The intramembrane protease SPPL2a promotes B cell development and controls endosomal traffic by cleavage of the invariant chain

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Der Einfluss der Prozessierung von CD74 durch die Intramembranprotease SPPL2a auf Transportprozesse und Signalwege in B-Zellen

The intramembrane protease SPPL2a mediates the processing of transmembrane proteins in type II orientation. As the first in vivo validated substrate of this protease CD74 (invariant chain) was revealed. It functions as chaperone in MHCII-mediated antigen presentation. In the late endosomal membrane, different proteases sequentially process CD74. SPPL2a mediates the turnover of the final membrane-bound N-terminal fragment (NTF) of CD74. Absence of this protease in mice causes an accumulation of CD74 NTF accompanied by a high abundance of intracellular vesicles which is associated with a B cell maturation arrest at the transitional 1 stage and major impairments in the endocytic system. However, the detailed mechanisms underlying these disturbances were unknown. In this study, a detailed analysis of B cells from SPPL2a-/- mice revealed major defects in endocytic trafficking. SPPL2a-deficient B cells show a delayed degradation of the fluid-phase cargo ovalbumin as well as a retention of endocytosed transferrin in intracellular vesicles as compared to wild type cells. Moreover, CD74 NTF accumulation causes enhanced MHCII levels at the cell surface and in intracellular vesicles. To clarify how the accumulated CD74 NTF leads to the B cell maturation block, the impact of SPPL2a-deficiency on signaling pathways which are essential for B cell survival and differentiation were analyzed. It could be demonstrated that tonic and B cell antigen receptor (BCR)-induced signaling via the PI3K/Akt pathway is severely compromised in SPPL2a-deficient B cells. These cells show an altered BCR trafficking leading to a reduction of surface localized IgM and a reduced signal transmission via the BCR and the tyrosine kinase Syk. Decreased IgM surface expression and the diminished activation of Syk in SPPL2a-/- B cells are interconnected processes...Die Intramembranprotease SPPL2a prozessiert Transmembranproteine in Typ II Orientierung. Das erste in vivo validierte Substrat dieser Protease ist das Molekül CD74, welches als Chaperon bei der MHCII-vermittelten Antigenpräsentation agiert. CD74 wird in späten Endosomen sequenziell prozessiert, wobei SPPL2a den Abbau des membran-ständigen N-terminalen Fragments (NTF) von CD74 reguliert. In Mäusen induziert die Defizienz dieser Protease die Akkumulation des CD74 NTFs sowie die Anreicherung von intrazellulären Vesikeln in B-Zellen. Dies führt zu einer Störung der B-Zellmaturierung im „Transitional 1 Stadium“ in der Milz und einer Beeinträchtigung des endocytischen Systems. Der genaue molekulare Mechanismus ist jedoch unklar. In dieser Arbeit wurden die Defekte des endosomalen Systems auf Grund der CD74 NTF Akkumulation in B-Zellen aus SPPL2a-/- Mäusen näher charakterisiert. Es konnte gezeigt werden, dass SPPL2a-defiziente B-Zellen im Vergleich zu Wildtyp-Zellen eine verlangsamte Degradierung des Fluid-Phase Cargo Ovalbumin sowie eine Retention von internalisiertem Transferrin in intrazellulären Vesikeln aufweisen. Des Weiteren zeigen SPPL2a-/- B-Zellen erhöhte MHCII Proteinmengen an der Zelloberfläche und in intrazellulären Kompartimenten. Um den Mechanismus des B-Zellreifungsdefekts zu untersuchen, wurde die Auswirkung der SPPL2a-Defizienz auf Signalwege untersucht, welche besonders für das Überleben und Reifen von B-Zellen verantwortlich sind. Hierbei wurde eine stark verminderte tonische und B cell antigen receptor (BCR)-induzierte Aktivierung des PI3K/Akt-Weges in SPPL2a-/- B-Zellen festgestellt. Weiterhin zeigen diese Zellen im Vergleich zu Wildtyp-Kontrollen einen veränderten Transport des BCR und damit einhergehend eine Reduktion von IgM an der Zelloberfläche. Als Folge ist die Signalübertragung über den BCR und die ..

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

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.</jats:p

Signal-peptide-peptidase-like 2a is required for CD74 intramembrane proteolysis in human B cells

The invariant chain (CD74) mediates targeting of the MHCII complex to endosomal compartments, where CD74 undergoes degradation allowing MHCII to acquire peptides. We demonstrated recently that intramembrane proteolysis of the final membrane-bound N-terminal fragment (NTF) of CD74 is catalysed by Signal-peptide-peptidase-like 2a (SPPL2a) and that this process is indispensable for development and function of B lymphocytes in mice. In SPPL2a(−/−) mice, homeostasis of these cells is disturbed by the accumulation of the unprocessed CD74 NTF. So far, evidence for this essential role of SPPL2a is restricted to mice. Nevertheless, inhibition of SPPL2a has been suggested as novel approach to target B cells for treating autoimmunity. Here, we characterize human B cell lines with a homozygous microdeletion on chromosome 15. We demonstrate that this deletion disrupts the SPPL2a genomic locus and leads to loss of SPPL2a transcript. Lymphoblastoid cell lines from patients with this deletion exhibit absence of SPPL2a at the protein level and show an accumulation of the CD74 NTF comparable to B cells from SPPL2a(−/−) mice. By this means, we present evidence that the role of SPPL2a in CD74 proteolysis is conserved in human B cells and provide support for modulation of SPPL2a activity as a therapeutic concept

The intramembrane proteases signal peptide peptidase-like 2a and 2b have distinct functions in vivo

We reported recently that the presenilin homologue signal peptide peptidase-like 2a (SPPL2a) is essential for B cell development by cleaving the N-terminal fragment (NTF) of the invariant chain (li, CD74). Based on this, we suggested that pharmacological modulation of SPPL2a may represent a novel approach to deplete B cells in autoimmune disorders. With regard to reported overlapping substrate spectra of SPPL2a and its close homologue, SPPL2b, we investigated the role of SPPL2b in CD74 NTF proteolysis and its impact on B and dendritic cell homeostasis. In heterologous expression experiments, SPPL2b was found to cleave CD74 NTF with an efficiency simliar to that of SPPL2a. For in vivo analysis, SPPL2b single-deficient and SPPL2a/SPPL2b double-deficient mice were generated and examined for CD74 NTF turnover/accumulation, B cell maturation and functionality, and dendritic cell homeostasis. We demonstrate that in vivo SPPL2b does not exhibit a physiologically relevant contribution to CD74 proteolysis in B and dendritic cells. Furthermore, we reveal that both proteases exhibit divergent subcellular localizations in B cells and different expression profiles in murine tissues. These findings suggest distinct functions of SPPL2a and SPPL2b and, based on a high abundance of SPPL2b in brain, a physiological role of this protease in the central nervous system