Difference between revisions of "Part:BBa K4040000"

Latest revision as of 03:41, 10 October 2021

Intracellular Domain of the MEGF10 Protein

Sequence and Features

Usage and Biology

MEGF10 is a membrane receptor involved in phagocytosis by macrophages and astrocytes of apoptotic cells. Receptor for C1q, an eat-me signal, that binds phosphatidylserine expressed on the surface of apoptotic cells[1]. It cooperates with ABCA1 within the process of engulfment. Promotes the formation of large intracellular vacuoles and may be responsible for the uptake of amyloid-beta peptides[2]. and is necessary for astrocyte-dependent apoptotic neuron clearance in the developing cerebellum[3]. MEGF10 plays role in muscle cell proliferation, adhesion and motility. Is also an essential factor in the regulation of myogenesis. Controls the balance between skeletal muscle satellite cells proliferation and differentiation through regulation of the notch signaling pathway[4]. It may also function in the mosaic spacing of specific neuron subtypes in the retina through homotypic retinal neuron repulsion. Mosaics provide a mechanism to distribute each cell type evenly across the retina, ensuring that all parts of the visual field have access to a full set of processing elements[5-10].

Figure 1. Structure of MEGF10.

Background and detail description

Used in our project

The synthetic receptors were constructed to contain an scFv derived from an antibody recognizing the virus spike protein, CR3022, which has been reported to bind with the receptor-binding domain of the SARS-CoV-2 S glycoprotein with high affinity, and the CD8 transmembrane domain present in the aCD19 CAR for T cells. For the cytoplasmic domains, we used the common g subunit of Fc receptors (CARg), MEGF10 (CARMEGF10), MERTK(CARMERTK) and CD3z (CARz) in our study[11].

These cytoplasmic domains are capable of promoting phagocytosis by macrophages.More details and experimental results can be found in CAR-MEGF10(BBa_K4040015).

Used in a CAR

It is found that CAR-PMegf10macrophages engulfed CD19 beads and beads containing 10% phosphatidylserine and the adhesion molecule ICAM-1 at a similar frequency (Figure 2). This indicates that the CAR-P is comparably efficient to the endogenous system[12].

Figure 2. J774A.1 macrophages expressing the aCD19 CAR-PMegf10(green) were fed beads of various sizes (magenta, diameter of bead indicated below image). The beads were covered in a supported lipid bilayer ligated to His-tagged CD19 extracellular domain and the number of beads engulfed per cell is reported below each image (magenta bars indicate CAR-PMegf10macrophages and pink bars indicate CAR-PGFP). The aCD19 CAR-PMegf10 macrophages were also incubated with 10 mm beads coated in phosphatidylserine (PS) and ICAM-1 (blue bar in graph, 51/390 cells engulfed a bead). The fraction of cells engulfing a CD19 bead is as follows: 135/169 CAR-PMegf10and 134/187 CAR-PGFPcells engulfed 2.5 um bead, 126/395 CAR-PMegf10 and 112/499 CAR-PGFPcells engulfed a 5 um bead, 48/377 CAR-PMegf10and 21/378 CAR-PGFPcells engulfed a 10 um bead, 120/706 CAR-PMegf10and 45/675 CAR-PGFPcells engulfed a 15 um bead, 194/760 CAR-PMegf10and 23/587 CAR-PGFPcells engulfed a 20 um bead (data is pooled from at least three separate experiments). Error bars denote 95% confidence intervals of the mean. *** indicates p<0.0001 respectively by Mann-Whitney test. All scale bars represent 5 mm.

To determine if the CAR-PMegf10initiates active signaling at the synapse between the macrophage and target, previous study has stained for phosphotyrosine. Macrophages expressing CAR-PMegf10 exhibited an increase in phosphotyrosine at the synapse, while macrophages expressing CAR-PGFPdid not show this enrichment (Figure 3). Consistent with previous reports, they found that F-actin also was enriched at the cell bead synapse (Figure 3). This result suggests that CARPMegf10 initiates engulfment through a localized signaling cascade involving tyrosine phosphorylation.[12]

Figure 3. A phosphorylated ITAM at the cell-target synapse drives engulfment. Macrophages expressing aCD19 CAR-PMegf10(green, top) or aCD19 CAR-PGFPwere incubated with CD19-ligated beads (position indicated with dotted line), fixed and stained for phosphotyrosine (magenta, top;greyscale, bottom). The fold enrichment of phosphotyrosine at the cell-bead synapse compared to the cell cortex is graphed on the right (n>=11; each dot represents one cell-bead synapse; lines represent the mean ±one standard deviation).

References

[1]Iram T, Ramirez-Ortiz Z, Byrne MH, Coleman UA, Kingery ND, Means TK, Frenkel D, El Khoury J. Megf10 Is a Receptor for C1Q That Mediates Clearance of Apoptotic Cells by Astrocytes.JNeurosci.2016 May 11;36(19):5185-92. doi: 10.1523/JNEUROSCI.3850-15.2016. PMID: 27170117; PMCID: PMC4863057.

[2]Singh TD, Park SY, Bae JS, Yun Y, Bae YC, Park RW, Kim IS. MEGF10 functions as a receptor for the uptake of amyloid-β. FEBS Lett. 2010 Sep 24;584(18):3936-42. doi: 10.1016/j.febslet.2010.08.050. Epub 2010 Sep 7. PMID: 20828568.

[3]Suzuki E, Nakayama M. MEGF10 is a mammalian ortholog of CED-1 that interacts with clathrin assembly protein complex 2 medium chain and induces large vacuole formation. Exp Cell Res. 2007 Oct 15;313(17):3729-42. doi: 10.1016/j.yexcr.2007.06.015. Epub 2007 Jul 3. PMID: 17643423.

[4]Iram T, Ramirez-Ortiz Z, Byrne MH, Coleman UA, Kingery ND, Means TK, Frenkel D, El Khoury J. Megf10 Is a Receptor for C1Q That Mediates Clearance of Apoptotic Cells by Astrocytes. J Neurosci. 2016 May 11;36(19):5185-92. doi: 10.1523/JNEUROSCI.3850-15.2016. PMID: 27170117; PMCID: PMC4863057.

[5]Suzuki E, Nakayama M. The mammalian Ced-1 ortholog MEGF10/KIAA1780 displays a novel adhesion pattern. Exp Cell Res. 2007 Jul 1;313(11):2451-64. doi: 10.1016/j.yexcr.2007.03.041. Epub 2007 Apr 19. PMID: 17498693.

[6]Suzuki E, Nakayama M. MEGF10 is a mammalian ortholog of CED-1 that interacts with clathrin assembly protein complex 2 medium chain and induces large vacuole formation. Exp Cell Res. 2007 Oct 15;313(17):3729-42. doi: 10.1016/j.yexcr.2007.06.015. Epub 2007 Jul 3. PMID: 17643423.

[7]Singh TD, Park SY, Bae JS, Yun Y, Bae YC, Park RW, Kim IS. MEGF10 functions as a receptor for the uptake of amyloid-β. FEBS Lett. 2010 Sep 24;584(18):3936-42. doi: 10.1016/j.febslet.2010.08.050. Epub 2010 Sep 7. PMID: 20828568.

[8]Logan CV, Lucke B, Pottinger C, Abdelhamed ZA, Parry DA, Szymanska K, Diggle CP, van Riesen A, Morgan JE, Markham G, Ellis I, Manzur AY, Markham AF, Shires M, Helliwell T, Scoto M, Hübner C, Bonthron DT, Taylor GR, Sheridan E, Muntoni F, Carr IM, Schuelke M, Johnson CA. Mutations in MEGF10, a regulator of satellite cell myogenesis, cause early onset myopathy, areflexia, respiratory distress and dysphagia (EMARDD). Nat Genet. 2011 Nov 20;43(12):1189-92. doi: 10.1038/ng.995. PMID: 22101682.

[9]Iram T, Ramirez-Ortiz Z, Byrne MH, Coleman UA, Kingery ND, Means TK, Frenkel D, El Khoury J. Megf10 Is a Receptor for C1Q That Mediates Clearance of Apoptotic Cells by Astrocytes. J Neurosci. 2016 May 11;36(19):5185-92. doi: 10.1523/JNEUROSCI.3850-15.2016. PMID: 27170117; PMCID: PMC4863057.

[10]Saha M, Mitsuhashi S, Jones MD, Manko K, Reddy HM, Bruels CC, Cho KA, Pacak CA, Draper I, Kang PB. Consequences of MEGF10 deficiency on myoblast function and Notch1 interactions. Hum Mol Genet. 2017 Aug 1;26(15):2984-3000. doi: 10.1093/hmg/ddx189. PMID: 28498977; PMCID: PMC6075367.

[11]Fu W, Lei C, Ma Z, Qian K, Li T, Zhao J, Hu S. CAR Macrophages for SARS-CoV-2 Immunotherapy. Front Immunol. 2021 Jul 23;12:669103. doi: 10.3389/fimmu.2021.669103. PMID: 34367135; PMCID: PMC8343226.

[12]Morrissey MA, Williamson AP, Steinbach AM, Roberts EW, Kern N, Headley MB, Vale RD. Chimeric antigen receptors that trigger phagocytosis. Elife. 2018 Jun 4;7:e36688. doi: 10.7554/eLife.36688. PMID: 29862966; PMCID: PMC6008046.