Smith-Lemli-Opitz syndrome (SLOS) is an inborn error of metabolism affecting the last step of cholesterol biosynthesis. It is characterized by a deficiency of the enzyme 7-dehydrocholesterol reductase and accumulation of 7-dehydrocholesterol (7DHC) in cells and body fluids. Given the similarities between 7DHC and cholesterol, 7DHC can be incorporated into cell membranes in lieu of cholesterol. Nevertheless, due to their structural differences and distinct affinity to other membrane components, this substitution alters membrane properties and one can expect to find abnormalities in membrane protein composition. In order to identify differences in membrane proteins that could facilitate our understanding of SLOS physiopathology, we isolated detergent-resistant membranes (DRMs) from the skeletal muscle of Dhcr7T93M/T93M mice and C57/BL6 controls and performed comparative proteomic analysis using iTRAQ for peptide quantification. A total of 133 proteins were identified in the DRM fraction: 17 (13%) proteins demonstrated increased expression in SLOS mice, whereas, 21 (16%) showed decreased expression. Characterization of functional point of view and bioenergetics pathway and transmembrane transport responded to the major differences between the two groups of animals.
- skeletal muscle
- detergent-resistant membranes (DRMs)
- Smith-Lemli-Opitz syndrome
- comparative proteomics
- mouse model
Smith-Lemli-Opitz syndrome (SLOS), OMIM #270400, is one of the nine known disorders associated with altered post-squalene cholesterol biosynthesis [1, 2]. This autosomal recessive genetic disease was first described in 1964, as a syndrome of cognitive impairment and multiple malformations . Thirty years were needed to further characterize the disorder as a metabolic disease and identify the underlying enzymatic defect. SLOS is caused by deficiency of 7-dehydrocholesterol redutase (7-DHCR, 3-hydroxysteroid redutase, EC.184.108.40.206) which catalyzes the conversion of 7-dehydrocholesterol (7DHC) to cholesterol, the terminal step of Kandutsch-Russell pathway [4, 5]. Consequently, SLOS patients typically show increased 7DHC and decreased serum and tissue cholesterol levels . In 1998, mutations of the 3β-hydroxysterol Δ7-reductase gene (
Effective cholesterol biosynthesis is especially critical at certain stages during development and continues to be important throughout life , since cholesterol is an essential lipid with multiple functions. Cholesterol is a major lipid component of membrane microdomains, which are crucial cell-surface dynamic structures responsible for many cellular signaling and communication events . Membrane domains can form through a number of mechanisms involving lipid-lipid and protein-lipid interactions. One type of membrane domain is the cholesterol-dependent membrane raft . Properties of these membrane domains have been primarily inferred from the study of detergent-resistant membranes (DRMs), composed by the non-ionic-detergent insoluble, low-buoyant density membranous fractions of cells . Although it was initially thought that such microdomains enriched in cholesterol exist exclusively in the plasma membrane, increasing evidence suggests that similar lipid microdomains (sometimes referred as raft-like microdomains) are also present in internal organelles [20, 21, 22] with some of them being involved in the crosstalk between organelles .
Proteomics constitutes a powerful tool to study complex biological mechanisms and to identify alterations in protein expression induced by changes in the environment, drugs, or disease states. As such, proteomics is now widely employed to help understand pathological processes induced by the disease [24, 25, 26, 27]. The effect of an inborn error of cholesterol synthesis on skeletal muscle has not previously been reported. In this chapter, we report a comparative analysis of protein expression in skeletal muscle DRMs isolated from
2. Material and methods
Butylhydroxytoluene (BHT), N,O-bis(trimethylsilyl) trifluoroacetamide (BSTFA), triethylammonium bicarbonate (TEAB), trifluoroacetic acid (TFA), α-cyano-4-hydroxycinnamic acid (α-CHCA), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), protease inhibitor cocktail, formic acid and urea were purchased from Sigma (Karlsruhe, Germany). RC DC Protein Assay kit for protein quantification was from BioRad lab (Hercules, USA). The iTRAQ kit was purchased from Applied Biosystems (Foster City, CA). Sequencing grade modified trypsin (bovine) was from ABSciex (ABSciex, USA). HPLC-grade acetonitrile (ACN, Riedel, Seelze, Germany) and Milli-Q grade water were also used. Rabbit raised polyclonal anti-caveolin 1 (ab2910) and anti-annexin A2 antibodies were purchased from Abcom, Cambridge, UK. Analytical reagent grade chemicals were used unless stated otherwise.
The T93M mutation  was backcrossed into C57/BL6 for three generations. Homozygous (T93M/T93M) mice are viable and fertile . Control C57/BL6 mice were obtained from IBMC Animal Centre from Oporto University.
All the animals were housed in plastic cages with free access to water and food (cholesterol free—chow -Mucedola, Ref: 4RF21). The animals were handled and maintained in controlled conditions according to international standards. Animals were euthanized using deep isoflurane anesthesia when they were 10 days old. Gastrocnemius and soleus muscles were dissected, submerged in ice-cold buffer (TRIS, HCl, pH 7.4, 10 mM mercaptoethanol, 0.28 M sucrose) and immediately frozen at −80°C.
2.3. DRMs extraction
Detergent-resistant membranes (DRMs) were isolated using cold Triton X-100 treatment followed by sucrose gradient centrifugation. In order to minimize individual variation, samples were analyzed as pools of several animals. The procedure was adapted from Kim et al. . Briefly, the samples were allowed to thaw slowly on ice, and tissue from three mice (approximately 300 mg) was minced with scissors, mixed with 700 µL of cold (4°C) lysis buffer (25 nM HEPES-HCl, pH 6,5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100 and protease inhibitor cocktail) and homogenized 30 times with a Poter homogenizer. The sample was maintained in an ice bath during homogenization and then incubated for 30 min at 4°C. Aliquots of this homogenate were collected for protein quantification and sterol analysis.
The resulting extract was mixed with an equal volume of cold sucrose 80% (w/v) to give a final sucrose concentration of 40%, transferred to the bottom of a ultra-centrifuge tube (Ultra-clear 14 × 89 mm, Beckman Ref 344059) and overlaid carefully with 6.0 mL 30% and 3.0 mL 5% cold sucrose solutions, containing 25 nM HEPES-HCl, pH 6.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100. Discontinuous sucrose gradients were centrifuged for 20 h, at 187,000 g on a Sorvall Ultra Pro 80 UC, swinging bucket SW41 at 4°C. The DRMs fraction, which collects at the interface of the 5 and 30% sucrose layers, was isolated, washed with 9 mL of modified HEPES buffer and centrifuged 30 min at 49,500 g at 4°C. After centrifugation, the supernatant was discarded and the pellet containing purified DRMs was suspended in phosphate buffered saline (PBS) and stored frozen at −80°C until further proteomic or sterol analysis.
2.4. Sterols analysis
For extraction of neutral sterols, 50 µL of sample was mixed with 50 µL of BHT 0.05% in methanol (antioxidant), 50 µL of epicoprostanol 0.10 mmol/L (internal standard) and saponified at 60°C for 1 h by 16 adding 1 mL of 4% (w/v) KOH in 90% ethanol. The samples were then diluted with 1 mL of deionized water and the lipids were extracted twice with 2 mL of hexane. The pooled hexane extracts were dried under a gentle nitrogen stream at room temperature and derivatized with 50 µL BSTFA in 50 µL pyridine at 60°C for 1 h . The trimethylsilylether derivatives of sterols were separated by GC–MS with a Supelco 28471_U SLB-5MS column (30 m × 0.32 mm i.d., 0.25 μm film thickness). The injector temperature was set at 270°C, and the splitless injection mode was used. The initial column temperature was 180°C for 1 min and programmed to increase at a rate of 20°C/min to 250°C and then increased again at 5°C/min rate till 300°C. The carrier gas was helium at a linear constant flow rate of 40 cm/s. The interface was programmed to 280°C, the quadrupole to 150°C and the ionization source to 230°C. After a solvent delay of 2.5 min, the eluted sterols were identified by their retention times (comparing with commercial standards) and respective mass spectra and quantified by selected ion monitoring (Table 1). The method was linear and reproducible for the range of amounts assayed.
2.5. Proteomic profile
2.5.1. Pellet solubilization and protein quantification
Each assay involved four sample pools: a wt-BL6 control and a
The protein concentration was determined by RC DC Protein Assay kit, using BSA to generate the calibration curve.
2.5.2. Sample preparation for mass spectrometry analysis
Reduction, alkylation and digestion steps were performed according to the protocol provided by the manufacturer. Briefly, 100 mg of DRM protein was mixed with TEAB buffer (1 M, pH 8.5) and an enhancer of enzymatic digestion RapiGest (Waters) to give a final concentration of 0.5 M and 0.1%, respectively. Samples were then treated with: (1) a reducing agent—TCEP 5 mM—used to break disulfide bonds within and between proteins, for 1 h at 37°C and (2) a cysteine (sulfhydryl group) blocking reagent—S-methyl methanethiosulfonate (MMTS) 10 mM—for 10 min, at room temperature. Then, 2 mg of trypsin was added to each sample and the digestion was performed for 18 h at 37°C. The digested tryptic specimens were dried using a Speed-Vac. iTRAQ labeling was carried out according to the instructions provided by the manufacturer.
2.5.3. LC-MS/MS analysis
Peptide mass spectra were obtained in the mass range 700–4500 Da on a MALDI-TOF/TOF mass spectrometer (4800 Proteomics Analyzer, Applied Biosystems, Foster City, CA, USA) in the positive ion reflector mode. The obtained spectra were processed and analyzed by the ProteinPilot® software (v4.0 AB Sciex, USA), which uses an algorithm for protein/peptide identification based on the comparison of MS/MS data against the SwissProt protein database .
Protein clustering was performed according to biological and molecular functions derived from the PANTHER classification system.
2.6. Immunocytochemistry assay
Immunocytochemistry assays were performed in order to confirm the presence of (1) caveolin-1, a protein marker of a subtype of membrane microdomains which are rich in cholesterol, designated caveolae (anti-caveolin-1 antibody dilution 1:500) and (2) annexin 2 another protein associated with enriched cholesterol microdomains, that was found increased in the SLO mouse model (antibody dilution 1:2000). A sequential incubation with a secondary biotinylated anti-rabbit antibody was performed and diaminobenzidine (which stains brown) was employed as chromogen. Skeletal muscle samples were then counterstained with hematoxylin.
3. Results and discussion
Sterols were extracted from two pools of biological samples: muscle homogenates and muscle DRMs, and then analyzed by GC-MS. Results are presented as the average ratios between sterols amounts. We observed a markedly elevated cholesterol/7DHC ratio in muscle homogenates from wild-type animals, reflecting the large amount of cholesterol and minimal levels of its precursor (7DHC), in controls. This ratio was also much greater than one in
Desmosterol is a cholesterol precursor by an alternative biosynthetic route, and there are no significant differences in desmosterol levels between controls and affected animals. This is also reflected in the 7DHC/desmosterol ratio. This parameter showed an enrichment of
In order to explore the protein changes on sarcolemma, due to decreased 7-DHCR activity, we analyzed DRMs utilizing iTRAQ labeling and LC-MS/MS.
A total of 133 unique proteins were identified. Those identified based on a single peptide and those with a protein score less than 2.5 fold were excluded. Then a cut-off of 30% was applied to iTRAQ average ratios allowing us to select proteins with an important variation in SLOS mice relatively to controls. Differential protein expression was specific and not just a general finding. Caveolin-1, a protein known to be expressed in cholesterol-rich membrane microdomains did not show differential expression (Figure 3).
Of the 133 identified proteins, we observed an altered expression of 38 (29%) proteins. Increased and decreased expression was observed for 17 and 21 proteins, respectively (Table 2). The replicate samples demonstrated a strong positive correlation (r = 0.90) and indicated good reproducibility (Figure 4).
|Total score||Sequence coverage %||Accession #||Name||Peptides (95%)||SLOS/wt-BL6|
|51.2||50||Q8R429||AT2A1_MOUSE||Sarcoplasmic/endoplasmic reticulum calcium ATPase 1||85||3.9|
|11.6||53||P07310||KCRM_MOUSE||Creatine kinase M-type||12||2.7|
|6.7||41||P27573||MYP0_MOUSE||Myelin protein P0||10||2.0|
|18.5||37||Q8K2B3||DHSA_MOUSE||Succinate dehydrogenase [ubiquinone] flavoprotein subunit, mitochondrial||18||1.9|
|17.4||59||P51881||ADT2_MOUSE||ADP/ATP translocase 2||24||1.9|
|29.5||56||P56480||ATPB_MOUSE||ATP synthase subunit beta, mitochondrial||35||1.8|
|4.1||46||Q9D3D9||ATPD_MOUSE||ATP synthase subunit delta, mitochondrial||4||1.6|
|4,6||31||Q8C7E7||STBD1_MOUSE||Starch-binding domain-containing protein 1||7||1.5|
|10.0||17||P70302||STIM1_MOUSE||Stromal interaction molecule 1||7||1.5|
|25.4||70||P48962||ADT1_MOUSE||ADP/ATP translocase 1||39||1.5|
|6.4||33||Q8VEM8||MPCP_MOUSE||Phosphate carrier protein, mitochondrial||9||1.4|
|19.1||59||Q03265||ATPA_MOUSE||ATP synthase subunit alpha, mitochondrial||27||1.3|
|3.0||47||Q9CQQ7||AT5F1_MOUSE||ATP synthase subunit b1, mitochondrial||14||1.3|
|4.0||19||Q5U458||DJC11_MOUSE||DnaJ homolog subfamily C member 11||3||0.8|
|23.3||45||Q8BMK4||CKAP4_MOUSE||Cytoskeleton-associated protein 4||26||0.8|
|5.5||50||Q9CR68||UCRI_MOUSE||Cytochrome b-c1 complex subunit Rieske, mitochondrial||9||0.7|
|4.2||82||P99028||QCR6_MOUSE||Cytochrome b-c1 complex subunit 6, mitochondrial||13||0.7|
|5.0||73||P56391||CX6B1_MOUSE||Cytochrome c oxidase subunit 6B1||9||0.6|
|4.4||53||Q9DCS9||NDUBA_MOUSE||NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 10||14||0.6|
|4.2||7||P99024||TBB5_MOUSE||Tubulin beta-5 chain||7||0.6|
|20.1||46||P08121||CO3A1_MOUSE||Collagen alpha-1(III) chain||25||0.6|
|26.0||56||Q9CZ13||QCR1_MOUSE||Cytochrome b-c1 complex subunit 1, mitochondrial||38||0.6|
|6.5||60||P97450||ATP5J_MOUSE||ATP synthase-coupling factor 6, mitochondrial||19||0.6|
|36.1||62||Q01149||CO1A2_MOUSE||Collagen alpha-2(I) chain||47||0.6|
|4.5||59||Q9CPQ1||COX6C_MOUSE||Cytochrome c oxidase subunit 6C||6||0.5|
|10.1||57||Q9D6J5||NDUB8_MOUSE||NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 8, mitochondrial||13||0.5|
|3.0||29||Q99LY9||NDUS5_MOUSE||NADH dehydrogenase [ubiquinone] iron-sulfur protein 5||2||0.5|
|5.9||13||P68134||ACTS_MOUSE||Actin, alpha skeletal muscle||5||0.4|
|8.8||46||P19783||COX41_MOUSE||Cytochrome c oxidase subunit 4 isoform 1, mitochondrial||12||0.4|
|2.6||3||P68369||TBA1A_MOUSE||Tubulin alpha-1A chain||2||0.2|
|3.0||4||Q08857||CD36_MOUSE||Platelet glycoprotein 4||2||0.2|
Most proteins showing an altered expression in DRMs preparations were found to participate in at least one of three main cellular processes: membrane trafficking, energy production and Ca2+ homeostasis.
Proteins with altered expression corresponded to a number of biological processes. We found alterations affecting several membrane transporters. Even though no results from skeletal muscle studies of SLOS are available, membrane trafficking abnormalities, in other cells harboring inborn errors of cholesterol biosynthesis, had already been reported. For example, in cultivated human skin fibroblasts from SLOS patients the membrane fluidity is altered, calcium permeability is augmented whereas folate uptake and membrane-bound Na+/K+ ATPase activity are markedly decreased . In agreement with these data, we now report decreased expression of phospholemman, a small plasma transmembrane protein that acts as a channel or channel regulator and modulates Na+/K+ ATPase activity [34, 35]. Further, we detected significantly decreased expression of another integral membrane glycoprotein associated with DRMs, the fatty acid translocase (FAT also called Cd36 or platelet glycoprotein 4) on sarcolemma of SLOS mice, responsible for the uptake of long chain fatty acids [36, 37, 38, 39]. Upregulated transporters include mitochondrial phosphate carrier protein (which transports inorganic phosphate into the mitochondrial matrix, essential for the aerobic synthesis of ATP) and ADP/ATP translocases 1 and 2 (that catalyzes the exchange of cytoplasmic ADP with mitochondrial ATP across the mitochondrial inner membrane).
These abnormalities of mitochondrial transporters may be indicative of more generalized defect in mitochondrial function and ATP production. Decreased expression of subunits corresponding to complexes I, III and IV of the oxidative phosphorylation (OXPHOS) system was observed, while four ATP-synthase subunits showed increased expression (ATP synthase mitochondrial subunits alpha, beta, delta and b1). Despite their mitochondrial origin, these proteins should not be considered as contaminants of DRMs preparations. Several biochemistry and proteomic studies had already shown the presence of complex I and ATP-synthase subunits in DRMs, [40, 41] and Poston showed that Triton X-114-resistant DRMs are also present in mitochondria and contain proteins that facilitate ATP production and export from this organelle , compatible with the increasing evidence of the presence of raft-like microdomains in mitochondria [21, 42].
Ca2+ is one of the most important signaling compounds involved in various cellular processes being intracellular Ca2+ levels tightly regulated by specialized proteins in the plasma membrane, sarcoplasmic reticulum (SR) and mitochondria . Recent studies suggest that membrane rafts are involved in coordinating the protein interactions required for proper Ca2+ exchange between the MAM and mitochondria . In the present study, we found increased expression of four membrane proteins related with Ca2+ homeostasis namely: annexin A2, sarcoplasmic/endoplasmic reticulum calcium ATPase 1 (SERCA1), calsequestrin-1 (CASQ1) and stromal interaction molecule 1 (STIM1).
Annexin A2 is a calcium-regulated membrane-binding protein, which holds two calcium ions. It has been proposed that it could play a key role in many processes including, (1) endocytosis and (2) exocytosis, (3) ion channel conductance, (4) link of F-actin cytoskeleton to the plasma membrane, (5) membrane organization, (6) formation of membrane cholesterol-rich microdomains and (7) regulation of cellular redox [44, 45, 46]. By LC-MS/MS, we found that annexin A2 was six times more abundant in DRMs obtained from
It is possible that the presence of 7DHC in SLOS mice membranes drives a higher annexin A2 incorporation in microdomains since 7DHC may promote microdomain formation . An alternate hypothesis would be that increased annexin A2 expression is related to its role in the regulation of redox potential. In fact, several data suggest an increase of oxidative stress in SLOS: (1) over a dozen of oxysterols have been produced from 7DHC by free radical oxidation in solution , (2) the oxysterol mixture derived from 7DHC free radical oxidation is biologically active and leads to morphological changes in Neuro2a cells treated with these oxysterols , (3) the synthesis of 3β,5α-dihydroxycholest-7-en-6-one (DHCEO), an oxysterol recently identified as a biomarker of 7DHC oxidation (in fibroblasts from SLOS patients and brain tissue), was found to be inhibited by an antioxidant compound in SLOS fibroblasts , (4) retinas from a SLOS rat model contain high levels of lipid hydroperoxides , (5) 7DHC peroxidation is a major source of oxysterols observed in cells  and (6) we previously identified a significant increase of the antioxidant enzyme superoxide dismutase mitochondrial in cultivated human SLOS fibroblasts. Considering these facts, we can hypothesize that there is an alteration of redox state of the cells in SLOS and annexin 2 is overexpressed as a part of cell strategy to compensate such a situation and protect cells’ biological compounds from peroxidation.
Another Ca2+ binding protein found upregulated in SLOS was SERCA1 an intracellular pump located in the SR of muscle cells, which catalyzes the hydrolysis of ATP coupled with the translocation of Ca2+ from the cytosol to the SR lumen, thus contributing to calcium sequestration involved in muscular excitation/contraction process. Another protein from this group is CASQ1, the major Ca2+-binding protein in the skeletal muscle SR. CASQ1 acts as an internal calcium store in muscle. The release of calcium bound to this protein through a calcium release channel triggers muscle contraction. Finally, stromal interaction molecule is a transmembrane protein essential for the activation of store-operated Ca2+ entry (SOCE), a major Ca2+ influx mechanism.
We also found integrin beta-1, overexpressed in affected mice. This microdomain-associated protein belongs to the integrin family which incorporates heterodimeric transmembrane proteins that function as major receptors for extracellular matrix proteins . Integrin beta-1, plays a role in the maintenance of the cytoarchitecture of mature muscle as well as in the functional integrity of the muscle cells and is present at the neuromuscular junctions in skeletal muscle ones . One wonders if its overexpression could be one of the factors which protect skeletal muscle from severe dysfunction in SLOS in opposition to other organs.
Ordered domains are formed when actin filaments attach to the plasma membrane . Kwik and collaborators found changes in the organization and activity of actin and actin-modifying proteins after cholesterol depletion, and Gangully and Chattopadhyay demonstrated that cholesterol depletion mimics the effect of cytoskeletal destabilization [55, 56]. Differences in myofilaments and cytoskeleton proteins were also suspected in our study.
Furthermore, some of the proteins identified were not so far, according to the available bibliography, associated with lipid-rafts or MAMs; nevertheless, they are involved in intracellular physical connections like cytoskeleton-associated protein 4 (CKAP4) which is a transmembrane protein that further to its function as receptor also links endoplasmic reticulum (ER) to the cytoskeleton . It is predictable that a membrane compact microdomain be involved in such function, in order to provide further support to the anchor. Such hypothesis is sustained by the fact that CKAP4 is a reversibly palmitoylated protein , and it is well described that palmitoylation of cytoplasmic proteins regulates the interaction of these soluble proteins with specific membranes or membrane domains. It is possible that palmitoylation controls the conformation of transmembrane segments, to modify the affinity of a membrane protein for specific membrane domains and to control protein–protein interactions .
Finally one should consider that both lipids and proteins for microdomains constructs are synthesized in the ER/Golgi before transport to the plasma membrane and, indeed, those proteins can be in a detergent-resistant, cholesterol-dependent state while residing there or in vesicle trafficking .
Our purpose was to contribute to the biological characterization of this
Proteomic analysis of DRMs of skeletal muscular samples clearly show differences between SLOS and wild-type mice, concerning proteins linked to membranes. The employed methodology allowed us to identify further alterations related with calcium homeostasis and membrane trafficking associated with SLOS and detected, for the first time, changes in mitochondrial energy metabolism in this mouse model.
To the best of our knowledge, this is the first research study focusing on the skeletal muscle of SLOS. The differential protein expression profile identified will open the way to comparative studies with more severally affected disease mice models as well as to similar approaches in human cells which may be helpful to uncover cellular mechanisms related to SLOS.
The authors are thankful to Prof Forbes D. Porter and Doctor Christopher A. Wassif from NIH who kindly provided the genetically modified mice model of SLOS used in this investigation. FDP and CAW are supported by the intramural research program of 30 the Eunice Kennedy Shriver National Institute of Child Health and Human Development.
Requimte received finnantial support from the European Union (FEDER funds POCI/01/0145/FEDER/007265) and National Funds (FCT/MEC, Fundação para a Ciência e Tecnologia and Ministério da Educação e Ciência) under the Partnership Agreement PT2020 UID/QUI/50006/2013.
This work received financial support from FCT/MEC through national funds and co-financed by FEDER, under the Partnership Agreement PT2020 (UID/MULTI/04378/2013 – POCI/01/0145/FERDER/007728).
Conflict of interest
The authors declare that there are no conflicts of interest.
|Cd36||cluster differentiation 36|
|iTRAQ||isobaric tagging reagents|
|FAT||fatty acid translocase|
|LCFAs||long-chain fatty acids|
|PBS||phosphate buffered saline|
|Dhcr7T93M/T93M||Smith-Lemli-Opitz syndrome mouse, homozygous for T93M mutation|
|STIM1||stromal interaction molecule 1|
|TEAB||bicarbonate salt of triethylamine|
|VDAC||voltage dependent anion-selective channels|