Binding of neural cell adhesion molecules (N-CAMs) to the cellular prion protein

Gerold Schmitt-Ulms, Giuseppe Legname, Michael A. Baldwin, Haydn L. Ball, Nicole Bradon, Patrick J. Bosque, Kathryn L. Crossin, Gerald M. Edelman, Stephen J. DeArmond, Fred E. Cohen, Stanley B. Prusiner

Research output: Contribution to journalArticlepeer-review

282 Scopus citations

Abstract

To identify molecular interaction partners of the cellular prion protein (PrPC), we sought to apply an in situ crosslinking method that maintains the microenvironment of PrPC. Mild formaldehyde crosslinking of mouse neuroblastoma cells (N2a) that are susceptible to prion infection revealed the presence of PrPC in high molecular mass (HMM) protein complexes of 200 to 225 kDa. LC/MS/MS analysis identified three murine splice-variants of the neural cell adhesion molecule (N-CAM) in the complexes, which isolate with caveolae-like domains (CLDs). Enzymatic removal of N-linked sugar moieties did not disrupt the complexes, arguing that the interaction of PrP with N-CAM occurs through amino acid side-chains. Additionally, similar levels of PrP/N-CAM complexes were found in N2a and prion-infected N2a (ScN2a) cells. With the use of an N-CAM-specific peptide library, the PrP-binding site was determined to comprise β-strands C and C′ within the two consecutive fibronectin type III (FNIII) modules found in proximity of the membrane-attachment site of N-CAM. As revealed by in situ crosslinking of PrP deletion mutants, the PrP face of the binding site is formed by the N terminus, helix A (residues 144-154) and the adjacent loop region of PrP. N-CAM-deficient (N-CAM-/-) mice that were intracerebrally challenged with scrapie prions succumbed to disease with a mean incubation period of 122 (±4.1, SEM) days, arguing that N-CAM is not involved in PrPSc replication. Our findings raise the possibility that N-CAM may join with PrPc in carrying out some as yet unidentified physiologic cellular function.

Original languageEnglish (US)
Pages (from-to)1209-1225
Number of pages17
JournalJournal of Molecular Biology
Volume314
Issue number5
DOIs
StatePublished - Dec 14 2001

Keywords

  • Cell adhesion
  • Formaldehyde crosslinking
  • Prion protein
  • Protein X
  • Scrapie

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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