Substrate determinants of signal peptide peptidase-like 2a (SPPL2a)-mediated intramembrane proteolysis of the invariant chain CD74

Research output: Contribution to journalResearch articleContributedpeer-review

Contributors

  • Susann Hüttl - , Kiel University (Author)
  • Felix Helfrich - , Kiel University (Author)
  • Torben Mentrup - , Institute of Physiological Chemistry, Kiel University (Author)
  • Sebastian Held - , Kiel University (Author)
  • Akio Fukumori - , German Center for Neurodegenerative Diseases (DZNE) (Author)
  • Harald Steiner - , German Center for Neurodegenerative Diseases (DZNE), Ludwig Maximilian University of Munich (Author)
  • Paul Saftig - , Kiel University (Author)
  • Regina Fluhrer - , German Center for Neurodegenerative Diseases (DZNE), Ludwig Maximilian University of Munich (Author)
  • Bernd Schröder - , Institute of Physiological Chemistry, Kiel University (Author)

Abstract

The presenilin homologue signal peptide peptidase-like 2a (SPPL2a) is an intramembrane protease of lysosomes/late endosomes which cleaves type II transmembrane proteins. We recently identified CD74, the invariant chain of the MHCII complex, as the first in vivo validated substrate of this protease. In endosomal compartments, CD74 undergoes sequential proteolysis leading to the generation of a membranebound N-terminal fragment (NTF) that requires cleavage by SPPL2a for its turnover. In SPPL2a -/- mice, this fragment accumulates in B-cells and significantly disturbs their maturation and functionality. To date, the substrate requirements of the protease SPPL2a have not been investigated. In the present study, we systematically analysed the molecular determinants of CD74 with regard to the intramembrane cleavage by SPPL2a. Using domain-exchange experiments, we demonstrate that the intracellular domain (ICD) of CD74 can be substituted without affecting cleavability by SPPL2a. Based on IP-MS analysis of the cleavage product, we report identification of the primary SPPL2a cleavage site between Y52 and F53 within the CD74 transmembrane segment. Furthermore, systematic alaninescanning mutagenesis of the transmembrane and membraneproximal parts of the CD74 NTF has been performed. We show that none of the analysed determinants within the CD74 NTF including the residues flanking the primary cleavage site are absolutely essential for SPPL2a cleavage. Importantly, we found that alanine substitution of helix-destabilizing glycines within the transmembrane segment and distinct residues within the luminal membrane-proximal segment led to a reduced efficiency of SPPL2a-mediated processing. Therefore we propose that elements within the transmembrane segment and the luminal juxtamembrane domain facilitate intramembrane proteolysis of CD74 by SPPL2a.

Details

Original languageEnglish
Pages (from-to)1405-1422
Number of pages18
JournalBiochemical journal
Volume473
Issue number10
Publication statusPublished - 15 May 2016
Peer-reviewedYes

External IDs

PubMed 26987812

Keywords

Keywords

  • CD74, Intramembrane proteolysis, Membrane protein degradation, Presenilin, Signal peptide peptidase-like protease, γ-secretase