Journal
JOURNAL OF MOLECULAR BIOLOGY
Volume 314, Issue 5, Pages 1209-1225Publisher
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1006/jmbi.2000.5183
Keywords
prion protein; protein X; formaldehyde crosslinking; cell adhesion; scrapie
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Funding
- NCRR NIH HHS [RR01614, RR12961] Funding Source: Medline
- NIA NIH HHS [AG10770, AG02132] Funding Source: Medline
- NINDS NIH HHS [NS14069, NS39837] Funding Source: Medline
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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 beta-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 144154) 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. (C) 2001 Academic Press.
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