Direct Capture of Functional Proteins from Mammalian Plasma Membranes into Nanodiscs

Mammalian plasma membrane proteins make up the largest class of drug targets yet are difficult to study in a cell free system because of their intransigent nature. Herein, we perform direct encapsulation of plasma membrane proteins derived from mammalian cells into a functional nanodisc library. Peptide fingerprinting was used to analyze the proteome of the incorporated proteins in nanodiscs and to further demonstrate that the lipid composition of the nanodiscs directly affects the class of protein that is incorporated. Furthermore, the functionality of the incorporated membrane proteome was evaluated by measuring the activity of membrane proteins: Na⁺/K⁺-ATPase and receptor tyrosine kinases. This work is the first report of the successful establishment and characterization of a cell free functional library of mammalian membrane proteins into nanodiscs

Table shows the percentage of proteins in each category in each cell membrane.

<p>Table 1 represents the calculations reflected in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183930#pone.0183930.g001" target="_blank">Fig 1</a>. HOS and POS are the non-metastatic human and canine cell lines and 143B and HMPOS are the metastatic human and canine cell lines respectively.</p

Immunohistochemistry of paired canine primary (left) and metastatic (right) in two different patient samples showing the expression of (A-B) CD44 and (C-D) CD147.

<p>p values for A and B p < 0.05 with metastatic > primary. p values for C p < 0.01 with metastatic > primary. Details of quantification are indicated in SI.</p

Table shows the relative emPAI of selected differentially regulated proteins in the human and canine osteosarcoma cells.

<p>HOS and POS are the non-metastatic human and canine cell lines and 143B and HMPOS are the metastatic human and canine cell lines respectively. emPAI of each protein is normalized to total emPAI. The ratios 143B/HOS and HMPOS/POS show fold change in expression.</p

Flow cytometry fluorescence levels showing the expression of (A) CD44 and (B) CD147.

<p>Cell pellet immunohistochemistry showing the expression of (C) CD44 and (D) CD147. HOS and POS are the non- metastatic human and canine cell lines and 143B and HMPOS are the metastatic human and canine cell lines respectively. Details of quantification are indicated in SI.</p

Fig shows global membrane proteomic composition across various cell lines classified according to function.

<p>The colors are as mentioned in the key. HOS and POS are the non- metastatic human and canine cell lines and 143B and HMPOS are the metastatic human and canine cell lines respectively.</p

Comparative proteomic investigation of metastatic and non-metastatic osteosarcoma cells of human and canine origin

<div><p>Osteosarcoma is the most common bone cancer in dogs and people. In order to improve clinical outcomes, it is necessary to identify proteins that are differentially expressed by metastatic cells. Membrane bound proteins are responsible for multiple pro-metastatic functions. Therefore characterizing the differential expression of membranous proteins between metastatic and non-metastatic clonal variants will allow the discovery of druggable targets and consequently improve treatment methodology. The objective of this investigation was to systemically identify the membrane-associated proteomics of metastatic and non-metastatic variants of human and canine origin. Two clonal variants of divergent <i>in vivo</i> metastatic potential from human and canine origins were used. The plasma membranes were isolated and peptide fingerprinting was used to identify differentially expressed proteins. Selected proteins were further validated using western blotting, flow cytometry, confocal microscopy and immunohistochemistry. Over 500 proteins were identified for each cell line with nearly 40% of the proteins differentially regulated. Conserved between both species, metastatic variants demonstrated significant differences in expression of membrane proteins that are responsible for pro-metastatic functions. Additionally, CD147, CD44 and vimentin were validated using various biochemical techniques. Taken together, through a comparative proteomic approach we have identified several differentially expressed cell membrane proteins that will help in the development of future therapeutics.</p></div

Western blot showing the expression of (A) CD44, (B) CD147 and (C) Vimentin.

<p>Lanes from left to right: ladder, HOS, 143B, POS and HMPOS. HOS and POS are the non-metastatic human and canine cell lines and 143B and HMPOS are the metastatic human and canine cell lines respectively.</p

Fig shows the relative GPCR activity as measured by cAMP concentration.

<p>The experiment was performed in biological triplicates (and technical duplicates) with n = 2 (total separate trials). **** p <0.0001 and ** p<0.01.</p

Flow cytometry fluorescence levels showing the expression of (A) CD44 and (B) CD147.

<p>Cell pellet immunohistochemistry showing the expression of (C) CD44 and (D) CD147. HOS and POS are the non- metastatic human and canine cell lines and 143B and HMPOS are the metastatic human and canine cell lines respectively. Details of quantification are indicated in SI.</p