 of its corresponding draft assembly 1349 fold. Moreover, benchmarking on different input contigs showed that this approach overall outperformed existing SLR scaffolders, providing longer contiguity and fewer misassemblies, especially for short contigs assembled by next-generation sequencing data. The open-source code of SLR-superscaffolder is available at https://github.com/BGI-Qingdao/SLR-superscaffolder. Conclusions SLR-superscaffolder can dramatically improve the contiguity of a draft assembly by integrating a hybrid assembly strategy.

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Alphoid DNA from different chromosomes forms de novo minichromosomes with high efficiency

Chromosome Research ◽

10.1007/s10577-005-0979-4 ◽

2005 ◽

Vol 13 (4) ◽

pp. 411-422 ◽

Cited By ~ 6

Author(s):

T. Kaname ◽

A. McGuigan ◽

A. Georghiou ◽

Y. Yurov ◽

K. Osoegawa ◽

...

Keyword(s):

High Efficiency ◽

De Novo ◽

Alphoid Dna

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Identification of Novel Conopeptides and Distinct Gene Superfamilies in the Marine Cone Snail Conus quercinus

Frontiers in Marine Science ◽

10.3389/fmars.2021.766792 ◽

2021 ◽

Vol 8 ◽

Author(s):

Han Zhang ◽

Lei Wang ◽

Xiang Yang ◽

Zhiwei Lian ◽

Yinbin Qiu ◽

...

Keyword(s):

Mass Spectrometry ◽

Prokaryotic Expression ◽

Disulfide Bonds ◽

De Novo ◽

Animal Experiments ◽

Cone Snail ◽

Active Peptides ◽

Neuroscience Research ◽

First Time ◽

Mass Spectrometry Mass Spectrometry

Conopeptides from the marine cone snails are a mixture of cysteine-rich active peptides, representing a unique and fertile resource for neuroscience research and drug discovery. The ConoServer database includes 8,134 conopeptides from 122 Conus species, yet many more natural conopeptides remain to be discovered. Here, we identified 517 distinct conopeptide precursors in Conus quercinus using de novo deep transcriptome sequencing. Ten of these precursors were verified at the protein level using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). The combined gene and protein analyses revealed two novel gene superfamilies (Que-MNCLQ and Que-MAMNV), and three other gene superfamilies (N, P, and I1) were reported for the first time in C. quercinus. From the Que-MAMNV superfamily, a novel conotoxin, Que-0.1, was obtained via cloning and prokaryotic expression. We also documented a new purification process that can be used to induce the expression of conopeptides containing multiple pairs of disulfide bonds. The animal experiments showed that Que-0.1 strongly inhibited neuroconduction; the effects of Que-1.0 were 6.25 times stronger than those of pethidine hydrochloride. In addition, a new cysteine framework (CC-C-C-C-C-C-CC-C-C-C-C-C) was found in C. quercinus. These discoveries accelerate our understanding of conopeptide diversity in the genus, Conus and supply promising materials for medical research.

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Abstract 17357: Direct Cardiac Reprogramming Using Combinatorial Modified mRNA

Circulation ◽

10.1161/circ.142.suppl_3.17357 ◽

2020 ◽

Vol 142 (Suppl_3) ◽

Author(s):

Keerat Kaur ◽

Asharee Mahmoud ◽

Hanna Girard ◽

Ann Anu Kurian ◽

Magdalena Zak ◽

...

Keyword(s):

Myocardial Infarction ◽

Gene Delivery ◽

High Efficiency ◽

De Novo ◽

Heart Function ◽

Lineage Tracing ◽

Dominant Negative ◽

Post Myocardial Infarction ◽

Transduction Efficiency

Introduction: Despite various clinical modalities, ischemic heart disease remains among the leading causes of mortality and morbidity worldwide. The elemental problem is the immense loss of cardiomyocytes (CMs) post-myocardial infarction (MI). Reprogramming non- cardiomyocytes (non-CMs) into cardiomyocyte (CM)-like cells in vivo is a promising strategy for cardiac regeneration: however, the traditional viral delivery method hampered its application into clinical settings due to low and erratic transduction efficiency. Methods: We used a modified mRNA (modRNA) gene delivery platform to deliver different stoichiometry of cardiac-reprogramming genes (Gata4, Mef2c, Tbx5 and Hand2) together with reprogramming helper genes (Dominant Negative (DN)-TGFβ, DN- Wnt8a and Acid ceramidase (AC)), named 7G, to induce direct cardiac reprogramming post myocardial infarction (MI). Results: Here, we identified 7G modRNA cocktail as an important regulator ofthe cardiac reprogramming. Cardiac transfection with 7G modRNA doubled cardiac reprogramming efficiency (57%) in comparison to Gata4, Mef2C and Tbx5 (GMT) alone (28%) in vitro . By inducing MI in our lineage tracing model, we showed that one-time delivery of the 7G-modRNA cocktail reprogrammed ~25% of the non-CMs in the scar area to CM- like cells. Furthermore, 7G modRNA treated mice showed significantly improved cardiac function, longer survival, reduced scar size and greater capillary density than control mice 28 days post-MI. We attributed the improvement in heart function post modRNA delivery of 7G or 7G with increased Hand2 ratio (7G-GMT Hx2) to significant upregulation of 15 key angiogenic factors without any signs of angioma or edema. Conclusions: 7G or 7G GMT HX2 modRNA cocktails boosts de novo CM-like cells and promotes cardiovascular regeneration post-MI. Thus, we highlight that this gene delivery approach not only has high efficiency but also high margin of safety for translation to clinics.

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