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. 2021 Jun 2:15:596976.
doi: 10.3389/fnins.2021.596976. eCollection 2021.

Discovery of a Highly Conserved Peptide in the Iron Transporter Melanotransferrin that Traverses an Intact Blood Brain Barrier and Localizes in Neural Cells

Chaahat S B Singh  1   2   3   4   5 , Brett A Eyford  1   2   3   4 , Thomas Abraham  6 , Lonna Munro  1   2   3   4 , Kyung Bok Choi  1   2   3   4 , Mark Okon  7 , Timothy Z Vitalis  8 , Reinhard Gabathuler  8   9 , Chieh-Ju Lu  1   2   3   4 , Cheryl G Pfeifer  1   2   3   4 , Mei Mei Tian  8 , Wilfred A Jefferies  1   2   3   4   5   10   11   12
Affiliations

Discovery of a Highly Conserved Peptide in the Iron Transporter Melanotransferrin that Traverses an Intact Blood Brain Barrier and Localizes in Neural Cells

Chaahat S B Singh et al. Front Neurosci. .

Abstract

The blood-brain barrier (BBB) hinders the distribution of therapeutics intended for treatment of diseases of the brain. Our previous studies demonstrated that that a soluble form of melanotransferrin (MTf; Uniprot P08582; also known as p97, MFI2, and CD228), a mammalian iron-transport protein, is an effective carrier for delivery of drug conjugates across the BBB into the brain and was the first BBB targeting delivery system to demonstrate therapeutic efficacy within the brain. Here, we performed a screen to identify peptides from MTf capable of traversing the BBB. We identified a highly conserved 12-amino acid peptide, termed MTfp, that retains the ability to cross the intact BBB intact, distributes throughout the parenchyma, and enter endosomes and lysosomes within neurons, astrocytes and microglia in the brain. This peptide may provide a platform for the transport of therapeutics to the CNS, and thereby offers new avenues for potential treatments of neuropathologies that are currently refractory to existing therapies.

Keywords: MTfp; blood-brain barrer; drug delivery and targeting; glial targeting; melanotransferrin; microglial targeting; neuronal targeting; peptide transport.

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Conflict of interest statement

Bioasis Technologies Inc., (BTI) is a University of British Columbia start-up company. TZV, RG, and MMT were employees and equity holders in BTI at the time this work was undertaken. WAJ was the founding scientist and an equity holder in BTI at the time this work was undertaken. The funding sources had no influence in the study design, data collection, analysis or interpretation of data, in the writing of the manuscript or on the decision to submit for publication. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Representative deconvolved images showing localization of MTfp in the brains of mice. Cell nuclei are blue (DAPI) and capillaries are green (FITC). (A) Fluorescence (red) in the brain for a mouse treated with PBS (i.e., background fluorescence), (B) Cy5 fluorescence in the brain after IV injection with RVGp-Cy5, (C) Cy5 fluorescence in the brain after IV injection of revMTfp-Cy5, (D) Cy5 fluorescence in the brain after IV injection of MTfp-Cy5, (E) brain distribution of Cy5 in wild type mice, where values indicate total volume of Cy5 fluorescence in each tissue normalized to tissue volume (VTABPV in Supplementary Table 1). Data are shown as individual points with the error bars indicating means ± SEM (n = 3, 3 sections/animal and 4–11 fields of view per section). **p < 0.01, ****p < 0.0001.
FIGURE 2
FIGURE 2
Representative 3D confocal images showing localization of various cell types and subcellular objects with MTfp in the mouse brain sections. Cell nuclei are in blue color (DAPI) and BBBs are in green color (FITC). (A–C) Cellular localization of MTFp and (A–E) subcellular localization of MTFp. (A) Localization of neuronal marker NeuN (Pink) with MTfp (Red), (B) localization of Astrocytes marker GFAP (Pink) with MTfp (Red), (C) localization of Microglia marker TMEM (Pink) with MTfp (Red), (D) localization of Lysosomal marker LAMP1 (Pink) with MTfp (Red), (E) localization of Endosomal marker EEA1 (Pink) with MTfp (Red). White arrows indicate co-localization of MTfp (red) and cells (pink). The graphs represent the extent of localization of various cell types and subcellular objects with MTfp in the CNS as a percentage of fluorescence volume fraction. Each animal is presented as an individual data point in the graph with the error bars indicating mean ± SEM.
FIGURE 3
FIGURE 3
Proton NMR showing amide to amide proximity. Cross-peak signals suggestive of a helical structure in the C-terminus of MTfp can be seen at 10 and 20°C but all signals in this region fade as temperature rises; possibly due to proton exchange with solvent. All images represent 5 mM MTfp in 25 mM sodium phosphate buffer at pH 7 with 5% D2O. (A) 10°C. (B) 20°C. (C) 30°C. (D) 37°C.
See this image and copyright information in PMC

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