Role of FcαR EC2 region in extracellular membrane localization

Phua, Ser-Xian; Lua, Wai-Heng; Gan, Samuel Ken‐En

doi:10.1080/15384101.2018.1444236

Cited by 8 publications

(9 citation statements)

References 6 publications

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“…Fab or Fv, and not on IgA. Thus, together with our other findings [26,27,45,46] also involving other proteins, we have shown the need to examine proteins as a whole for more comprehensive holistic investigations, especially on allostery. In particular to the context of whole antibody, we found the hinge rigidity and the allosteric communications between the distant constant and variable regions both to contribute to antigen binding.…”

Section: Discussionsupporting

confidence: 54%

Allosteric Effects between the Antibody Constant and Variable Regions: A Study of IgA Fc Mutations on Antigen Binding

Lua

Ling

et al. 2018

Preprint

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Therapeutics antibodies have increasingly shifted the paradigm of disease treatments, from small molecules to biologics, especially in cancer therapy. Despite the increasing number of antibody candidates, much remains unknown about the antibody and how its various regions interact. In fact, the constant region can govern effects that might be useful in reducing the unwanted consequences resulted from systemic circulation. For this reason, apart from the commonly used IgG isotypes, IgA antibodies are promising therapeutics drugs, given its localized mucosal effects. While the antibody Fc effector cell activity has been well explored, recent research has shown evidences that the constant region of the antibody can also influence antigen binding, challenging the conventional idea of region-specific antibody functions. To further investigate this, we analyzed the IgA antibody constant and its allosteric effects onto the antigen binding regions, using recombinant Pertuzumab IgA1 and IgA2 variants. We found mutations in the C-region to reduce Her2 binding, and our computational structural analysis showed that such allosteric communications were highly dependent on the antibody hinge, providing the evidence to consider antibodies as a whole protein rather than a sum of functional regions.

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Section: Discussionsupporting

confidence: 54%

Allosteric Effects between the Antibody Constant and Variable Regions: A Study of IgA Fc Mutations on Antigen Binding

Lua

Ling

et al. 2018

Preprint

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“…It should also be noted that many of these previous studies were performed on antibody fragments, e.g., Fab or Fv, or on IgG, and not on the whole IgA. Thus, together with our other findings [25,26,46,47] also involving other proteins, we have shown the need to examine proteins as a whole for comprehensive holistic investigations, especially on allostery. In the context of the whole antibody, we found that the hinge rigidity and the allosteric communications between the distant constant and variable regions both contribute to antigen binding.…”

Section: Discussionmentioning

confidence: 49%

Allosteric Effects between the Antibody Constant and Variable Regions: A Study of IgA Fc Mutations on Antigen Binding

Lua

Ling

et al. 2018

Antibodies

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Therapeutic antibodies have shifted the paradigm of disease treatments from small molecules to biologics, especially in cancer therapy. Despite the increasing number of antibody candidates, much remains unknown about the antibody and how its various regions interact. Recent findings showed that the antibody constant region can govern localization effects that are useful in reducing side effects due to systemic circulation by the commonly used IgG isotypes. Given their localized mucosal effects, IgA antibodies are increasingly promising therapeutic biologics. While the antibody Fc effector cell activity has been a focus point, recent research showed that the Fc could also influence antigen binding, challenging the conventional idea of region-specific antibody functions. To investigate this, we analysed the IgA antibody constant region and its distal effects on the antigen binding regions using recombinant Pertuzumab IgA1 and IgA2 variants. We found that mutations in the C-region reduced Her2 binding experimentally, and computational structural analysis showed that allosteric communications were highly dependent on the antibody hinge, providing strong evidence that we should consider antibodies as whole proteins rather than a sum of functional regions.

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“…In-house competent E. coli DH5a cells were chemically transformed 16 with two ampicillin-resistant plasmids, pTT5 17 and pET21d (Thermo Fisher Scientific, Singapore). These plasmids hold the following gene inserts: HIV-1 protease (Hprot, accession number: AY622223.1), HIV-1 Gag (Hgag 18 ), human CD89 (Fc fragment of IgA receptor FCAR, accession number: NM_002000.4, 19,20 ), and human CD32 (Fc fragment of IgG receptor IIa FCG2A, accession number: NM_001136219.3 21 ).…”

Section: Bacterial Strains and Plasmidsmentioning

confidence: 99%

To Plate or to Simply Unfreeze, That Is the Question for Optimal Plasmid Extraction

Thean¹,

Ong²,

Heng³

et al. 2021

J Biomol Tech

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Many molecular biology applications require fast plasmid DNA extraction, spurring multiple studies on how to speed up the process. It is regularly instructed in standard laboratory protocols to plate out frozen glycerol bacterial stocks prior to bacteria incubation in liquid media and subsequent plasmid extraction, although the rationale for this is often unexplained (other than for the isolation of single colonies). Given the commonality and importance of this laboratory operation, such a practice is time-consuming and laborious. To study the impact of this practice and the alternative direct culturing method, we investigated the association between bacterial cell mass and its potential influence on plasmid yields from the 2 methods. Our results showed no difference with preplating for 7 out of 8 plasmid constructs used in the study, suggesting that direct glycerol recovery would not lead to poorer plasmid yields. The findings support the rationale for direct glycerol recovery for plasmid extraction, without the need of an intermediate preplating step.

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Role of FcαR EC2 region in extracellular membrane localization

Cited by 8 publications

References 6 publications

Allosteric Effects between the Antibody Constant and Variable Regions: A Study of IgA Fc Mutations on Antigen Binding

Allosteric Effects between the Antibody Constant and Variable Regions: A Study of IgA Fc Mutations on Antigen Binding

Allosteric Effects between the Antibody Constant and Variable Regions: A Study of IgA Fc Mutations on Antigen Binding

To Plate or to Simply Unfreeze, That Is the Question for Optimal Plasmid Extraction

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