Biophysical and biochemical characterization of a VHH-based IgG-like bi- and trispecific antibody platform.
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ABSTRACT:
SUBMITTER: Pekar L
PROVIDER: S-EPMC7531565 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Evolving specific molecular recognition function of proteins requires strategic navigation of a complex mutational landscape. Protein scaffolds aid evolution via a conserved platform on which a modular paratope can be evolved to alter binding specificity. Although numerous protein scaffolds have been discovered, the underlying properties that permit binding evolution remain unknown. We present an algorithm to predict a protein scaffold's ability to evolve novel binding function based upon computationally calculated biophysical parameters. The ability of 17 small proteins to evolve binding functionality across seven discovery campaigns was determined via magnetic activated cell sorting of 1010 yeast-displayed protein variants. Twenty topological and biophysical properties were calculated for 787 small protein scaffolds and reduced into independent components. Regularization deduced which extracted features best predicted binding functionality, providing a 4/6 true positive rate, a 9/11 negative predictive value, and a 4/6 positive predictive value. Model analysis suggests a large, disconnected paratope will permit evolved binding function. Previous protein engineering endeavors have suggested that starting with a highly developable (high producibility, stability, solubility) protein will offer greater mutational tolerance. Our results support this connection between developability and evolvability by demonstrating a relationship between protein production in the soluble fraction of Escherichia coli and the ability to evolve binding function upon mutation. We further explain the necessity for initial developability by observing a decrease in proteolytic stability of protein mutants that possess binding functionality over nonfunctional mutants. Future iterations of protein scaffold discovery and evolution will benefit from a combination of computational prediction and knowledge of initial developability properties.
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Project description:Antibodies raised in Indian rhesus macaques [Macaca mulatta (MM)] in many preclinical vaccine studies are often evaluated in vitro for titer, antigen-recognition breadth, neutralization potency, and/or effector function, and in vivo for potential associations with protection. However, despite reliance on this key animal model in translation of promising candidate vaccines for evaluation in first in man studies, little is known about the properties of MM immunoglobulin G (IgG) subclasses and how they may compare to human IgG subclasses. Here, we evaluate the binding of MM IgG1, IgG2, IgG3, and IgG4 to human Fc gamma receptors (Fc?R) and their ability to elicit the effector functions of human Fc?R-bearing cells, and unlike in humans, find a notable absence of subclasses with dramatically silent Fc regions. Biophysical, in vitro, and in vivo characterization revealed MM IgG1 exhibited the greatest effector function activity followed by IgG2 and then IgG3/4. These findings in rhesus are in contrast with the canonical understanding that IgG1 and IgG3 dominate effector function in humans, indicating that subclass-switching profiles observed in rhesus studies may not strictly recapitulate those observed in human vaccine studies.
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Project description:Background The critical role of antibody Fc-mediated effector functions in immune defense has been widely reported in various viral infections. These effector functions confer cellular responses through engagement with innate immune cells. The precise mechanism(s) by which immunoglobulin G (IgG) Fc domain and cognate receptors may afford protection are poorly understood, however, in the context of HIV/SHIV infections. Many different in vitro assays have been developed and utilized to measure effector functions, but the extent to which these assays capture distinct antibody activities has not been fully elucidated. Results In this study, six Fc-mediated effector function assays and two biophysical antibody profiling assays were performed on a common set of samples from HIV-1 infected and vaccinated subjects. Biophysical antibody profiles supported robust prediction of diverse IgG effector functions across distinct Fc-mediated effector function assays. While a number of assays showed correlated activities, supervised machine learning models indicated unique antibody features as primary contributing factors to the associated effector functions. Additional experiments established the mechanistic relevance of relationships discovered using this unbiased approach. Conclusions In sum, this study provides better resolution on the diversity and complexity of effector function assays, offering a clearer perspective into this family of antibody mechanisms of action to inform future HIV-1 treatment and vaccination strategies. Supplementary Information The online version contains supplementary material available at 10.1186/s12977-021-00579-9.
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Project description:Filoviruses (family Filoviridae) include five ebolaviruses and Marburg virus. These pathogens cause a rapidly progressing and severe viral disease with high mortality rates (generally 30-90%). Outbreaks of filovirus disease are sporadic and, until recently, were limited to less than 500 cases. However, the 2013-2016 epidemic in western Africa, caused by Ebola virus (EBOV), illustrated the potential of filovirus outbreaks to escalate to a much larger scale (over 28,000 suspected cases). mAbs against the envelope glycoprotein represent a promising therapeutic platform for managing filovirus infections. However, mAbs that exhibit neutralization or protective properties against multiple filoviruses are rare. Here we examined a panel of engineered bi- and trispecific antibodies, in which variable domains of mAbs that target epitopes from multiple filoviruses were combined, for their capacity to neutralize viral infection across filovirus species. We found that bispecific combinations targeting EBOV and Sudan virus (another ebolavirus), provide potent cross-neutralization and protection in mice. Furthermore, trispecific combinations, targeting EBOV, Sudan virus, and Marburg virus, exhibited strong neutralization potential against all three viruses. These results provide important insights into multispecific antibody engineering against filoviruses and will inform future immunotherapeutic discoveries.
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Project description:Cytokine-like protein 1 (CYTL1) is a small widely expressed secreted protein lacking significant primary sequence homology to any other known protein. CYTL1 expression appears to be highest in the hematopoietic system and in chondrocytes; however, maintenance of cartilage in mouse models of arthritis is its only reported function in vivo. Despite lacking sequence homology to chemokines, CYTL1 is predicted by computational methods to fold like a chemokine, and has been reported to function as a chemotactic agonist at the chemokine receptor CCR2 in mouse monocyte/macrophages. Nevertheless, since chemokines are defined by structure and chemokine receptors are able to bind many non-chemokine ligands, direct determination of the CYTL1 tertiary structure will ultimately be required to know whether it actually folds as a chemokine and therefore is a chemokine. Towards this goal, we have developed a method for producing functional recombinant human CYTL1 in bacteria, and we provide new evidence about the biophysical and biochemical properties of recombinant CYTL1. Circular dichroism analysis showed that, like chemokines, CYTL1has a higher content of beta-sheet than alpha-helix secondary structure. Furthermore, recombinant CYTL1 promoted calcium flux in chondrocytes. Nevertheless, unlike chemokines, CYTL1 had limited affinity to proteoglycans. Together, these properties further support cytokine-like properties for CYTL1 with some overlap with the chemokines.
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Project description:?-glutamylcysteine synthetase (Gcs) is a vital enzyme catalyzing the first and rate limiting step in the trypanothione biosynthesis pathway, the ATP-dependent ligation of L-Glutamate and L-Cysteine to form gamma-glutamylcysteine. The Trypanothione biosynthesis pathway is unique metabolic pathway essential for trypanosomatid survival rendering Gcs as a potential drug target. Here we report the cloning, expression, purification and characterization of L. donovani Gcs. Three other constructs of Gcs (GcsN, GcsC and GcsT) were designed on the basis of S. cerevisiae and E. coli Gcs crystal structures. The study shows Gcs possesses ATPase activity even in the absence of substrates L-glutamate and L-Cysteine. Divalent ions however plays an indispensable role in LdGcs ATPase activity. Isothermal titration calorimetry and fluorescence studies illustrates that L. donovani Gcs binds substrate in order ATP >L-glutamate>L-cysteine with Glu 92 and Arg 498 involved in ATP hydrolysis and Glu 92, Glu 55 and Arg 498 involved in glutamate binding. Homology modeling and molecular dynamic simulation studies provided the structural rationale of LdGcs catalytic activity and emphasized on the possibility of involvement of three Mg2+ ions along with Glutamates 52, 55, 92, 99, Met 322, Gln 328, Tyr 397, Lys 483, Arg 494 and Arg 498 in the catalytic function of L. donovani Gcs.
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Project description:Enolase, which catalyses the conversion of 2-phospho-D-glycerate to phosphoenolpyruvate, is an important enzyme in the classic glycolysis pathway in cells. Enolase is highly conserved in organisms from bacteria to humans, indicating its importance in cells. Thus, enolase is a good target for developing new drugs. In the last decade, new functions of this enzyme have been found. Helicobacter pylori is a common human pathogen that causes gastric diseases and even gastric cancer. In this study, the sequence of H. pylori enolase (HpEno) was analysed; the conservation (at least partial) of binding sites for cofactor, plasminogen, and host extracellular RNA, as well as catalytic site, indicates that HpEno should be capable of performing the functions. Recombinant HpEno was overexpressed and purified from E. coli. Compared to the enolases from other species, HpEno had similar characteristics for its secondary structure. The temperature-induced profiles indicate that HpEno is quite stable to temperature, compared to other homologs. Regarding the kinetics of the unfolding reaction, we found that the activation enthalpy associated with the thermal unfolding reaction is equivalent to the reported activation enthalpy for yeast enolase, indicating a similar scaffold and kinetic stability. Although a wide range of experimental conditions were assayed, it was not possible to detect any enzymatic activity of HpEno. To prove the lack of activity, still a much wider range of experiments should be carried out.
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Project description:The human enzyme D-3-phosphoglycerate dehydrogenase (hPHGDH) catalyzes the reversible dehydrogenation of 3-phosphoglycerate (3PG) into 3-phosphohydroxypyruvate (PHP) using the NAD+/NADH redox cofactor, the first step in the phosphorylated pathway producing L-serine. We focused on the full-length enzyme that was produced in fairly large amounts in E. coli cells; the effect of pH, temperature and ligands on hPHGDH activity was studied. The forward reaction was investigated on 3PG and alternative carboxylic acids by employing two coupled assays, both removing the product PHP; 3PG was by far the best substrate in the forward direction. Both PHP and α-ketoglutarate were efficiently reduced by hPHGDH and NADH in the reverse direction, indicating substrate competition under physiological conditions. Notably, neither PHP nor L-serine inhibited hPHGDH, nor did glycine and D-serine, the coagonists of NMDA receptors related to L-serine metabolism. The investigation of NADH and phosphate binding highlights the presence in solution of different conformations and/or oligomeric states of the enzyme. Elucidating the biochemical properties of hPHGDH will enable the identification of novel approaches to modulate L-serine levels and thus to reduce cancer progression and treat neurological disorders.
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Project description:The modulation of mRNA turnover has been increasingly recognized as a hotpoint for gene expression regulation at the post-transcriptional level. In eukaryotic cells, most mRNAs are degraded via the deadenylation-dependent pathway, in which the removal of the poly(A) tail is the initial and rate-limiting step. Caf1, a deadenylase specifically degrades poly(A) from the 3'-end, is highly conserved from yeast to mammalians. Caf1s in higher plants have been shown to be involved in plant development and stress response. However, little is known about the biochemical and biophysical properties of Caf1s in plants. In this research, we cloned the crcaf1 gene from Chlamydomonas reinhardtii and studied the properties of the recombinant proteins. The results showed that CrCaf1 was a deadenylase with conserved sequence motifs, structural features, and catalytic properties of the Caf1 family. CrCaf1 degraded poly(A) in a distributive mode with the optimal reacting conditions at pH 7 and 35°C. CrCaf1 had similar activity when coordinated with Mg(2+) and Mn(2+), while the enzyme bound to Ca(2+) or Zn(2+) was almost inactivated. Zn(2+) could induce CrCaf1 aggregation with the disruption of the native structure, while Mg(2+), Mn(2+) and Ca(2+) could stabilize CrCaf1 against thermal denaturation by reducing protein aggregation. Among the various metal ions, Mn(2+) showed the strongest protective effect on CrCaf1 stability, implying that Mn(2+) might play a role in regulating CrCaf1 stability in the C. reinhardtii cells under some stressed conditions. These findings provide a starting point for further investigation of the physiological functions of CrCaf1 in C. reinhardtii.
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Project description:Transmissible gastroenteritis coronavirus (TGEV) is one of the most destructive agents, responsible for the enteric infections that are lethal for suckling piglets, causing enormous economic loss to the porcine fostering industry every year. Although it has been known that TGEV spiker protein is essential for the viral entry for many years, the detail knowledge of the TGEV fusion protein core is still very limited. Here, we report that TGEV fusion core (HR1-SGGRGG-HR2), in vitro expressed in GST prokaryotic expression system, shares the typical properties of the trimer of coiled-coil heterodimer (six alpha-helix bundle), which has been confirmed by a combined series of biochemical and biophysical evidences including size exclusion chromatography (gel-filtration), chemical crossing, and circular diagram. The 3D homologous structure model presents its most likely structure, extremely similar to those of the coronaviruses documented. Taken together, TGEV spiker protein belongs to the class I fusion protein, characterized by the existence of two heptad-repeat (HR) regions, HR1 and HR2, and the present knowledge about the truncated TGEV fusion protein core may facilitate in the design of the small molecule or polypeptide drugs targeting the membrane fusion between TGEV and its host.
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