Skip to main content
Advertisement
  • Neurology.org
  • Journals
    • Neurology
    • Clinical Practice
    • Genetics
    • Neuroimmunology & Neuroinflammation
  • Specialty Sites
    • Equity, Diversity, and Inclusion
    • Innovations in Care Delivery
    • Without Borders
  • Collections
    • Topics A-Z
    • Residents & Fellows
    • Infographics
    • Patient Pages
    • Null Hypothesis
    • Translations
  • Podcast
  • CME
  • About
    • About the Journals
    • Contact Us
    • Editorial Board
  • Authors
    • Submit a Manuscript
    • Author Center

Advanced Search

Main menu

  • Neurology.org
  • Journals
    • Neurology
    • Clinical Practice
    • Genetics
    • Neuroimmunology & Neuroinflammation
  • Specialty Sites
    • Equity, Diversity, and Inclusion
    • Innovations in Care Delivery
    • Without Borders
  • Collections
    • Topics A-Z
    • Residents & Fellows
    • Infographics
    • Patient Pages
    • Null Hypothesis
    • Translations
  • Podcast
  • CME
  • About
    • About the Journals
    • Contact Us
    • Editorial Board
  • Authors
    • Submit a Manuscript
    • Author Center
  • Home
  • Articles
  • Issues

User menu

  • My Alerts
  • Log in

Search

  • Advanced search
Neurology Genetics
Home
A peer-reviewed clinical and translational neurology open access journal
  • My Alerts
  • Log in
Site Logo
  • Home
  • Articles
  • Issues

Share

December 2018; 4 (6) ArticleOpen Access

Molecular pathogenesis of human CD59 deficiency

Netanel Karbian, Yael Eshed-Eisenbach, Adi Tabib, Hila Hoizman, B. Paul Morgan, Ora Schueler-Furman, Elior Peles, Dror Mevorach
First published October 29, 2018, DOI: https://doi.org/10.1212/NXG.0000000000000280
Netanel Karbian
From the Rheumatology Research Center (N.K., A.T., H.H., D.M.), Center of Rare Diseases, and Department of Medicine, Hadassah-Hebrew University Medical Center, Jerusalem; The Weizmann Institute (Y.E.-E., E.P.), Rehovot, Israel; Systems Immunity Research Institute (B.P.M.), Cardiff University, Cardiff, Wales, UK; and Hebrew University (O.S.-F., D.M.), Jerusalem, Israel.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Yael Eshed-Eisenbach
From the Rheumatology Research Center (N.K., A.T., H.H., D.M.), Center of Rare Diseases, and Department of Medicine, Hadassah-Hebrew University Medical Center, Jerusalem; The Weizmann Institute (Y.E.-E., E.P.), Rehovot, Israel; Systems Immunity Research Institute (B.P.M.), Cardiff University, Cardiff, Wales, UK; and Hebrew University (O.S.-F., D.M.), Jerusalem, Israel.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Adi Tabib
From the Rheumatology Research Center (N.K., A.T., H.H., D.M.), Center of Rare Diseases, and Department of Medicine, Hadassah-Hebrew University Medical Center, Jerusalem; The Weizmann Institute (Y.E.-E., E.P.), Rehovot, Israel; Systems Immunity Research Institute (B.P.M.), Cardiff University, Cardiff, Wales, UK; and Hebrew University (O.S.-F., D.M.), Jerusalem, Israel.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Hila Hoizman
From the Rheumatology Research Center (N.K., A.T., H.H., D.M.), Center of Rare Diseases, and Department of Medicine, Hadassah-Hebrew University Medical Center, Jerusalem; The Weizmann Institute (Y.E.-E., E.P.), Rehovot, Israel; Systems Immunity Research Institute (B.P.M.), Cardiff University, Cardiff, Wales, UK; and Hebrew University (O.S.-F., D.M.), Jerusalem, Israel.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
B. Paul Morgan
From the Rheumatology Research Center (N.K., A.T., H.H., D.M.), Center of Rare Diseases, and Department of Medicine, Hadassah-Hebrew University Medical Center, Jerusalem; The Weizmann Institute (Y.E.-E., E.P.), Rehovot, Israel; Systems Immunity Research Institute (B.P.M.), Cardiff University, Cardiff, Wales, UK; and Hebrew University (O.S.-F., D.M.), Jerusalem, Israel.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Ora Schueler-Furman
From the Rheumatology Research Center (N.K., A.T., H.H., D.M.), Center of Rare Diseases, and Department of Medicine, Hadassah-Hebrew University Medical Center, Jerusalem; The Weizmann Institute (Y.E.-E., E.P.), Rehovot, Israel; Systems Immunity Research Institute (B.P.M.), Cardiff University, Cardiff, Wales, UK; and Hebrew University (O.S.-F., D.M.), Jerusalem, Israel.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Elior Peles
From the Rheumatology Research Center (N.K., A.T., H.H., D.M.), Center of Rare Diseases, and Department of Medicine, Hadassah-Hebrew University Medical Center, Jerusalem; The Weizmann Institute (Y.E.-E., E.P.), Rehovot, Israel; Systems Immunity Research Institute (B.P.M.), Cardiff University, Cardiff, Wales, UK; and Hebrew University (O.S.-F., D.M.), Jerusalem, Israel.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Dror Mevorach
From the Rheumatology Research Center (N.K., A.T., H.H., D.M.), Center of Rare Diseases, and Department of Medicine, Hadassah-Hebrew University Medical Center, Jerusalem; The Weizmann Institute (Y.E.-E., E.P.), Rehovot, Israel; Systems Immunity Research Institute (B.P.M.), Cardiff University, Cardiff, Wales, UK; and Hebrew University (O.S.-F., D.M.), Jerusalem, Israel.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Full PDF
Citation
Molecular pathogenesis of human CD59 deficiency
Netanel Karbian, Yael Eshed-Eisenbach, Adi Tabib, Hila Hoizman, B. Paul Morgan, Ora Schueler-Furman, Elior Peles, Dror Mevorach
Neurol Genet Dec 2018, 4 (6) e280; DOI: 10.1212/NXG.0000000000000280

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Permissions

Make Comment

See Comments

Downloads
522

Share

  • Article
  • Figures & Data
  • Info & Disclosures
Loading

Article Figures & Data

Figures

  • Figure 1
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 1 Sequence and structure of CD59 WT and mutants

    (A) The sequences of WT CD59, point mutations Cys64Tyr and Asp24Val, and frameshift mutations Asp24Valfs* and Ala16Alafs*: The mature membrane surface CD59 primary sequence consists of 77 residues after removal of a 25-residue N-terminal signal sequence and a C-terminal GPI-anchoring signal (not shown). Colored arrows and boxes represent β-strands and α-helices, respectively, as observed in WT CD59 (see B). Point mutations are marked with an asterisk. The sequence of the 2 frameshift mutants reveals significantly shortened proteins that lack most of the CD59 sequence, in particular the GPI-anchoring signal. (B) The structure of CD59 highlights the vicinity of the point mutations to known sites of activity and interaction. The mutated Cys64 and Asp24 positions are highlighted as spheres and labeled. The 3 characterized interfaces of CD59 are marked by numbered crescents: (1) The classic site characterized originally described by Bodian et al.21 with the central Trp40 residue shown in sticks, (2) a loop spanning residues 20–24 that modulates CD59 activity20 (colored in white), and (3) the edge β-strand that interacts with ILY18 (to the left of the crescent). Disulfide bridges that stabilize CD59 are shown in sticks. Two solved structures (2j8b and 2uwr) are shown, highlighting the relative flexibility of the C-terminal helix (colored in magenta). (C) The frameshift mutants contain only a small part of the original CD59 sequence: The structure of CD59 is shown, with the region with a sequence in common with the Ala16Alafs* and Asp24Valfs* mutants colored in yellow and yellow-white, respectively. The I-TASSER model for the Asp24Valfs* sequence aligns 2 additional β-strands (colored in green, ending with the sphere at CD59 position Trp40), before it diverges, and the remaining part (in blue) is unique to CD59. GPI = glycosyl phosphatidylinositol; WT = wild type.

  • Figure 2
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 2 Western blot profile

    See Methods for details. Anti-myc was used in all assays for detection. (A) Western blot expression pattern of WT and mutants Cys64Tyr, Asp24Val, Asp24Valfs*, and Ala16Alafs*, as well as GFP. Anti-myc was used for detection (upper panel) and anti-tubulin as a loading control (lower panel). (B) Western blot expression pattern of WT and Cys64Tyr mutant and GFP after using the Endo H enzyme. (C) Western blot expression pattern of conditioned medium. (D) Proteosomal degradation profile. Transfected cells treated with bortezomib (Velcade) and lysate sample were separated on 15% polyacrylamide gels. PVDF membranes were used for protein transfer. Anti-tubulin was used as a loading control (lower panel). PVDF = polyvinylidene difluoride; WT = wild type.

  • Figure 3
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 3 Surface and intracellular localization of hCD59 mutants

    (A) Characterization of WT and mutant Cys64Tyr, Asp24Val, Asp24Valfs*, and Ala16Alafs* constructs by myc antibody (red) and DAPI (blue). Staining with myc tag and secondary antibodies was added after fixation without Triton. (B) Characterization of WT and Asp24Valfs* and Ala16Alafs* CD59 mutants by anti-myc antibodies with or without Triton permeabilization. Differential recognition of WT and the Asp24Valfs* and Ala16Alafs* CD59 mutants by anti-myc. In both procedures, anti-myc detected the WT constructs with or without Triton, but in the mutant construct, ER staining was seen only with Triton. (C) Characterization of WT and Asp24Valfs* and Ala16Alafs* CD59 mutants by anti-myc and PDI antibodies. Upper panel: myc antibody (red); middle panel: PDI antibody (green) and DAPI (blue); and lower panel: merge. Cells were treated with methanol for permeabilization. Scale bars = 50 μm. PDI = protein disulfide isomerase; WT = wild type.

  • Figure 4
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 4 Mutant detection by anti-CD59 antibodies using flow cytometry and fluorescent microscopy

    (A) A panel of anti-CD59 antibodies in Cys64Tyr and healthy control lymphoblasts: MEM43, HC1, BRIC229, YTH53.1, A35, 1.39, and rabbit polyclonal antibodies, assayed by flow cytometry. (B) Staining of WT and mutant constructs Cys64Tyr, Asp24Val, Asp24Valfs*, and Ala16Alafs* by BRIC229 or MEM43f (red), DAPI (blue). Procedures were performed by live staining without fixation and Triton treatment and assayed by fluorescent microscopy. Scale bars = 50 μm.

  • Figure 5
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 5 Lysis functional assay in hCD59 mutants

    WT and mutant constructs Cys64Tyr, Asp24Val, Asp24Valfs*, and Ala16Alafs* were transfected to CHO cells. Forty-eight hours after transfection, cells were marked by calcein AM, and calcein fluorescence of supernatants was read using the Cytation 3 Cell Imaging Multi-Mode Reader with the excitation filter set at 485 nm and emission filter at 530 nm. Percent lysis for each well was calculated as calcein release/total calcein loading (*p < 0.05, **p < 0.002, ANOVA and Student t test). WT = wild type.

Letters: Rapid online correspondence

No comments have been published for this article.
Comment

NOTE: All contributors' disclosures must be entered and current in our database before comments can be posted. Enter and update disclosures at http://submit.ng.neurology.org. Exception: replies to comments concerning an article you originally authored do not require updated disclosures.

  • Stay timely. Submit only on articles published within the last 8 weeks.
  • Do not be redundant. Read any comments already posted on the article prior to submission.
  • 200 words maximum.
  • 5 references maximum. Reference 1 must be the article on which you are commenting.
  • 5 authors maximum. Exception: replies can include all original authors of the article.
  • Submitted comments are subject to editing and editor review prior to posting.

More guidelines and information on Letters

Compose Comment

More information about text formats

Plain text

  • No HTML tags allowed.
  • Web page addresses and e-mail addresses turn into links automatically.
  • Lines and paragraphs break automatically.
Author Information
NOTE: The first author must also be the corresponding author of the comment.
First or given name, e.g. 'Peter'.
Your last, or family, name, e.g. 'MacMoody'.
Your email address, e.g. higgs-boson@gmail.com
Your role and/or occupation, e.g. 'Orthopedic Surgeon'.
Your organization or institution (if applicable), e.g. 'Royal Free Hospital'.
Publishing Agreement
NOTE: All authors, besides the first/corresponding author, must complete a separate Letters Submission Form and provide via email to the editorial office before comments can be posted.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.

Vertical Tabs

You May Also be Interested in

Back to top
  • Article
    • Abstract
    • Glossary
    • Methods
    • Results
    • Discussion
    • Author contributions
    • Study funding
    • Disclosure
    • Acknowledgment
    • Footnotes
    • References
  • Figures & Data
  • Info & Disclosures

Related Articles

  • No related articles found.

Topics Discussed

  • All Genetics
  • Peripheral neuropathy
  • Guillain-Barre syndrome
  • Chronic inflammatory demyelinating polyneuropathy
  • Childhood stroke

Alert Me

  • Alert me when eletters are published
Advertisement
Neurology Genetics: 7 (2)

Articles

  • Articles
  • Issues
  • Popular Articles

About

  • About the Journals
  • Ethics Policies
  • Editors & Editorial Board
  • Contact Us
  • Advertise

Submit

  • Author Center
  • Submit a Manuscript
  • Information for Reviewers
  • AAN Guidelines
  • Permissions

Subscribers

  • Subscribe
  • Sign up for eAlerts
  • RSS Feed
Site Logo
  • Visit neurology Template on Facebook
  • Follow neurology Template on Twitter
  • Visit Neurology on YouTube
  • Neurology
  • Neurology: Clinical Practice
  • Neurology: Genetics
  • Neurology: Neuroimmunology & Neuroinflammation
  • AAN.com
  • AANnews
  • Continuum
  • Brain & Life
  • Neurology Today

Wolters Kluwer Logo

Neurology: Genetics | Online ISSN: 2376-7839

© 2021 American Academy of Neurology

  • Privacy Policy
  • Feedback
  • Advertise