title: Substrate determinants of signal peptide peptidase-like 2a (SPPL2a)-mediated intramembrane proteolysis of the invariant chain CD74.
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 membrane-bound 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 alanine-scanning mutagenesis of the transmembrane and membrane-proximal 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.
authors.affiliation: Biochemical Institute, Christian Albrechts University of Kiel, Otto-Hahn-Platz 9, D-24118 Kiel, Germany.
authors.forename: Susann
authors.initials: S
authors.lastname: Hüttl
authors.affiliation: Biochemical Institute, Christian Albrechts University of Kiel, Otto-Hahn-Platz 9, D-24118 Kiel, Germany.
authors.forename: Felix
authors.initials: F
authors.lastname: Helfrich
authors.affiliation: Biochemical Institute, Christian Albrechts University of Kiel, Otto-Hahn-Platz 9, D-24118 Kiel, Germany.
authors.forename: Torben
authors.initials: T
authors.lastname: Mentrup
authors.affiliation: Biochemical Institute, Christian Albrechts University of Kiel, Otto-Hahn-Platz 9, D-24118 Kiel, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Held
authors.affiliation: DZNE-German Center for Neurodegenerative Diseases, Munich, Feodor-Lynen-Strasse 17, D-81377 Munich, Germany.
authors.forename: Akio
authors.initials: A
authors.lastname: Fukumori
authors.affiliation: DZNE-German Center for Neurodegenerative Diseases, Munich, Feodor-Lynen-Strasse 17, D-81377 Munich, Germany Biomedizinisches Centrum (BMC), Ludwig Maximilians University of Munich, Feodor-Lynen-Strasse 17, D-81377 Munich, Germany.
authors.forename: Harald
authors.initials: H
authors.lastname: Steiner
authors.affiliation: Biochemical Institute, Christian Albrechts University of Kiel, Otto-Hahn-Platz 9, D-24118 Kiel, Germany.
authors.forename: Paul
authors.initials: P
authors.lastname: Saftig
authors.affiliation: DZNE-German Center for Neurodegenerative Diseases, Munich, Feodor-Lynen-Strasse 17, D-81377 Munich, Germany Biomedizinisches Centrum (BMC), Ludwig Maximilians University of Munich, Feodor-Lynen-Strasse 17, D-81377 Munich, Germany.
authors.forename: Regina
authors.initials: R
authors.lastname: Fluhrer
authors.affiliation: Biochemical Institute, Christian Albrechts University of Kiel, Otto-Hahn-Platz 9, D-24118 Kiel, Germany baschroeder@biochem.uni-kiel.de.
authors.forename: Bernd
authors.initials: B
authors.lastname: Schröder
mesh_terms: D000818:Animals; D000944:Antigens, Differentiation, B-Lymphocyte / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016282:Aspartic Acid Endopeptidases / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D019084:Fluorescent Antibody Technique, Indirect; D000949:Histocompatibility Antigens Class II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D059748:Proteolysis
chemical_list: D000944:Antigens, Differentiation, B-Lymphocyte; D000949:Histocompatibility Antigens Class II; D008565:Membrane Proteins; C045084:invariant chain; D016282:Aspartic Acid Endopeptidases; C524670:SPPL2a protein, human; C568855:SPPL2a protein, mouse; C460085:signal peptide peptidase
keywords: CD74; intramembrane proteolysis; membrane protein degradation; presenilin; signal peptide peptidase-like protease; γ-secretase