Biology subjects

Gan, S. K.

Publications and source records attributed to Gan, S. K..

10 recordsLinked to original sources

bioRxiv2023

The Influence of Variable-Heavy (VH) Chain Families on IgG2, 3, 4 on FcγRs and Antibody Superantigens Protein G and L Binding using Biolayer Interferometry

BackgroundAs the most abundant immunoglobulin in blood and the most common human isotype used for therapeutic monoclonal antibodies, the engagement and subsequent activation of its Fc receptors by IgGs are crucial for antibody function. While generally assumed to be relatively constant within subtypes, recent studies have shown the antibody variable regions to exert distal effects of modulating antibody-receptor interactions on many antibody isotypes. Such effects are also expected for IgG and its subtypes with the in-depth understanding of these V-region effects highly relevant for engineering antibodies, antibody purifications, and understanding to how robust the microbial immune evasion proteins are. MethodsIn this study, we created a panel of IgG2/IgG3/IgG4 antibodies by changing the VH family (VH1-7) frameworks while retaining the complementarity determining regions of Pertuzumab and measured the interaction of the IgGs with Fc{gamma}RIa, Fc{gamma}RIIaH167, Fc{gamma}RIIaR167, Fc{gamma}RIIb/c, Fc{gamma}RIIIaF176, Fc{gamma}RIIIaV176, Fc{gamma}RIIIbNA1, and Fc{gamma}RIIIbNA2 receptors alongside antibody superantigens proteins L and G using biolayer interferometry. ResultsThe library of 21 IgGs demonstrated that the VH frameworks influenced receptor binding sites on the constant region of the subtypes significantly, providing non-canonical interactions and non-interactions. However, there was minimal influence on the binding of bacterial B-cell superantigens Proteins L and G on the IgGs, showing their robustness against V-region effects. ConclusionsThese results demonstrate the importance of the V-regions during humanization of therapeutic antibodies that can confer or diminish FcR-dependent immune responses, while remaining both suitable and susceptible to the binding by bacterial antibody superantigens in antibody purification and be present with normal flora. STATEMENT OF SIGNIFICANCEIgGs are the predominant isotype for clinical and research applications. Despite the vast amount of research to study it, particularly on IgG1, there remains a gap in understanding how the variable regions and the receptor binding sites can influence one another in the other IgG subtypes, across the IgG subtypes with different hinges and makeup. This study investigates the effect of these variable regions on the engagement of receptors and also how bacterial antibody superantigens present in microflora and used in antibody purification can exert distal effects.

immunology↗

bioRxiv2022

Augmenting the Signal Peptide of the Ag43 Autotransporter for the improved heterologous display of sfGFP using Fluorescence-Activated Cell Sorting (FACs)-assisted natural selection.

Protein display, secretion and export in prokaryotes are essential for utilizing microbial systems as engineered living materials for medicines, biocatalysts, and protein factories. To select for improved signal peptides for Escherichia coli protein display, we utilized error-prone polymerase chain reaction (epPCR) coupled with single-cell sorting and microplate titer to generate, select, and detect improved Ag43 signal peptides. Through three rounds of mutagenesis and selection using green fluorescence from the 56 kDa sfGFP-beta-lactamase, we isolated clones that increased surface display from 1.4 to 3 folds as detected by the microplate plate-reader and native SDS-PAGE assays. To establish that the protein was displayed extracellularly, we trypsinised the bacterial cells to release the surface displayed proteins for analysis. This workflow demonstrated a fast and high-throughput method leveraging on epPCR and single-cell sorting to rapidly augment bacterial surface display, a method that could be applied to other bacterial proteins.

synthetic biology↗

bioRxiv2022

The contributory effects of the VH families and CH regions of multimeric IgM on its interaction with Fc(mu)R, and antigen.

As the primary response antibody with increasing interest as a therapeutic antibody format, IgM is also the largest antibody structure among the five major human isotypes. Spontaneously forming pentamer and hexamers, IgM has avidity effects that could compensate for weaker interactions, although steric hindrances can occur for certain epitopes. With recent evidence of the heavy chain constant region affecting antigen binding and the VH families of the V-regions affecting FcR engagement found on other isotypes, we investigated CDR-grafted Trastuzumab and Pertuzumab VH1-7 IgMs for biolayer interferometry. From our panel of the 14 IgM variants, the V-regions holistically affected FcR binding, and the IgM C-region modulated Her2 engagements with contributions from the V-regions and influences from protein L binding at the V{kappa}. These findings revealed the oligomerization effect of IgMs to play a significant role in both FcR and antigen binding that is distinct from the other isotypes that can guide the development and protein en-gineering of IgM therapeutics.

immunology↗

bioRxiv2021

More than meets the Kappa for Antibody Superantigen Protein L (PpL)

Immunoglobulin superantigens play an important role in the affinity purification of antibodies and underlie the microbiota-immune axis at mucosal areas Focussing on the Staphylococcal Protein A (SpA), Streptococcal Protein G (SpG), and the Finegoldia Protein L (PpL) that were previously thought to bind to only specific regions of human antibodies, a systematic and holistic analysis of the antibody regions using 63 antibody permutations involving six V{kappa} and seven VH region IgG1 revealed showed novel PpL-antibody interactions. While SpA and SpG showed relatively consistent interactions with the antibodies, our findings showed PpL binding to certain VH-V{kappa}2, 5 and 6 interactions had contribution by other antibody regions. The findings of this have implications on PpL-based affinity antibody purifications and antibody design as well as provides novel insights to PpL-based microbiota-immune axis effects.

immunology↗

bioRxiv2021

Engaging the A-Class Antibody: Variable-Heavy (VH) region influencing IgA1 engagement of FcαRI and superantigen proteins G, A, and L.

Interest in IgA as an alternative therapeutic and diagnostic antibody has increased over the years, yet much remains to be investigated especially given their importance in activating immune cells in blood and in mucosal immunity. Recent whole antibody-based investigations have shown significant distal effects between the variable (V) and constant (C)-regions that can be mitigated by the different hinge regions of the human IgA subtypes A1 and A2. Diving deeper into the mechanisms underlying this, systematic VH manipulations retaining the CDRs were performed on a panel of 28 IgA1s and A2s across the Trastuzumab and Pertuzumab models, revealed distal effects on FcRI binding. Further insights from structural modelling showed these effects to also be mitigated by the differing glycosylation patterns in IgA1 and 2 to explain reversal of trends of IgA1s and 2s effected by slight changes in the CDRs. IgAs bound at the Fc showed similar trends but magnitudes better binding to Her2 with that bound by ppL, showing that ppL can sterically hinder Her2 antigen binding. Contrary to canonical knowledge, we found strong evidence of IgAs binding SpG that was narrowed to be at the CH2-3 region, and that the likely binding with SpA was beyond VH3 FWR and most likely at the CH1. VH1 was found to be the most suitable framework (FWRs) for CDR-grafting for both IgA1 and 2. With relevance to interactions with the microbiome at mucosal surfaces, mechanistic insight of how these IgAs can interact bacterial superantigens proteins G, A, and L are also discovered for potential future interventions. One Sentence SummaryAn insight into the mechanism of distal V-region effects on FCAR and superantigens proteins G, A, and L by both IgA1 and A2.

immunology↗

bioRxiv2020

Antibody Signal peptides and essential amino acid counts on recombinant antibody production

Boosting the production of recombinant therapeutic antibodies is crucial in both academic and industry settings. In this work, we investigated the usage of varying signal peptides by antibody genes and their roles in recombinant transient production. Comparing myeloma and the native signal peptides of both heavy and light chains in 168 antibody permutation variants, we performed a systematic analysis, finding amino acids counts to be involved in antibody production to construct a model for predicting co-transfection transient recombinant antibody production rates using the HEK293 system. The findings also provide insights into the usage of the large repertoire of antibody signal peptides.

molecular biology↗

bioRxiv2020

The Structural Basis for Gag Non-Cleavage Site Mutations in Determining HIV-1 Viral Fitness

The high mutation rate in retroviruses is one of the leading causes of drug resistance. In human immunodeficiency virus type-1 (HIV-1), synergistic mutations in its protease and the protease substrate - the Group-specific antigen (Gag) polyprotein - work together to confer drug resistance against protease inhibitors and compensate the mutations affecting viral fitness. Some Gag mutations can restore Gag-protease binding, yet most Gag-protease correlated mutations occur outside of the Gag cleavage site. To investigate the molecular basis for this, we now report multiscale modelling approaches to investigate various sequentially cleaved Gag products in the context of clinically relevant mutations that occur outside of the cleavage sites, including simulations of the largest Gag proteolytic product in its viral membrane-bound state. We found that some mutations, such as G123E and H219Q, involve direct interaction with cleavage site residues to influence their local environment, while certain mutations in the matrix domain lead to the enrichment of lipids important for Gag targeting and assembly. Collectively, our results reveal why non-cleavage site mutations have far-reaching implications outside of Gag proteolysis, with important consequences for drugging Gag maturation intermediates and tackling protease inhibitor resistance.

microbiology↗

bioRxiv2020

Plastic agriculture using worms: Augmenting polystyrene consumption and using frass for plant growth towards a zero-waste circular economy

Polystyrene (PS) is one of the major plastics contributing to environmental pollution with its durability and resistance to natural biodegradation. Recent research showed that meal-worms (Tenebrio molitor) and superworms (Zophobas morio) are naturally able to consume PS as a carbon food source and degrade them without observable toxic effects. In this study, we explored the effects of possible food waste contamination and use of worm frass as potential plant fertilizers. We found that small amounts of sucrose and bran increased PS consumption and that the worm frass alone could support dragon fruit cacti (Hylocereus undatus) growth, with superworm frass in particular, supporting better growth and rooting than mealworm frass and control media over a fortnight. As known fish and poultry feed, these finding present worms as a natural solution to simultaneously tackle both the global plastic problem and urban farming issue in a zero-waste sustainable bioremediation cycle.

ecology↗

bioRxiv2019

Probability of Change in Life: amino acid changes in single nucleotide substitutions

Mutations underpin the processes in life, be it beneficial or detrimental. While mutations are assumed to be random in the bereft of selection pressures, the genetic code has underlying computable probabilities in amino acid phenotypic changes. With a wide range of implications including drug resistance, understanding amino acid changes is important. In this study, we calculated the probabilities of substitutions mutations in the genetic code leading to the 20 amino acids and stop codons. Our calculations reveal an enigmatic in-built self-preserving organization of the genetic code that averts disruptive changes at the physicochemical properties level. These changes include changes to start, aromatic, negative charged amino acids and stop codons. Our findings thus reveal a statistical mechanism governing the relationship between amino acids and the universal genetic code.

molecular biology↗

bioRxiv2019

Inhibiting HIV-1 and MMLV Reverse Transcriptase: The potential of an Allosteric Broad-Spectrum Inhibitor

HIV drug resistance continues to demand for alternative drug targets. Since Reverse Transcriptase (RT) is unique and critical for the virus life cycle, it is a rational target that is likely to have less off-target effects in humans. Serendipitously, we found two chemical compound scaffolds from the NCI Diversity Set V that inhibited the HIV1- RT catalytic activity. Computational structural analyses and subsequent experimental testing demonstrated that one of the two chemical scaffolds binds to a novel location in the HIV-1 RT p51 subunit, interacting with residue Y183 that has no known association with previously reported drug resistance. This finding leads to the notion of a novel druggable site on p51 for a new class of non-nucleoside RT Inhibitors that may inhibit HIV-1 RT allosterically. Although inhibitory activity was shown experimentally only to be in the hundreds micromolar range, the scaffolds serve as a proof-of-concept of targeting HIV RT p51, with the possibility for medical chemistry methods to be applied to improve the inhibitory activity, towards a functioning drug.

microbiology↗