Bovine antibodies with ultra long H3 Complementarity Determining Regions

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

Nature Communications ◽

10.1038/s41467-021-27103-z ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Hristo L. Svilenov ◽

Julia Sacherl ◽

Ulrike Protzer ◽

Martin Zacharias ◽

Johannes Buchner

Keyword(s):

Disulfide Bonds ◽

High Efficiency ◽

De Novo ◽

Human Antibody ◽

Antigen Binding ◽

Antibody Structure ◽

Alpha Variant ◽

Complementarity Determining Regions ◽

Antibody Design ◽

Stalk Length

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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Signatures of selection in the human antibody repertoire: Selective sweeps, competing subclones, and neutral drift

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1814213116 ◽

2019 ◽

Vol 116 (4) ◽

pp. 1261-1266 ◽

Cited By ~ 31

Author(s):

Felix Horns ◽

Christopher Vollmers ◽

Cornelia L. Dekker ◽

Stephen R. Quake

Keyword(s):

B Cell ◽

Binding Affinity ◽

Antibody Repertoire ◽

Human Antibody ◽

Selective Sweeps ◽

Human Immune System ◽

Evolutionary Processes ◽

Neutral Drift ◽

Cell Lineages ◽

Antibody Repertoires

Antibodies are created and refined by somatic evolution in B cell populations, which endows the human immune system with the ability to recognize and eliminate diverse pathogens. However, the evolutionary processes that sculpt antibody repertoires remain poorly understood. Here, using an unbiased repertoire-scale approach, we show that the population genetic signatures of evolution are evident in human B cell lineages and reveal how antibodies evolve somatically. We measured the dynamics and genetic diversity of B cell responses in five adults longitudinally before and after influenza vaccination using high-throughput antibody repertoire sequencing. We identified vaccine-responsive B cell lineages that carry signatures of selective sweeps driven by positive selection, and discovered that they often display evidence for selective sweeps favoring multiple subclones. We also found persistent B cell lineages that exhibit stable population dynamics and carry signatures of neutral drift. By exploiting the relationship between B cell fitness and antibody binding affinity, we demonstrate the potential for using phylogenetic approaches to identify antibodies with high binding affinity. This quantitative characterization reveals that antibody repertoires are shaped by an unexpectedly broad spectrum of evolutionary processes and shows how signatures of evolutionary history can be harnessed for antibody discovery and engineering.

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Age-associated changes in the circulating human antibody repertoire are upregulated in autoimmunity

Immunity & Ageing ◽

10.1186/s12979-020-00193-x ◽

2020 ◽

Vol 17 (1) ◽

Author(s):

Aaron Arvey ◽

Michael Rowe ◽

Joseph Barten Legutki ◽

Gang An ◽

Anantha Gollapudi ◽

...

Keyword(s):

Autoimmune Disease ◽

Regression Model ◽

Serum Antibody ◽

Antibody Repertoire ◽

Human Antibody ◽

Humoral Immune ◽

Peptide Microarrays ◽

Highly Correlated ◽

Circulating Antibody

Abstract Background The immune system undergoes a myriad of changes with age. While it is known that antibody-secreting plasma and long-lived memory B cells change with age, it remains unclear how the binding profile of the circulating antibody repertoire is impacted. Results To understand humoral immunity changes with respect to age, we characterized serum antibody binding to high density peptide microarrays in a diverse cohort of 1675 donors. We discovered thousands of peptides that bind antibodies in age-dependent fashion, many of which contain di-serine motifs. Peptide binding profiles were aggregated into an “immune age” by a machine learning regression model that was highly correlated with chronological age. Applying this regression model to previously-unobserved donors, we found that a donor’s predicted immune age is longitudinally consistent over years, suggesting it could be a robust long-term biomarker of humoral immune ageing. Finally, we assayed serum from donors with autoimmune disease and found a significant association between “accelerated immune ageing” and autoimmune disease activity. Conclusions The circulating antibody repertoire has increased binding to thousands of di-serine peptide containing peptides in older donors, which can be represented as an immune age. Increased immune age is associated with autoimmune disease, acute inflammatory disease severity, and may be a broadly relevant biomarker of immune function in health, disease, and therapeutic intervention.

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Understanding the human antibody repertoire

mAbs ◽

10.1080/19420862.2020.1729683 ◽

2020 ◽

Vol 12 (1) ◽

pp. 1729683 ◽

Cited By ~ 8

Author(s):

Anthony R Rees

Keyword(s):

Antibody Repertoire ◽

Human Antibody

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Characteristics of quinine- and quinidine-induced antibodies specific for platelet glycoproteins IIb and IIIa

Blood ◽

10.1182/blood.v77.12.2668.2668 ◽

1991 ◽

Vol 77 (12) ◽

pp. 2668-2676 ◽

Cited By ~ 63

Author(s):

GP Visentin ◽

PJ Newman ◽

RH Aster

Keyword(s):

Immune Response ◽

Platelet Membrane ◽

The Other ◽

Membrane Glycoprotein ◽

Igg Antibodies ◽

Drug Induced ◽

Disulfide Bonding ◽

Platelet Membrane Glycoprotein ◽

Drug Dependent

Abstract Recent studies have shown that antibodies characteristic of quinine- and quinidine-induced thrombocytopenia sometimes recognize the platelet membrane glycoprotein (GP) complex IIb/IIIa in addition to their well known target, GPIb/IX. We have investigated the frequency with which drug-induced antibodies bind to GPIIb/IIIa and the nature of their target epitopes. In studies of sera from 13 patients sensitive to quinidine or quinine, we found that 10 contained IgG antibodies specific for both GPIb/IX and GPIIb/IIIa, two reacted with GPIb/IX alone, and one reacted with GPIIb/IIIa alone. In all cases, the presence of drug was required for binding of IgG to target GPs. By immunoabsorption, we found that each of five polyspecific sera contained at least two different antibodies, one reactive with GPb/IX and the other with GPIIb/IIIa. Further studies with eight drug- dependent antibodies (DDAb) specific for GPIIb/IIIa showed that three recognized the GPIIb/IIIa complex only, one recognized GPIIb alone, and three recognized GPIIIa alone. The eighth serum appeared to bind to both GPIIIa alone and to an epitope determined by the GPIIb/IIIa complex. The three antibodies specific for GPIIIa alone also reacted with GPIIIa deglycosylated with endo-H, and with the major (61 Kd) fragment obtained by chymotryptic digestion of GPIIIa but failed to react with reduced GPIIIa. These findings demonstrate that, in drug- induced, immunologic thrombocytopenia, the anti-platelet immune response is typically directed against epitopes on both GPIb/IX and GPIIb/IIIa. The three DDAb we studied that were specific for GPIIIa alone recognize epitopes resistant to chymotrypsin and endo-H treatment that are dependent on intrachain disulfide bonding.

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Human antibody repertoire frequently includes antibodies to a bacterial biofilm associated protein

PLoS ONE ◽

10.1371/journal.pone.0219256 ◽

2019 ◽

Vol 14 (7) ◽

pp. e0219256 ◽

Cited By ~ 2

Author(s):

Stefan Ryser ◽

Edgar Tenorio ◽

Angeles Estellés ◽

Lawrence M. Kauvar

Keyword(s):

Bacterial Biofilm ◽

Antibody Repertoire ◽

Human Antibody

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REDOX Reaction at ASK1-Cys250 Is Essential for Activation of JNK and Induction of Apoptosis

Molecular Biology of the Cell ◽

10.1091/mbc.e09-03-0211 ◽

2009 ◽

Vol 20 (16) ◽

pp. 3628-3637 ◽

Cited By ~ 65

Author(s):

Philippe J. Nadeau ◽

Steve J. Charette ◽

Jacques Landry

Keyword(s):

Oxidative Stress ◽

Disulfide Bonds ◽

Reductase Activity ◽

The Other ◽

Activation Loop ◽

Cysteine Oxidation ◽

Jnk Pathway ◽

Induction Of Apoptosis ◽

Thiol Reductase ◽

Jnk Activation

ASK1 cysteine oxidation allows JNK activation upon oxidative stress. Trx1 negatively regulates this pathway by reducing the oxidized cysteines of ASK1. However, precisely how oxidized ASK1 is involved in JNK activation and how Trx1 regulates ASK1 oxidoreduction remains elusive. Here, we describe two different thiol reductase activities of Trx1 on ASK1. First, in H2O2-treated cells, Trx1 reduces the various disulfide bonds generated between cysteines of ASK1 by a rapid and transient action. Second, in untreated cells, Trx1 shows a more stable thiol reductase activity on cysteine 250 (Cys250) of ASK1. After H2O2 treatment, Trx1 dissociates from Cys250, which is not sufficient to activate the ASK1-JNK pathway. Indeed, in untreated cells, a Cys250 to alanine mutant of ASK1 (C250A), which cannot bind Trx1, does not constitutively activate JNK. On the other hand, in H2O2-treated cells, this mutant (C250A) fails to activate JNK and does not induce apoptosis, although it remains fully phosphorylated on Threonine 838 (Thr838) in its activation loop. Overall, our data show that Cys250 is essential for H2O2-dependent signaling downstream from ASK1 but at a step subsequent to the phosphorylation of ASK1 Thr838. They also clarify the thiol reductase function of Trx1 on ASK1 activity.

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Isolation and Composition of the Seed Globulins of Winged Bean, Psophocarpus tetragonolobus (L.) DC

Functional Plant Biology ◽

10.1071/pp9780357 ◽

1978 ◽

Vol 5 (3) ◽

pp. 357 ◽

Cited By ~ 11

Author(s):

JM Gillespie ◽

RJ Blagrove

Keyword(s):

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Winged Bean ◽

The Other ◽

Bean Seed ◽

Psophocarpus Tetragonolobus ◽

Disulfide Bonding ◽

Single Protein ◽

Seed Globulins ◽

Sulfur Containing

The amino acid composition of winged bean seed meal is similar to that of soybean but their storage globulins are quite different. Winged bean proteins are soluble to the extent of 60% at the pH of a meal-water slurry (pH 6.6), 80% at pH 11 but only 12% at pH 5. However, the proteins are soluble to the extent of 80% from pH 5 to 9 in 10% NaCl rising to 90% at pH 11. There are no satisfactory ways of recovering all the proteins from solution by simple changes in pH or ionic strength. Winged bean seed contains major proteins with sedimentation coefficients of 2 S and 6 S. Electrophoresis on cellulose acetate resolves three globulin fractions which we have named psophocarpins A, B, and C. The proteins from these electrophoretic regions have been isolated and partially purified. Psophocarpin A is essentially a single protein comparatively rich in sulfur-containing amino acids while the other fractions are composed of a number of related components which have not been separated. When examined by SDS-polyacrylamide gel electrophoresis, the globulin fractions differed in the kind of subunit proteins they contain and in the extent of disulfide bonding. The 40 000 mol. wt subunit of psophocarpin A contains disulfide bonded chains of mol. wt 16 000 and 24 000. The proteins corresponding to the other electrophoretic regions are more complex.

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