ABSTRACT
Human inborn errors of IFN-γ immunity underlie mycobacterial diseases. We describe patients with Mycobacterium bovis (BCG) disease who are homozygous for loss-of-function mutations of SPPL2A. This gene encodes a transmembrane protease that degrades the N-terminal fragment (NTF) of CD74 (HLA invariant chain) in antigen-presenting cells. The CD74 NTF therefore accumulates in the HLA class II+ myeloid and lymphoid cells of SPPL2a-deficient patients. This toxic fragment selectively depletes IL-12- and IL-23-producing CD1c+ conventional dendritic cells (cDC2s) and their circulating progenitors. Moreover, SPPL2a-deficient memory TH1* cells selectively fail to produce IFN-γ when stimulated with mycobacterial antigens in vitro. Finally, Sppl2a-/- mice lack cDC2s, have CD4+ T cells that produce small amounts of IFN-γ after BCG infection, and are highly susceptible to infection with BCG or Mycobacterium tuberculosis. These findings suggest that inherited SPPL2a deficiency in humans underlies mycobacterial disease by decreasing the numbers of cDC2s and impairing IFN-γ production by mycobacterium-specific memory TH1* cells.
Subject(s)
Aspartic Acid Endopeptidases/genetics , Aspartic Acid Endopeptidases/metabolism , Dendritic Cells/immunology , Membrane Proteins/metabolism , Mycobacterium Infections/immunology , Mycobacterium bovis/physiology , Mycobacterium tuberculosis/physiology , Th1 Cells/immunology , Tuberculosis/immunology , Animals , Antigens, Differentiation, B-Lymphocyte/metabolism , Cells, Cultured , HLA Antigens/metabolism , Histocompatibility Antigens Class II/metabolism , Humans , Immunity , Immunologic Memory , Infant , Interferon-gamma/metabolism , Lymphadenopathy , Male , Membrane Proteins/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Mutation/genetics , Mycobacterium Infections/genetics , VaccinationABSTRACT
Human genetic variants that introduce an AG into the intronic region between the branchpoint (BP) and the canonical splice acceptor site (ACC) of protein-coding genes can disrupt pre-mRNA splicing. Using our genome-wide BP database, we delineated the BP-ACC segments of all human introns and found extreme depletion of AG/YAG in the [BP+8, ACC-4] high-risk region. We developed AGAIN as a genome-wide computational approach to systematically and precisely pinpoint intronic AG-gain variants within the BP-ACC regions. AGAIN identified 350 AG-gain variants from the Human Gene Mutation Database, all of which alter splicing and cause disease. Among them, 74% created new acceptor sites, whereas 31% resulted in complete exon skipping. AGAIN also predicts the protein-level products resulting from these two consequences. We performed AGAIN on our exome/genomes database of patients with severe infectious diseases but without known genetic etiology and identified a private homozygous intronic AG-gain variant in the antimycobacterial gene SPPL2A in a patient with mycobacterial disease. AGAIN also predicts a retention of six intronic nucleotides that encode an in-frame stop codon, turning AG-gain into stop-gain. This allele was then confirmed experimentally to lead to loss of function by disrupting splicing. We further showed that AG-gain variants inside the high-risk region led to misspliced products, while those outside the region did not, by two case studies in genes STAT1 and IRF7. We finally evaluated AGAIN on our 14 paired exome-RNAseq samples and found that 82% of AG-gain variants in high-risk regions showed evidence of missplicing. AGAIN is publicly available from https://hgidsoft.rockefeller.edu/AGAIN and https://github.com/casanova-lab/AGAIN.
Subject(s)
RNA Splice Sites , RNA Splicing , Humans , Introns , Mutation , GenomeABSTRACT
Dectin-1 is a C-type lectin-receptor involved in sensing fungi by innate immune cells. Encoded by the Clec7a gene, Dectin-1 exists in two major splice isoforms, Dectin-1a and 1b, which differ in the presence of a membrane-proximal stalk domain. As reported previously, this domain determines degradative routes for Dectin-1a and 1b leading to the generation of a stable N-terminal fragment exclusively from Dectin-1a. Here, we narrow down the responsible part of the stalk and demonstrate the stabilisation of the Dectin-1a N-terminal fragment in tetraspanin-enriched microdomains. C57BL/6 and BALB/c mice show divergent Dectin-1 isoform expression patterns, which are caused by a single nucleotide polymorphism in exon 3 of the Clec7a gene, leading to a non-sense Dectin-1a mRNA in C57BL/6 mice. Using backcrossing, we generated mice with the C57BL/6 Clec7a allele on a BALB/c background and compared these to the parental strains. Expression of the C57BL/6 allele leads to the exclusive presence of the Dectin-1b protein. Furthermore, it was associated with higher Dectin-1 mRNA expression, but less Dectin-1 at the cell surface according to flow cytometry. In neutrophils, this altered ROS production induced by Dectin-1 model ligands, while cellular responses in macrophages and dendritic cells were not significantly influenced by the Dectin-1 isoform pattern.
ABSTRACT
Signal peptide peptidase-like 2a (SPPL2a) is an aspartyl intramembrane protease essential for degradation of the invariant chain CD74. In humans, absence of SPPL2a leads to Mendelian susceptibility to mycobacterial disease, which is attributed to a loss of the dendritic cell (DC) subset conventional DC2. In this study, we confirm depletion of conventional DC2 in lymphatic tissues of SPPL2a-/- mice and demonstrate dependence on CD74 using SPPL2a-/- CD74-/- mice. Upon contact with mycobacteria, SPPL2a-/- bone marrow-derived DCs show enhanced secretion of IL-1ß, whereas production of IL-10 and IFN-ß is reduced. These effects correlated with modulated responses upon selective stimulation of the pattern recognition receptors TLR4 and Dectin-1. In SPPL2a-/- bone marrow-derived DCs, Dectin-1 is redistributed to endosomal compartments. Thus, SPPL2a deficiency alters pattern recognition receptor pathways in a CD74-dependent way, shifting the balance from anti- to proinflammatory cytokines in antimycobacterial responses. We propose that in addition to the DC reduction, this altered DC functionality contributes to Mendelian susceptibility to mycobacterial disease upon SPPL2a deficiency.
Subject(s)
Aspartic Acid Endopeptidases/metabolism , Cell Membrane/metabolism , Dendritic Cells/immunology , Membrane Proteins/metabolism , Mycobacterium bovis/physiology , Animals , Antigens, Differentiation, B-Lymphocyte/genetics , Aspartic Acid Endopeptidases/genetics , Cattle , Cells, Cultured , Cytokines/metabolism , Genetic Predisposition to Disease , Histocompatibility Antigens Class II/genetics , Humans , Immunity , Immunomodulation , Membrane Proteins/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Toll-Like Receptor 4/immunology , Tuberculosis, BovineABSTRACT
Small mammals exhibit seasonal changes in intestinal morphology and function via increased intestine size and resorptive surface and/or nutrient transport capacity to increase energy yield from food during winter. This study investigated whether seasonal or acute acclimation to anticipated or actual energetic challenges in Djungarian hamsters also resulted in higher nutrient resorption capacities owing to changes in small intestine histology and physiology. The hamsters show numerous seasonal energy-saving adjustments in response to short photoperiod. As spontaneous daily torpor represents one of these adjustments related to food quality and quantity, it was hypothesized that the hamsters' variable torpor expression patterns are influenced by their individual nutrient uptake capacity. Hamsters under short photoperiod showed longer small intestines and higher mucosal electrogenic transport capacities for glucose relative to body mass. Similar observations were made in hamsters under long photoperiod and food restriction. However, this acute energetic challenge caused a stronger increase of glucose transport capacity. Apart from that, neither fasting-induced torpor in food-restricted hamsters nor spontaneous daily torpor in short photoperiod-exposed hamsters clearly correlated with mucosal glucose transport capacity. Both seasonally anticipated and acute energetic challenges caused adjustments in the hamsters' small intestine. Short photoperiod appeared to induce an integration of these and other acclimation processes in relation to body mass to achieve a long-term adjustment of energy balance. Food restriction seemed to result in a more flexible, short-term strategy of maximizing energy uptake possibly via mucosal glucose transport and reducing energy consumption via torpor expression as an emergency response.
Subject(s)
Acclimatization , Phodopus , Animals , Cricetinae , Energy Metabolism , Intestines , Photoperiod , SeasonsABSTRACT
Members of the GxGD-type intramembrane aspartyl proteases have emerged as key players not only in fundamental cellular processes such as B-cell development or protein glycosylation, but also in development of pathologies, such as Alzheimer's disease or hepatitis virus infections. However, one member of this protease family, signal peptide peptidase-like 2c (SPPL2c), remains orphan and its capability of proteolysis as well as its physiological function is still enigmatic. Here, we demonstrate that SPPL2c is catalytically active and identify a variety of SPPL2c candidate substrates using proteomics. The majority of the SPPL2c candidate substrates cluster to the biological process of vesicular trafficking. Analysis of selected SNARE proteins reveals proteolytic processing by SPPL2c that impairs vesicular transport and causes retention of cargo proteins in the endoplasmic reticulum. As a consequence, the integrity of subcellular compartments, in particular the Golgi, is disturbed. Together with a strikingly high physiological SPPL2c expression in testis, our data suggest involvement of SPPL2c in acrosome formation during spermatogenesis.
Subject(s)
Aspartic Acid Endopeptidases/metabolism , Membrane Proteins/metabolism , SNARE Proteins/metabolism , Acrosome/metabolism , Animals , Biocatalysis , Down-Regulation , Glycomics , Glycoproteins/metabolism , Glycosyltransferases/metabolism , Golgi Apparatus/metabolism , HEK293 Cells , Humans , Male , Mice, Inbred C57BL , Models, Biological , Protein Transport , Proteolysis , Spermatids/metabolism , Subcellular Fractions/metabolism , Substrate SpecificityABSTRACT
Signal peptide peptidase (SPP) and the four homologous SPP-like (SPPL) proteases constitute a family of intramembrane aspartyl proteases with selectivity for type II-oriented transmembrane segments. Here, we analyse the physiological function of the orphan protease SPPL2c, previously considered to represent a non-expressed pseudogene. We demonstrate proteolytic activity of SPPL2c towards selected tail-anchored proteins. Despite shared ER localisation, SPPL2c and SPP exhibit distinct, though partially overlapping substrate spectra and inhibitory profiles, and are organised in different high molecular weight complexes. Interestingly, SPPL2c is specifically expressed in murine and human testis where it is primarily localised in spermatids. In mice, SPPL2c deficiency leads to a partial loss of elongated spermatids and reduced motility of mature spermatozoa, but preserved fertility. However, matings of male and female SPPL2c-/- mice exhibit reduced litter sizes. Using proteomics we identify the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2)-regulating protein phospholamban (PLN) as a physiological SPPL2c substrate. Accumulation of PLN correlates with a decrease in intracellular Ca2+ levels in elongated spermatids that likely contribute to the compromised male germ cell differentiation and function of SPPL2c-/- mice.
Subject(s)
Aspartic Acid Endopeptidases/metabolism , Calcium-Binding Proteins/metabolism , Cell Membrane/enzymology , Germ Cells/metabolism , Membrane Proteins/metabolism , Amino Acid Sequence , Animals , Aspartic Acid Endopeptidases/chemistry , Calcium/metabolism , Endoplasmic Reticulum/metabolism , Female , HEK293 Cells , HeLa Cells , Homeostasis , Humans , Male , Membrane Proteins/chemistry , Mice , Organ Specificity , Spermatids/metabolism , Substrate Specificity , Testis/enzymologyABSTRACT
Intramembrane proteolysis describes the cleavage of substrate proteins within their hydrophobic transmembrane segments. Several families of intramembrane proteases have been identified including the aspartyl proteases Signal peptide peptidase (SPP) and its homologues, the SPP-like (SPPL) proteases SPPL2a, SPPL2b, SPPL2c and SPPL3. As presenilin homologues, they employ a similar catalytic mechanism as the well-studied γ-secretase. However, SPP/SPPL proteases cleave transmembrane proteins with a type II topology. The characterisation of SPP/SPPL-deficient mouse models has highlighted a still growing spectrum of biological functions and also promoted the substrate discovery of these proteases. In this review, we will summarise the current hypotheses how phenotypes of these mouse models are linked to the molecular function of the enzymes. At the cellular level, SPP/SPPL-mediated cleavage events rather provide specific regulatory switches than unspecific bulk proteolysis. By this means, a plethora of different cell biological pathways is influenced including signal transduction, membrane trafficking and protein glycosylation.
Subject(s)
Aspartic Acid Endopeptidases/metabolism , Amyloid Precursor Protein Secretases/metabolism , Animals , Aspartic Acid Endopeptidases/chemistry , Humans , Membrane Proteins/metabolism , Protein Transport , Proteolysis , Signal Transduction , Substrate SpecificityABSTRACT
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.
Subject(s)
Antigens, Differentiation, B-Lymphocyte/immunology , B-Lymphocytes/immunology , Genes, MHC Class II , Histocompatibility Antigens Class II/immunology , Animals , Antigens, Differentiation, B-Lymphocyte/metabolism , Aspartic Acid Endopeptidases/deficiency , Aspartic Acid Endopeptidases/genetics , Aspartic Acid Endopeptidases/metabolism , Cathepsins/deficiency , Cathepsins/genetics , Cathepsins/metabolism , Cell Differentiation , Endosomes/immunology , Endosomes/physiology , Histocompatibility Antigens Class II/metabolism , Membrane Proteins/deficiency , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Signal TransductionABSTRACT
Signal peptide peptidase (SPP) and the four homologous SPP-like proteases SPPL2a, SPPL2b, SPPL2c and SPPL3 are GxGD-type intramembrane-cleaving proteases (I-CLIPs). In addition to divergent subcellular localisations, distinct differences in the mechanistic properties and substrate requirements of individual family members have been unravelled. SPP/SPPL proteases employ a catalytic mechanism related to that of the γ-secretase complex. Nevertheless, differential targeting of SPP/SPPL proteases and γ-secretase by inhibitors has been demonstrated. Furthermore, also within the SPP/SPPL family significant differences in the sensitivity to currently available inhibitory compounds have been reported. Though far from complete, our knowledge on pathophysiological functions of SPP/SPPL proteases, in particular based on studies in mice, has been significantly increased over the last years. Based on this, inhibition of distinct SPP/SPPL proteases has been proposed as a novel therapeutic concept e.g. for the treatment of autoimmunity and viral or protozoal infections, as we will discuss in this review. This article is part of a Special Issue entitled: Proteolysis as a Regulatory Event in Pathophysiology edited by Stefan Rose-John.
Subject(s)
Amyloid Precursor Protein Secretases/genetics , Membrane Proteins/genetics , Peptides/genetics , Proteolysis , Amino Acid Sequence/genetics , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Aspartic Acid Endopeptidases/antagonists & inhibitors , Aspartic Acid Endopeptidases/genetics , Humans , Membrane Proteins/antagonists & inhibitors , Peptides/antagonists & inhibitors , Peptides/metabolism , Substrate SpecificityABSTRACT
Protein N-glycosylation is involved in a variety of physiological and pathophysiological processes such as autoimmunity, tumour progression and metastasis. Signal peptide peptidase-like 3 (SPPL3) is an intramembrane-cleaving aspartyl protease of the GxGD type. Its physiological function, however, has remained enigmatic, since presently no physiological substrates have been identified. We demonstrate that SPPL3 alters the pattern of cellular N-glycosylation by triggering the proteolytic release of active site-containing ectodomains of glycosidases and glycosyltransferases such as N-acetylglucosaminyltransferase V, ß-1,3 N-acetylglucosaminyltransferase 1 and ß-1,4 galactosyltransferase 1. Cleavage of these enzymes leads to a reduction in their cellular activity. In line with that, reduced expression of SPPL3 results in a hyperglycosylation phenotype, whereas elevated SPPL3 expression causes hypoglycosylation. Thus, SPPL3 plays a central role in an evolutionary highly conserved post-translational process in eukaryotes.
Subject(s)
Aspartic Acid Endopeptidases/metabolism , Gene Expression Regulation , Glycoside Hydrolases/metabolism , Glycosyltransferases/metabolism , Polysaccharides/metabolism , Glycosylation , Humans , Protein Processing, Post-TranslationalABSTRACT
During regulated intramembrane proteolysis (RIP) a membrane-spanning substrate protein is cleaved by an ectodomain sheddase and an intramembrane cleaving protease. A cytoplasmic intracellular domain (ICD) is liberated, which can migrate to the nucleus thereby influencing transcriptional regulation. Signal peptide peptidase-like (SPPL) 2a and 2b have been implicated in RIP of type II transmembrane proteins. Even though SPPL2a might represent a potential pharmacological target for treatment of B-cell-mediated autoimmunity, no specific and potent inhibitors for this enzyme are currently available. We report here on the first quantitative cell-based assay for measurement of SPPL2a/b activity. Demonstrating the failure of standard Gal4/VP16 reporter assays for SPPL2a/b analysis, we have devised a novel system employing ß-galactosidase (ßGal) complementation. This is based on detecting nuclear translocation of the proteolytically released substrate ICDs, which results in specific restoration of ßGal activity. Utilizing this potentially high-throughput compatible new setup, we demonstrate nuclear translocation of the ICDs from integral membrane protein 2B (ITM2B), tumor necrosis factor (TNF) and CD74 and identify secreted frizzled-related protein 2 (SFRP2) as potential transcriptional downstream target of the CD74 ICD. We show that the presented assay is easily adaptable to other intramembrane proteases and therefore represents a valuable tool for the functional analysis and development of new inhibitors of this class of enzymes.
Subject(s)
Aspartic Acid Endopeptidases/metabolism , Cell Nucleus/metabolism , Nuclear Localization Signals , Active Transport, Cell Nucleus , Adaptor Proteins, Signal Transducing , Amino Acid Sequence , Antigens, Differentiation, B-Lymphocyte/metabolism , Aspartic Acid Endopeptidases/chemistry , HEK293 Cells , HeLa Cells , Histocompatibility Antigens Class II/metabolism , Humans , Membrane Glycoproteins/metabolism , Molecular Sequence Data , Protein Structure, Tertiary , Proteolysis , Tumor Necrosis Factor-alpha/metabolismABSTRACT
The invariant chain (CD74) is well known for its essential role in antigen presentation by mediating assembly and subcellular trafficking of the MHCII complex. Beyond this, CD74 has also been implicated in a number of processes independent of MHCII. These include the regulation of endosomal trafficking, cell migration and cellular signalling as surface receptor of the pro-inflammatory cytokine macrophage migration inhibitory factor (MIF). In several forms of cancer, CD74 is up-regulated and associated with enhanced proliferation and metastatic potential. In this review, an overview of the diverse biological functions of the CD74 protein is provided with a particular focus on how these may be regulated. In particular, proteolysis of CD74 will be discussed as a central mechanism to control the actions of this important protein at different levels.
Subject(s)
Antigen Presentation/immunology , Antigens, Differentiation, B-Lymphocyte/immunology , Histocompatibility Antigens Class II/immunology , Molecular Chaperones/immunology , Animals , Antigens, Differentiation, B-Lymphocyte/metabolism , Histocompatibility Antigens Class II/metabolism , Humans , Models, Immunological , Molecular Chaperones/metabolism , Neoplasm Metastasis , Neoplasms/metabolism , Neoplasms/pathology , Receptors, Immunologic/immunology , Receptors, Immunologic/metabolism , Up-RegulationABSTRACT
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.
Subject(s)
Antigens, Differentiation, B-Lymphocyte/metabolism , Aspartic Acid Endopeptidases/metabolism , B-Lymphocytes/metabolism , Histocompatibility Antigens Class II/metabolism , Membrane Proteins/metabolism , Receptors, Antigen, B-Cell/metabolism , Signal Transduction , Animals , Antigens, Differentiation, B-Lymphocyte/chemistry , Apoptosis/genetics , Aspartic Acid Endopeptidases/deficiency , Aspartic Acid Endopeptidases/genetics , B-Lymphocytes/cytology , B-Lymphocytes/immunology , Cell Differentiation , Cell Membrane/metabolism , Endocytosis/genetics , Endocytosis/immunology , Forkhead Box Protein O1 , Forkhead Transcription Factors/metabolism , Histocompatibility Antigens Class II/chemistry , Immunoglobulin M/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Lymphocyte Activation , MAP Kinase Signaling System , Membrane Proteins/deficiency , Membrane Proteins/genetics , Mice , Mice, Knockout , NF-kappa B/metabolism , NF-kappa B p52 Subunit/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation , Precursor Cells, B-Lymphoid/cytology , Precursor Cells, B-Lymphoid/metabolism , Protein Binding , Protein Interaction Domains and Motifs , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Syk KinaseABSTRACT
Signal peptide peptidase-like 3 (Sppl3) is a Golgi-resident intramembrane-cleaving protease that is highly conserved among multicellular eukaryotes pointing to pivotal physiological functions in the Golgi network which are only beginning to emerge. Recently, Sppl3 was shown to control protein N-glycosylation, when the key branching enzyme N-acetylglucosaminyltransferase V (GnT-V) and other medial/trans Golgi glycosyltransferases were identified as first physiological Sppl3 substrates. Sppl3-mediated endoproteolysis releases the catalytic ectodomains of these enzymes from their type II membrane anchors. Protein glycosylation is a multistep process involving numerous type II membrane-bound enzymes, but it remains unclear whether only few of them are Sppl3 substrates or whether Sppl3 cleaves many of them and thereby controls protein glycosylation at multiple levels. Therefore, to systematically identify Sppl3 substrates we used Sppl3-deficient and Sppl3-overexpression cell culture models and analyzed them for changes in secreted membrane protein ectodomains using the proteomics "secretome protein enrichment with click sugars (SPECS)" method. SPECS analysis identified numerous additional new Sppl3 candidate glycoprotein substrates, several of which were biochemically validated as Sppl3 substrates. All novel Sppl3 substrates adopt a type II topology. The majority localizes to the Golgi network and is implicated in Golgi functions. Importantly, most of the novel Sppl3 substrates catalyze the modification of N-linked glycans. Others contribute to O-glycan and in particular glycosaminoglycan biosynthesis, suggesting that Sppl3 function is not restricted to N-glycosylation, but also functions in other forms of protein glycosylation. Hence, Sppl3 emerges as a crucial player of Golgi function and the newly identified Sppl3 substrates will be instrumental to investigate the molecular mechanisms underlying the physiological function of Sppl3 in the Golgi network and in vivo. Data are available via ProteomeXchange with identifier PXD001672.
Subject(s)
Aspartic Acid Endopeptidases/metabolism , Golgi Apparatus/metabolism , Glycosylation , HEK293 Cells , HumansABSTRACT
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.
Subject(s)
Antigens, Differentiation, B-Lymphocyte/metabolism , Aspartic Acid Endopeptidases/metabolism , Histocompatibility Antigens Class II/metabolism , Membrane Proteins/metabolism , Animals , Antigens, Differentiation, B-Lymphocyte/chemistry , Antigens, Differentiation, B-Lymphocyte/genetics , Aspartic Acid Endopeptidases/genetics , Cell Line, Tumor , Fluorescent Antibody Technique, Indirect , Histocompatibility Antigens Class II/chemistry , Histocompatibility Antigens Class II/genetics , Humans , Membrane Proteins/genetics , Mice , Mice, Knockout , ProteolysisABSTRACT
The lysosomal membrane was thought for a long time to primarily act as a physical barrier separating the luminal acidic milieu from the cytoplasmic environment. Meanwhile, it has been realized that unique lysosomal membranes play essential roles in a number of cellular events ranging from phagocytosis, autophagy, cell death, virus infection to membrane repair. This review provides an overview about the most interesting emerging functions of lysosomal membrane proteins and how they contribute to health and disease. Their importance is exemplified by their role in acidification, transport of metabolites and ions across the membrane, intracellular transport of hydrolases and the regulation of membrane fusion events. Studies in patient cells, non-mammalian model organisms and knockout mice contributed to our understanding of how the different lysosomal membrane proteins affect cellular homeostasis, developmental processes as well as tissue functions. Because these proteins are central for the biogenesis of this compartment they are also considered as attractive targets to modulate the lysosomal machinery in cases where impaired lysosomal degradation leads to cellular pathologies. We are only beginning to understand the complex composition and function of these proteins which are tightly linked to processes occurring throughout the endocytic and biosynthetic pathways.
Subject(s)
Intracellular Membranes/metabolism , Lysosomal Membrane Proteins/metabolism , Lysosomes/metabolism , Adaptor Proteins, Vesicular Transport/metabolism , Animals , Humans , Intracellular Membranes/chemistry , Lysosomes/physiology , Membrane Transport Proteins/metabolism , PhagocytosisABSTRACT
Northern ungulates acclimatize to winter conditions with restricted food supply and unfavorable weather conditions by reducing energy expenditure and voluntary food intake. We investigated in a study on red deer whether rates of peptide and glucose transport in the small intestines are also reduced during winter as part of the thrifty phenotype of winter-acclimatized animals, or whether transport rates are increased during winter in order to exploit poor forage more efficiently. Our results support the latter hypothesis. We found in a feeding experiment that total energy intake was considerably lower during winter despite ad libitum feeding. Together with reduced food intake, mass of visceral organs was significantly lower and body fat reserves were used as metabolic fuel in addition to food. However, efficacy of nutrient absorption seemed to be increased simultaneously. Extraction of crude protein from forage was higher in winter animals, at any level of crude protein intake, as indicated by the lower concentration of crude protein in feces. In line with these in vivo results, Ussing chamber experiments revealed greater electrogenic responses to both peptides and glucose in the small intestines of winter-acclimatized animals, and peptide uptake into jejunal brush-border membrane vesicles was increased. We conclude that reduced appetite of red deer during winter avoids energy expenditure for unproductive search of scarcely available food and further renders the energetically costly maintenance of a large gut and visceral organs unnecessary. Nevertheless, extraction of nutrients from forage is more efficient in the winter to attenuate an inevitably negative energy balance.
Subject(s)
Deer/physiology , Eating/physiology , Energy Intake/physiology , Energy Metabolism/physiology , Feeding Behavior , Seasons , Animals , Appetite/physiologyABSTRACT
Mast cell (MC) activation through the high-affinity IgE receptor FcεRI leads to the release of mediators involved in immediate-type allergic reactions. Although Abs against the tetraspanins CD63 and CD81 inhibit FcεRI-induced MC degranulation, the intrinsic role of these molecules in FcεRI-induced MC activation is unknown. In MCs, CD63 is expressed at the cell surface and in lysosomes (particularly secretory lysosomes that contain allergic mediators). In this study, we investigated the role of CD63 in MC using a CD63 knockout mouse model. CD63-deficiency did not affect in vivo MC numbers and tissue distribution. Bone marrow-derived MC developed normally in the absence of CD63 protein. However, CD63-deficient bone marrow-derived MC showed a significant decrease in FcεRI-mediated degranulation, but not PMA/ionomycin-induced degranulation, as shown by ß-hexosaminidase release assays. The secretion of TNF-α, which is both released from granules and synthesized de novo upon MC activation, was also decreased. IL-6 secretion and production of the lipid mediator leukotriene C4 were unaffected. There were no ultrastructural differences in granule content and morphology, late endosomal/lysosomal marker expression, FcεRI-induced global tyrosine phosphorylation, and Akt phosphorylation. Finally, local reconstitution in genetically MC-deficient Kit(w/w-v) mice was unaffected by the absence of CD63. However, the sites reconstituted with CD63-deficient MC developed significantly attenuated cutaneous anaphylactic reactions. These findings demonstrate that the absence of CD63 results in a significant decrease of MC degranulation, which translates into a reduction of acute allergic reactions in vivo, thus identifying CD63 as an important component of allergic inflammation.