Univ. Prof. Dr. Paul Saftig

Paul Saftig
Paul Saftig
Molecular Cell Biology and Transgenic Research
Biology of lysosomes: Lysosomes, lysosomal membrane proteins, hydrolases
and lysosomal storage disease
Lysosomes are organelles of eukaryotic cells involved in the turnover of various macromolecules. One of their major tasks is the degradation of extracellular material as well as intracellular components that are delivered to lysosomes by endocytosis or autophagy, respectively. Since the products of lysosomal catabolism and certain cytosolic compounds which are destined for degradation have to be transported across the 7-10 nm thick lysosomal membrane it is easy to envisage that this membrane must possess a number of highly specialized proteins. Furthermore, it has to withstand the luminal milieu with an acididc pH of less than 5 and 50 potent hydrolases, maintaining a tight barrier towards the surrounding cytosolic environment. Therefore, lysosomal membrane proteins (LMPs) are usually highly glycosylated probably forming a continuous glycoprotein layer at the luminal side of the lysosomal membrane.
Figur 1 Figure 1: Schematic view of a lysosome and of the ly­so­so­mal mem­brane as the interface to regulate com­mu­ni­ca­tion between lysosomal lumen and the cytosol.
Lysosomal membrane proteins are depicted and their di­verse roles in maintaining the lysosomal mi­cro­en­vi­ron­ment and in controlling transport processes across the ly­so­somal membrane are in­di­cated. The usually short cy­to­plasmic tails contain necessary information for the traffic­king of these proteins but may also be in­volved in the re­gu­la­ti­on of lysosomal motility and im­port of cytosolic pro­te­ins as was shown for LAMP-2A in case of chaperone me­di­ated autophagy. A model of the LAMP-1 structure as de­scribed in is indicated showing the compact character of the ly­so­so­mal "gly­co­ca­lyx". LIMP-2/SCARB2 mediates transport of the β-glucocerebrosidase to the lysosome where re­cep­tor and ligand dissociate. CD63 was shown to be involved in fusion events with the plasma mem­brane. DIRC2 is an ex­am­ple of the newly identified LMPs possibly in­volved in electrogenic transport across the lysosomal membrane. DIRC2 is also subject to cathepsin-L-mediated proteolysis. Cystine is transported through cys­ti­no­sin. The mul­ti-sub­unit v-type H+ ATPase mediates trans­port of protons into the ly­so­somal lumen and the chloride channel ClC-7 drives chloride anion transport into the lysosome using the proton gradient. The ami­no­terminus and transmembrane domain of the Ostm1 ß-subunit of ClC-7 are re­quired for ClC-7 Cl(-)/H(+)-exchange, whereas the Ostm1 transmembrane domain suffices for its ClC-7-dependent trafficking to lysosomes. Both LAMP-proteins (LAMP-1 and LAMP-2) par­ti­ci­pate in autophagic pathways and based on lysosomal localization ex­pe­ri­ments in LAMP-deficient fibroblasts it was proposed that they are also needed to control the motility of lysosomes though dynein-me­di­ated transport along microtubules. (Taken from Schwake M., Schröder, B., Saftig, P. Traffic 2013)
The most abundant type-1 transmembrane proteins of the lysosomal membrane are the lysosomal associated membrane proteins LAMP-1 and LAMP-2 with more than 10 used glycosylation sites. Based on computational analysis it is estimated that the thickness of the lysosomal glycoprotein coat is around 8nm. This is considerable lower than glycocalyces at the cell surface. The specialized glycoprotein layer may be important to regulate the stability and integrity of the lysosome. It may indirectly modulate the fusion of lysosomes with phagosomes, autophagosomes or with the plasma membrane during exocytosis.
Whereas the transmembrane segments of lysosomal membrane proteins are putatively involved in direct transport events across the membrane the usually rather short cytosolic parts of these proteins mediate contact to cytosolic proteins and proteins on other organelles. The latter may explain why certain lysosomal membrane proteins are needed for lysosomal motility, chaperone mediated autophagy, lysosomal exocytosis, membrane repair, MHC class II-dependent antigen presentation, phagocytosis and macroautophagy.
Similar to well-known and in the meantime partially treatable deficiencies of lysosomal hydrolases leading to typical lysosomal storage disorders, mutations in genes encoding for different lysosomal membrane proteins have been shown to cause clinical manifestations ranging from severe visceral symptoms to neurodegeneration. To date, more than 12 different disorders due to defects in LMPs have been recognized and the number is likely to increase since proteomic approaches have identified more than 100 different LMPs. It is therefore likely that hitherto unrecognized diseases will be linked to mutated integral lysosomal membrane proteins.
Development of a therapy for the lysosomal storage disorder alpha-Mannosidosis
Figur 2 Figure 2: The European ALPHA-MAN net­work is coordinated through activities in our lab. Correction of oligosaccharide sto­rage and improvement of cognitive func­tions in patients treated 12 month with recombinant mannosidase is pre­sen­ted (extraceted from J. Met. Inh. Dis. 2013).

The lysosomal storage disorder (LSD) alpha-Mannosidosis is a rare genetic disease affecting less than 500 people worldwide and according to the EU regulations, designated as an "orphan" disease. Alpha-Mannosidosis is caused by an enyzme defect due to mutations in the gene for lysosomal acid alpha-Mannosidase (LAMAN) affecting the lysosomal and cellular glycoprotein catabolism with severe consequences for the organism. In humans, LAMAN deficiency results in progressive mental retardation, skeletal changes, hearing loss and recurrent infections and many patients die during early childhood. Today, the most promising therapy for lysosomal storage disorders including alpha-Mannosidosis is Enzyme Replacement Therapy (ERT) where the respective enzyme lacking in the patient is produced by recombinant approaches and then introduced into the blood stream, from where it is internalized by the cells and reaches the lysosomes. replacing the missing endogenous enzyme. ERT products are on the market today for a number of LSD including Gaucher, Fabry, Pompe disease and the Mucopolysaccharidoses MPSI, II and VI and clinical trials are underway for a number of others. To date, no real treatment for alpha-Mannosidosis is available. Since children are born healthy, an early initiated therapy shortly after birth could dramatically improve their life expectancy and quality of life. Since pharmaceutical interest in this disease is low, two EU sponsored projects (EURAMAN and HUE-MAN) within the 5th and 6th framework program, respectively have worked towards developing the recombinant human enzyme (rhLAMAN) as a therapeutic agent for patients suffering from alpha-Mannosidosis and are now the basis for clinical trials in alpha-Mannosidosis.

The promising results of these two previous networks in general, but especially the achievements of the HUE-MAN project, including i) the large scale production of the recombinant human enzyme, ii) the evaluation of disease progression in alpha-Mannosidosis patients, iii) the determination of clinical endpoints through the natural history study and iv) the development of an effective ERT protocol in pre-clinical mouse studies, are the basis for us to propose the ALPHA-MAN project within the 7th framework program.

The main objectives of the ALPHA-MAN network (PI in Kiel: Judith Blanz) will be to transfer and expand the information and knowledge gained from the many years of work from the previous EURAMAN and HUE-MAN projects, to enable us to perform "First in Man" clinical trials in alpha-Mannosidosis patients, using the medicinal enzyme product rhLAMAN as the therapeutic agent and furthermore to improve the knowledge regarding i) long term "chronic dosing" and ii) mechanism of lysosomal enzyme transfer across the Blood Brain Barrier, in a newly established immune-tolerant mouse model. The final goal of ALPHA-MAN is to make a future treatment for ALL alpha-Mannosidosis patients available and thereby dramatically improve their life expectancy and quality of life. In addition, ALPHA-MAN will greatly increase the knowledge about the mechanism of how lysosomal enzymes can cross the blood brain barrier, which is also of great medical importance for the treatment of other neurodegenerative disorders.

The lysosomal sorting receptor LIMP-2/SCARB2
LIMP-2 (PI: Michael Schwake), also known as SCARB2 belongs to the CD36 family of scavenger receptors and spans the lysosomal membrane twice, with the N- and C-terminus located in the cytosol and a highly glycosylated loop within the lysosomal lumen. Multiple ligands have been described for LIMP-2, including thrombospondin, viruses and ß-glucocerebrosidase (GBA; the enzyme defective in Gaucher disease (GD)) underscoring the complex physiological function of this lysosomal membrane protein. GBA utilizes LIMP-2 for its transport to lysosomes independently of the mannose-6-phosphat pathway. Binding of LIMP-2 to GBA occurs early in the biosynthetic pathway already in the ER at neutral pH and is dependent on a stretch of sixteen amino acids (residues 152 to 167) with high probability to form an amphipathic helix. The GBA/LIMP-2 protein complex is transported from the Trans-Golgi Network (TGN) and endosomes directly to lysosomes, where GBA dissociates from LIMP-2 due to the low pH. Lack of LIMP-2 results in the missorting of GBA to the extracellular space and ER associated degradation. However, since no lipid-laden macrophages ("Gaucher cells" a pathologic hallmark of GD) could be observed in LIMP-2-deficient mice or AMRF patients it is likely that GBA may be taken up from the extracellular space or may be transported to lysosomes in leucocytes via still unknown pathways.
Figur 3 Figure 3: Unravelling the func­tions of the lysosomal mem­brane protein LIMP-2/SCARB2.
(A) LIMP-2 is involved in ly­so­so­mal transport of beta glu­co­ce­re­bro­si­da­se through a he­li­cal domain in its lu­mi­nal part (ami­no acid 152 to 167). Virus binding occurs in the luminal region between amino acid 144 and 151. The memb­rane pro­xi­mal N-ter­mi­nal part was also shown to be res­pon­si­ble for an en­large­ment of en­do­so­mes/ly­so­somes after he­te­ro­lo­gous ex­pres­sion of LIMP-2. (B) Apart from these func­tions LIMP-2 may par­ti­ci­pate in func­tions in the ureter and kidney, in the inner ear and in the myelinization of pe­ripheral nerves as revealed by knockout mouse studies. Deficiency of LIMP-2 leads to Action Myoclonus Renal Failure Syndrome, characterized by renal failure and epilepsy and intraneuronal accumulations in the CNS. (Taken from Schwake M., Schröder, B., Saftig, P. Traffic 2013)
In a very recent study we could contribute to the understanding about the structure of LIMP-2. We provided evidence supporting a model whereby lipidic constituents of the ligands attached to the receptor surface are handed of to the membrane through a tunnel which is part of the structure of LIMP-2 but also of the related proteins CD36 and SR-B1.
Figur 4
Figure 4: Structure of LIMP-2 (Nicolai et al. 2013, Nature).
Discovery and elucidation of the functions of hitherto unknown
lysosomal membrane proteins
The identification of new lysosomal membrane proteins by subproteomic approaches (PI: Bernd Schröder), in which new members of the lysosomal membrane are being investigated is another focus in the lab. The functional characterization of these new members of the lysosomal membrane is performed using biochemical and mouse genetic approaches. In this context the in vivo role of the signalpeptide-peptidase-like proteins (SPPL2a) could be revealed (PI: Bernd Schröder). In order to analyse functions of SPPL2a and SPPL2b in a complex in vivo system, mouse lines deficient in either of the two proteases have been generated. Evidence is provided that regulation of the aminoterminal fragment of CD74/invariant chain 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.
C.2 Proteolysis in or at membranes
Proteolytical processing of membrane-bound molecules is emerging as a fundamental mechanism for controlling the strength and timing of cell-to-cell communication. Proteins belonging to the ‘A Disintegrin And Metalloproteinase’ (ADAM) family are membrane-anchored proteases that are able to cleave the extracellular domains of several membrane-bound proteins in a process known as ‘ectodomain shedding’. Substrates for ADAMs include growth factors, cytokines, chemokines and adhesion molecules. Therfore many ADAM proteins play important roles in cell-cell adhesion, extracellular and intracellular signaling, cell differentiation and cell proliferation. It has been shown that ADAMs are widely expressed and are of required during developmental processes, by regulating cell-cell and cell-matrix interactions and by modulating differentiation, migration or receptor-ligand-activated signaling. In most cases, ectodomain shedding leads to the modulation of signaling activity on host and neighbouring cells either through downregulation of cell surface receptors or increased liberation of soluble ligands such as tumour necrosis factor-alpha (TNF-alpha) or epidermal growth factor receptor (EGFR)-ligands. Dysregulation of a properly regulated shedding activity is a critical factor in the development of complex pathologies such as cancer, cardiovascular disease, inflammation and neurodegeneration.
Figure 6
Figure 6: The complex regulation of ADAM-proteases. ADAMs (in pale blue) can be regulated at the level of transcription, trans­la­tion and through posttranslational processes (taken from Weber & Saftig 2012; Development)
The best-studied processing events carried out by the ADAM family member 10 (ADAM10) are the proteolytic processing of the Notch-1 receptor and the processing of the amyloid precursor protein (APP). In Alzheimer's disease (AD) the ADAM10-mediated a-secretase activity towards APP is a mechanism to prevent the excessive production of the neurotoxic amyloid beta (Ab) peptide. The processing of APP by ADAM10 occurs within the peptide sequence of Ab. This cleavage also generates a soluble N-terminal APP fragment (sAPPa) with apparent neurotrophic and neuroprotective functions. Enhancement of ADAM10 expression was suggested as a suitable therapeutic approach for the treatment of AD. Currently clinical trials are performed that address the question if induction of ADAM10 expression by retinoic acid derivatives is beneficial. Interestingly, recent data suggest that ADAM10 represents an additional AD risk gene since mutations in the prodomain of ADAM10 cause a shift towards amyloidogenesis in transgenic mice. Our previous studies demonstrated an essential role of this protease in the development of the embryonic brain and in neuronal network functions of adult neurons. ADAM10 achieves these functions by utilizing unique postsynaptic substrates in the central nervous system, in particular synaptic cell adhesion molecules. We also found that ADAM10 plays a crucial role mainly mediated by Notch-dependent processes in angiogenesis, in the immune system and in skin development and homeostasis. Using a yeast split-ubiquitin based screening approach we identified the tetraspanin family member 15 (TSPAN15) as a specific binding partner of ADAM10. This interaction occurs already in the ER and triggers the transport of the activated form of ADAM10 to the cell surface. This in turn leads to an increased shedding of ADAM10 substrates.
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Letzte Änderung / Last change: Juni 16, 2020