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

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Highlights

  • A human 15q21.2 microdeletion leads to loss of SPPL2a transcript and protein.

  • In human B cells, CD74 N-terminal fragment is cleaved by SPPL intramembrane proteases.

  • CD74 N-terminal fragment accumulates in patient-derived SPPL2a-deficient B cell lines.

  • In human B cells, SPPL2a is indispensable for turnover of CD74 N-terminal fragment.

Abstract

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.

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

References (19)

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