Signal-peptide-peptidase-like 2a is required for CD74 intramembrane proteolysis in human B cells
Introduction
The intramembrane protease Signal-peptide-peptidase-like 2a (SPPL2a) resides in lysosomes and late endosomes [1] and has been implicated in the processing of type 2 transmembrane proteins [2] including TNFα [3], [4], the Fas ligand [5], the Bri2 protein [6] and the invariant chain (CD74) of the MHCII complex [7]. Among these, only the latter has been confirmed in vivo. We recently demonstrated that the intramembrane cleavage of CD74 by SPPL2a is an essential process for development and functionality of B lymphocytes in mice [7]. This was based on a distinct phenotype of SPPL2a−/− mice that is characterized by a developmental arrest of B cells at the transitional stage 1 (T1) which was recovered to a significant degree by additional ablation of CD74 in SPPL2a-CD74 double-deficient mice [7]. This clearly identified the N-terminal fragment (NTF) of CD74, which accumulates in the absence of SPPL2a, as the causative element of this B cell phenotype. Key results from this work were independently confirmed by two other laboratories [8], [9].
Based on these studies, SPPL2a was suggested to represent a putative therapeutic target. Apart from an impairment of tooth enamel generation [10] in addition to the described B cell phenotype, absence of SPPL2a appeared to be well tolerated in mice. Thus, pharmacological inhibition of SPPL2a may represent a novel small-molecule based approach to deplete and/or modulate B cells. This concept would require that the described conclusions on the importance of SPPL2a for proteolysis of CD74 and homeostasis of B cells are also valid in humans. However, all experimental data available to date are derived from mice.
Here, we provide initial data on the role of SPPL2a in human B cells. We made use of cell lines derived from two siblings with a 192 kb homozygous deletion on chromosome 15q21.2 [11]. Chromosomal microarray (CMA) analysis indicated that the deletion included the exon 1 of the SPPL2a genomic locus. We show here that this homozygous 15q21.2 deletion disrupts SPPL2a expression. Using lymphoblastoid cell lines derived from these patients, we demonstrate that SPPL2a-deficiency leads to a massive accumulation of CD74 NTF in lysosomal/late endosomal compartments thus confirming that the requirement of SPPL2a for CD74 intramembrane proteolysis is conserved in humans.
Section snippets
Cell culture
Primary skin fibroblasts and peripheral blood mononuclear cells were obtained from the two siblings with homozygous deletions and their family members, who either carried a heterozygous deletion or did not have the deletion [11], after obtaining written informed consent and approval by the institutional review board. Epstein–Barr virus (EBV) transformation of primary lymphocytes was performed according to standard procedures. Fibroblasts were maintained in DMEM (PAA) with l-glutamine
CD74 NTF undergoes intramembrane proteolysis in human B cells
In previous studies in mice [7], [8], [9] SPPL2a was found to process an NTF of CD74 that comprises about 80 residues and remains from the sequential degradation of the luminal domain by different endosomal proteases (Fig. 1A). To assess if also in human B cells intramembrane proteolysis serves as degradation route for the CD74 NTF, we incubated the human B cell-derived Burkitt lymphoma cell line Raji in the presence of the SPP/SPPL inhibitors (Z-LL)2-ketone or inhibitor X and analyzed
Acknowledgments
The authors thank Sebastian Held for excellent technical assistance as well as Erin Riggs, Brian Bunke and Dawn Kunig from Emory University for coordination of patient cell lines and Florian Oyen and Tobias Obser, UKE Hamburg, for help with related experimental work. Furthermore, we are grateful to Guido Looft, UKE Hamburg, and Dr. Michael Schwake, University of Bielefeld, for providing Raji cells and an antibody against LIMP-2, respectively. This work was supported by the Deutsche
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Signal peptide peptidase-like 2 proteases: Regulatory switches or proteasome of the membrane?
2022, Biochimica et Biophysica Acta - Molecular Cell ResearchCitation Excerpt :Consequently, B lymphocytes and dendritic cells (DC) as the major CD74-expressing cells accumulate high amounts of this fragment in absence of SPPL2a. This was concordantly observed in three different SPPL2a knockout or mutant mouse models [50–52] as well as in immortalised and primary cells from human SPPL2a-deficient patients [46,53]. Importantly, SPPL2a-deficiency impacts on the differentiation and functionality of the named cell types in a CD74-dependent way (Fig. 2).
Signal peptide peptidase and SPP-like proteases - Possible therapeutic targets?
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2017, European Journal of Cell BiologyCitation Excerpt :EBV-immortalised B cells from these patients accumulated significant amounts of the CD74 NTF equivalent to the situation in mice. This confirms that the role of SPPL2a in CD74 NTF degradation is conserved (Schneppenheim et al., 2014a). Since no information about the immune status of the respective patients was available, the phenotypic consequences of a CD74 NTF accumulation on B cell and dendritic cell homeostasis in humans currently remain elusive.
Intramembrane proteolysis within lysosomes
2016, Ageing Research ReviewsCitation Excerpt :However, no clinical information on the immunological status of these patients was available. Therefore, effects on B and dendritic cell functionality in humans that may be induced by SPPL2a deficiency and impaired CD74 NTF turnover currently remain elusive (Schneppenheim et al., 2014a). Nevertheless, the concept of achieving B and/or dendritic cell depletion with a small molecule-based inhibitor is attractive since it exhibits a number of practical and economic advantages over the currently employed biologic agents like the antibody rituximab (Townsend et al., 2010).
The multifaceted roles of the invariant chain CD74 - More than just a chaperone
2016, Biochimica et Biophysica Acta - Molecular Cell Research