The Influence of MHC Class II on B Cell Defects Induced by Invariant Chain/CD74 N-Terminal Fragments.

The invariant chain (CD74) mediates assembly and targeting of MHC class II (MHCII) complexes. In endosomes, CD74 undergoes sequential degradation by different proteases, including cathepsin S (CatS) and the intramembrane protease signal peptide peptidase-like 2a (SPPL2a). In their absence, CD74 N-terminal fragments (NTFs) accumulate. In SPPL2a-/- B cells, such an NTF impairs endosomal trafficking and BCR signal transduction. In mice, this leads to a loss of splenic B cells beyond the transitional stage 1. To gain insight into CD74 determinants and the role of MHCII, we compared B cells from CatS-/- , SPPL2a-/- , and SPPL2a-MHCII double-deficient mice. We assessed differentiation of B cells in bone marrow and spleen and analyzed their endosomal morphology, BCR expression, and signal transduction. We demonstrate that MHCII is dispensable for the B cell phenotype of SPPL2a-/- mice, further supporting a CD74-intrinsic effect. Despite significant vacuolization of endosomal compartments similar to SPPL2a-/- B cells, CatS-/- traditional stage 1 B cells show unimpaired degradation of endocytic cargo, have intact BCR signaling, and do not exhibit any relevant defects in maturation. This could indicate that CD74 NTF-induced structural changes of endosomes are not directly involved in these processes. We further found that the block of CD74 degradation in CatS-/- B cells is incomplete, so that NTF levels are significantly lower than in SPPL2a-/- B cells. This suggests a dose dependency and threshold for the CD74 NTF-associated impairment of B cell signaling and maturation. In addition, different functional properties of the longer, MHCII-bound CD74 NTF could contribute to the milder phenotype of CatS-/- B cells.

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.

Processing of CD74 by the Intramembrane Protease SPPL2a Is Critical for B Cell Receptor Signaling in Transitional B Cells.

The invariant chain (CD74), a chaperone in MHC class II-mediated Ag presentation, is sequentially processed by different endosomal proteases. We reported recently that clearance of the final membrane-bound N-terminal fragment (NTF) of CD74 is mediated by the intramembrane protease signal peptide peptidase-like (SPPL)2a, a process critical for B cell development. In mice, SPPL2a deficiency provokes the accumulation of this NTF in endocytic vesicles, which leads to a B cell maturation arrest at the transitional 1 stage. To define the underlying mechanism, we analyzed the impact of SPPL2a deficiency on signaling pathways involved in B cell homeostasis. We demonstrate that tonic as well as BCR-induced activation of the PI3K/Akt pathway is massively compromised in SPPL2a(-/-) B cells and identify this as major cause of the B cell maturation defect in these mice. Altered BCR trafficking induces a reduction of surface IgM in SPPL2a-deficient B cells, leading to a diminished signal transmission via the BCR and the tyrosine kinase Syk. We provide evidence that in SPPL2a(-/-) mice impaired BCR signaling is to a great extent provoked by the accumulating CD74 NTF, which can interact with the BCR and Syk, and that impaired PI3K/Akt signaling and reduced surface IgM are not directly linked processes. In line with disturbances in PI3K/Akt signaling, SPPL2a(-/-) B cells show a dysregulation of the transcription factor FOXO1, causing elevated transcription of proapoptotic genes. We conclude that SPPL2a-mediated processing of CD74 NTF is indispensable to maintain appropriate levels of tonic BCR signaling to promote B cell maturation.

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 catalyzed 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 similar 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.

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

Regulated intramembrane proteolysis is a central cellular process involved in signal transduction and membrane protein turnover. The presenilin homologue signal-peptide-peptidase-like 2a (SPPL2a) has been implicated in the cleavage of type 2 transmembrane proteins. We show that the invariant chain (li, CD74) of the major histocompatability class II complex (MHCII) undergoes intramembrane proteolysis mediated by SPPL2a. B lymphocytes of SPPL2a(-/-) mice accumulate an N-terminal fragment (NTF) of CD74, which severely impairs membrane traffic within the endocytic system and leads to an altered response to B cell receptor stimulation, reduced BAFF-R surface expression, and accumulation of MHCII in transitional developmental stage T1 B cells. This results in significant loss of B cell subsets beyond the T1 stage and disrupted humoral immune responses, which can be recovered by additional ablation of CD74. Hence, we provide evidence that regulation of CD74-NTF levels by SPPL2a is indispensable for B cell development and function by maintaining trafficking and integrity of MHCII-containing endosomes, highlighting SPPL2a as a promising pharmacological target for depleting and/or modulating B cells.