L1CAM is a transmembrane protein expressed on neurons that was presumed to be found on neuron-derived extracellular vesicles (NDEVs) in human biofluids. We developed a panel of single-molecule array assays to evaluate the use of L1CAM for NDEV isolation. We demonstrate that L1CAM is not associated with extracellular vesicles in human plasma or cerebrospinal fluid and therefore recommend against its use as a marker in NDEV isolation protocols.
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The data supporting the findings of this study are available within the paper and its Extended Data files. Source data are provided with this paper.
The custom Python code used in this study is available as Supplementary Software.
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We thank A. Ng for help with stem cell differentiation and J. Van Deun for help with DGC. We also thank the Taplin Biological Mass Spectrometry Facility at Harvard Medical School and the Harvard Center for Mass Spectrometry for help with proteomic experiments. This work was supported by funding from the Chan Zuckerberg Initiative Neurodegeneration Challenge Network (to D.R.W., G.M.C., A.S.C.-P.), Good Ventures (to D.R.W.), the NIH Center for Excellence in Genomic Science (to G.M.C., RM1HG008525), the Howard Hughes Medical Institute (to A.R.) and the Klarman Cell Observatory (to A.R.). These funding agencies had no role in conceptualization, design, data collection, analysis, decision to publish or preparation of the manuscript.
D.R.W. is a founder and equity holder of Quanterix. A.R. is an SAB member of Thermo Fisher Scientific, Neogene Therapeutics, Asimov and Syros Pharmaceuticals. A.R. is a cofounder of and equity holder in Celsius Therapeutics and an equity holder in Immunitas. From 1 August 2020, A.R. is an employee of Genentech. G.M.C. is a founder, consultant or advisory board member to companies listed here: http://arep.med.harvard.edu/gmc/tech.html. These companies had no influence over any aspect of this research. We have filed intellectual property on methods for EV analysis and isolation. M.N., D.T.-O. and D.R.W. filed a provisional patent for the measurement of EVs using single-molecule arrays as described in this study. Additionally, D.T.-O., E.J.K.K., A.R. and G.M.C. filed intellectual property relating to the identification and use of new candidate markers for NDEV isolation.
Peer review information Nina Vogt was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
(a) Schematic overview of a Proteinase K protection assay analyzing the integrity of EVs after fractionation with SEC, using four different conditions: a No treatment, two-hour incubation, lyse with Triton X, one-hour incubation b Proteinase K application, two-hour incubation, lyse with Triton X, one-hour incubation. c No treatment, one-hour delay, add PMSF for one hour, lyse with Triton X, one-hour incubation. d Proteinase K application, one-hour incubation, add PMSF to inhibit Proteinase K for one hour, lyse with Triton X, one-hour incubation. (b) Alix and Albumin concentrations after the Proteinase K protection assay, as measured with Simoa. Data represent the average of two technical replicates of the Simoa assay measurements from a single experiment with one sample. This experiment was conducted 3 times with similar results.
Transmission Electron Microscopy of a. Fraction 9 and b. Fraction 12 from plasma fractionated using SEC and negatively stained with uranyl formate. Representative images are shown at 6000x magnification (top) and 20,000x magnification (bottom). Arrows indicate ‘cup-shaped’ EVs. This experiment was conducted once.
Mass spectrometry analysis shows peptides mapping to different parts of the L1CAM protein. Full length sequence of L1CAM displayed with peptides detected by mass spectrometry shown in green. Blue box indicates L1CAM transmembrane domain and red box indicates amino acid sequence encoded by Exon 25. Top: full length recombinant L1CAM protein standard shows peptides matching an isoform which includes Exon 25. Bottom: Mass Spectrometry of L1CAM immunocaptured from human plasma shows peptides matching the cytosolic domain at the C terminus (emphasized with black arrow). This experiment was conducted once.
(a) L1CAM intro-exon gene structure including Exon 25, which contains the only transmembrane domain. Alternative splicing skipping Exon 25 (L1CAM isoform without a transmembrane domain) would lead to transcripts with an exon-exon junction across Exon 24 and Exon 26. (b) Reads from GTEx RNA-Seq data of human Tibial Artery loaded in Integrative Genome Browser (IGV) aligning to Exon 25 of L1CAM, which contain the transmembrane domain (highlighted in red). Aligned junction reads supporting the skipping of Exon 25 are indicated with black arrows.
Extended Data Fig. 5 Analysis of reads from GTEx RNA-Seq Data indicating Exon 25 skipping in alternative splicing of L1CAM.
Fraction of reads mapping to L1CAM isoform supporting skipping of L1CAM Exon 25 (junction reads spanning Exon 24 and Exon 26) vs. inclusion of Exon 25 from RNA-Seq GTEx data of various human organs.
Concentration of Alpha Synuclein recombinant protein captured with control (mIgG) and L1CAM (UJ12) antibodies in a pull-down experiment. Data shown is the average of two technical replicates from a single experiment.
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Norman, M., Ter-Ovanesyan, D., Trieu, W. et al. L1CAM is not associated with extracellular vesicles in human cerebrospinal fluid or plasma. Nat Methods 18, 631–634 (2021). https://doi.org/10.1038/s41592-021-01174-8