Abstract
The Myo/Nog cell lineage was discovered in the chick embryo and is also present in adult mammalian tissues. The cells are named for their expression of mRNA for the skeletal muscle specific transcription factor MyoD and bone morphogenetic protein inhibitor Noggin. A third marker for Myo/Nog cells is the cell surface molecule recognized by the G8 monoclonal antibody (mAb). G8 has been used to detect, track, isolate and kill Myo/Nog cells. In this study, we screened a membrane proteome array for the target of the G8 mAb. The array consisted of >5,000 molecules, each synthesized in their native confirmation with appropriate post-translational modifications in a single clone of HEK-293T cells. G8 mAb binding to the clone expressing brain-specific angiogenesis inhibitor 1 (BAI1) was detected by flow cytometry, re-verified by sequencing and validated by transfection with the plasmid construct for BAI1. Further validation of the G8 target was provided by enzyme-linked immunosorbent assay. The G8 epitope was identified by screening a high-throughput, site directed mutagenesis library designed to cover 95–100% of the 954 amino acids of the extracellular domain of the BAI1 protein. The G8 mAb binds within the third thrombospondin repeat of the extracellular domain of human BAI1. Immunofluorescence localization experiments revealed that G8 and a commercially available BAI1 mAb co-localize to the subpopulation of Myo/Nog cells in the skin, eyes and brain. Expression of the multi-functional BAI1 protein in Myo/Nog cells introduces new possibilities for the roles of Myo/Nog cells in normal and diseased tissues.
Highlights
The Myo/Nog lineage was discovered in the epiblast of the chick embryo blastocyst [1,2,3]
We previously reported that G8+/Noggin+/MyoD+ Myo/Nog cells are present in low numbers in the inner and outer layers of the retina and choroid of rats and mice where they were distinct from Iba1+ microglia, GFAP+ Muller glial cells, calretinin+ ganglion cells, Chx10+ bipolar cells and photoreceptors [16, 17]
Expression of brain-specific angiogenesis inhibitor 1 (BAI1) in macrophages was recently called into question [31], previous detection of BAI1 protein in other lineages differs from its more restricted distribution mapped with G8 and R&D BAI1 monoclonal antibody (mAb) in our experiments
Summary
The Myo/Nog lineage was discovered in the epiblast of the chick embryo blastocyst [1,2,3]. The cells were identified by their co-expression of mRNA for the skeletal muscle specific transcription factor MyoD and bone morphogenetic protein (BMP) inhibitor Noggin [1,2,3]. A third marker of Myo/Nog cells is the cell surface molecule recognized by the G8 monoclonal. Myo/Nog cells express brain-specific angiogenesis 1 by Genisphere, LLC, https://genisphere.com/. Employees of Genisphere, including JB, LC, LG and RG, participated in study design, data collection and analysis, the decision to publish and preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section. The funder provided support in the form of salaries for authors
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