Investigation of Expression Levels of Ribosomal Proteins in Four Healthy Individuals
A
Diamond Balckfan Anemia Study
Sara Lahsaee
Degree project inapplied biotechnology, Master ofScience (2years), 2009 Examensarbete itillämpad bioteknik 45 hp tillmasterexamen, 2009
Biology Education Centre and Department ofGenetics and Pathology, Uppsala University Supervisors: Anne-Sophie Fröjmark and Jens Schuster
2 Table of Content
Summary……… 3
Introduction……… 4
Dimond Blackfan Anemia……….. 4
Inheritence and Genetics……… ……… 4
Erythropoiesis and cellular mechanism in DBA………... 4
CD34 molecules……….. 5
Glycophorin A………..……….. 6
Ribosomes………...……… 6
Molecular findings in DBA……… 6
Hypothesis……… 6
Western Blotting………... 7
Aim of the study……….. 8
Results………... 9
Optimization of antibodies ……… 9
Analysis of protein expression levels in four healthy individuals…..……… 9
Selection of cells………. ……….. 9
Protein prerartion……….. 9
Small subunit proteins and. large subunit proteins………... 10
Discussion………. 12
Ribosomal proteins are highly expressed in more differentiated erythroids……… 12
Ribosomal proteins in four different individuals……….. 12
Small subunit proteins vs. large subunit proteins………. 12
Future perspective………..………….. 13
Materials and Methods………. 15
Sample preparation………... 15
Cell separation………... 15
Protein extraction………... 15
Western blotting………... 15
Protein separation and transfer………. 15
Blocking the membrane….………... 15
Primary antibodies………... 15
Secondary antibodies……… 16
Membrane scanning……….. 16
Acknowledgement………. 17
References………. 18
Appendix 1……… 20
Appendix 2……… 23
3
Summary
Diamond Blackfan Anemia (DBA) is a severe hypoplastic anemia which typically presents with red blood cell aplasia. It is characterized by macrocytic anemia, reticulocytopenia and decrease or absence of red blood cell precursors in the bone marrow. Physical anomalies are common and there is an increased predisposition to cancer. The gene encoding ribosomal protein S19 (RPS19) is mutated in approximately 25% of the DBA cases. Since cloning and sequencing of human 19q13 breakpoint region associated with DBA resulted in discovery of RPS19 as one of the disease-causing genes, several other ribosomal proteins have been found to be mutated in DBA.
This indicates that haploinsufficiency for ribosomal proteins can give rise to DBA. This study was designed to investigate the levels of different ribosomal proteins in erythroid cells. It has been suggested that bone marrow cells express ribosomal protein at different levels in a tissue- specific manner and that some ribosomal proteins are rate limiting for ribosome assembly.
Analysis the relative amounts of ribosomal proteins at different cell fractions as well as between different individuals will help to prepare a protein “profile”. This profile could on one hand be a representative of relative amounts of ribosomal proteins to each other in healthy bone marrow and on the other hand be used as a reference for ribosomal protein expression levels in DBA affected tissues.
Bone marrow was sampled from four healthy control individuals at the Karolinska hospital, Stockholm, Sweden. The bone marrow cells were purified by immunomagnetic cell separation in three different erythroid fractions (GPA- (early), CD34- (intermediate) and GPA+ (late) cells).
Protein extracts were analyzed by western blot. The relative amounts of five ribosomal proteins were compared in the different cell fractions.
The results suggested that the expression levels of the five selected ribosomal proteins tended to increase during erythropoiesis. The ratio of small subunit proteins to large subunit proteins (SSU/LSU) was significantly higher in GpA- cells than in GpA+ cells, suggesting a relatively higher expression of large subunit proteins in more differentiated erythroid cells.
4
Introduction
Diamond Blackfan Anemia
Diamond Blackfan Anemia (DBA; OMIM#205900) is classified as one of the inherited bone marrow failure syndromes (IBMFS) commonly associated with aplastic anemia and a family history (Ellis & Lipton 2008). DBA was first reported in 1936 by H. W. Joseph as an anemia of infancy and early childhood (Ellis & Lipton 2008). The disease was described in more detail 1938 when K.Diamond and K. D. Blackfan stated: "…. there may be congenital insufficiency of red marrow tissue and inability on the part of the hematopoietic system to respond to the need for more blood as the erythrocytes wear out" (Flygare and Karlsson 2007). The diagnostic criteria for the disease were published by Diamond et al in 1976. DBA is characterized by selective marrow erythroid hypoplasia during the first year of life with a range of normal to slightly decreased number of neutrophils, reticulocytopenia, macrocytic anemia and variable platelet counts. While absent or decreased erythroid precursors is defined as the main clinical feature, no change in number of white blood cells has been reported in DBA patients (Naithani et al.
2006; Ellis & Lipton 2008). In addition to anemia, a heterogeneous mixture of physical abnormalities has been diagnosed in more than 50% of patients (Flygare & Karlsson 2007).
Craniofacial abnormalities, thumb malformations and short stature are common, while congenital cardiac and urogenital defects are observed to a lesser extent (Ellis & Massey 2006; Ellis &
Lipton 2008)
Elevated levels of fetal hemoglobin (HbF) and erythroid adenosine deaminase (eADA) activity are observed in some blood disorders including DBA whereas no deficiency of vitamin B12, vitamin B9 (folate), iron and erythropoietin has been reported in DBA patients (Ellis & Lipton 2008). In some DBA population elevated level of folic acid, vitamin B12 and erythropoietin (Epo) are observed (Campagnoli et al. 2008).
Inheritance and Genetics
To date DBA is the first and only human disorder found to be caused by mutations in ribosomal protein genes (Ellis and Massey 2006). Although most of these are spontaneous mutations, familial cases have been reported. In all cases the mode of inheritance is autosomal dominant.
The most common gene mutated in DBA patients encodes the ribosomal protein RPS19 (Draptchinskaia et al. 1999). This gene is reported to be mutated in approximately 25% of DBA patients (Draptchinskaia et al. 1999; Ellis et al. 2006; Boria et al. 2008). Mutations in additional ribosomal proteins that have been identified to be associated with DBA include RPS7, RPS24 RPS17, RPL35, RPL5 and RPL11 (Cmejla et al. 2009; Cmejla et al. 2007; Gazda et al. 2006;
Boria et al. 2008 and Gazda et al 2008). Most DBA cases are sporadic with an incidence of 5-7 per 100,000 live births. Male and females are affected equally (Flygare & Karlsson 2007).
Erythropoiesis and cellular mechanism in DBA
The term erythropoiesis (erythro = red blood cell, and poiesis = to make) refers to the process during which red blood cells (RBCs) are produced from their precursors. In adults this process
5 normally occurs in the red bone marrow (the central part of bone which is responsible for making red blood cells). During early stages of fetal development this process takes place in the yolk sac while spleen and liver are the responsible organs during the third and the forth months of development. Figure 1 illustrates the process of erythropoiesis and the main cell types produced in the maturation from bone marrow stem cells to mature erythrocytes. Erythrocytes are mature form of red blood cells differentiated from specific stem cells in the bone marrow.
Differentiation occurs through several stages upon stimulation by specific factors.
Figure 1. Erythropoiesis lineage. Erythrocytes are differentiated from specific stem cells in the bone marrow through several stages upon stimulation of specific factors. Progenitor cells basically develop in two phases: burst- forming unit erythroids (BFU-E) followed by colony-forming erythroids (CFU-E) upon erythropoietin stimulation while other factors drive progenitor cells to the next stage where they produce high amount of hemoglobin. CD34 is the main protein expressed on the surface of progenitor cells while more differentiated erythroids express Glycophorin A. Glycophorin expressing cells are shown by GpA+ while GpA- refers to the group of cells which do not express this glycoprotein.
The hematopoietic growth factor erythropoietin (Epo) is the trigger in manufacturing red blood cells in the bone marrow. Erythropoietin stimulation triggers cell proliferation at burst forming unit erythroids (BFU-E) while other factors drive progenitor cells to the next stage with high production of hemoglobin. CD34 is the main protein expressed on the surface of progenitor cells while more differentiated erythroids express glycophorin A. The progenitor cells develop in two phases: erythroid burst-forming units (BFU-E) followed by erythroid colony-forming units (CFU-E). BFU-E differentiate into CFU-E following erythropoietin stimulation, and then further differentiate into erythroblasts when stimulated by specific growth factors. Reticulocytes are the most differentiated red blood cells in the bone marrow (figure 1). Reticulocytes lose their nuclei when they leave the bone marrow and circulate through the body in the blood stream, where they develop into mature erythrocytes. Patients with DBA display a block in the erythropoiesis, more precisely at the BFU-E to CFU-E stage (Flygare & Karlsson 2007).
CD34
CD34 is a 115-kDa transmembrane glycoprotein present on the surface of certain cells, e.g.
hematopoietic progenitor cells and vascular endothelium in the human body. This molecule is a member of a family named “cluster of differentiation” (abbreviated as CD) which refers to the procedure used to analyze antibodies reacting with leukocyte cell surface molecules. This protein is normally considered as a marker for hematopoietic cells in bone marrow (figure 1). Bone
6 marrow progenitor cells display CD34 on their surface while the mature erythroblasts lack this glycoprotein (Furness & McNagny 2006).
Glycophorin A
Glycophorin A (GpA) is a 131 amino acid protein located in the cell membrane. In human, mature red blood cells express this protein on their surface. In this way the red blood progenitor cells which express glycophorin A (GpA+ cells) are recognizable from GpA- cells which are the less differentiated form of erythroid cells (Andersson et al. 1981).
Ribosomes
As the main organelles involved in protein synthesis, ribosomes are ribonucleoprotein particles present in the cytoplasm of all types of cells. Ribosomal genes are transcribed in the nucleus and translated in the cytoplasm. The proteins are then transported back to the nucleolus. Composed of protein and RNA, ribosomes play the main role in protein synthesis, the process called translation. Ribosomes are organelles where mRNAs and tRNAs come together to translate the mRNA into polypeptide chains that are primary structures of proteins. Ribosomes exist in both eukaryotic and prokaryotic cells. Ribosomes are made of two conserved subunits, the small subunit (SSU) and the large subunit (LSU), ranging in size from 2.5 million Daltons in bacteria to more than 4 million Daltons in eukaryotic cells. Approximately 60% of this mass is RNA and about 40% is protein. (Alberts et al 2002)
Molecular Findings in DBA
As mentioned above, RPS19 has been shown to be mutated in one forth of DBA patients while mutations in other ribosomal proteins are found in smaller fractions of the cases (Draptchinskaia et al 1999; Campagnoli et al 2008).
The mutations in RPS19 include point mutations such as transitions, insertions and deletions which result in missense, nonsense, frame shift and splice site alteration. Large deletions and chromosomal rearrangements also have been reported in rare cases (Gazda et al. 2004; Cmejla et al. 2009). All identified mutations and variants linked to DBA are listed in the Diamond- Blackfan Anemia mutation database (http://www.dbagenes.unito.it). Mutations in RPS19 have been shown to be linked with cell cycle arrest (Kuramitsu et al. 2008), apoptosis (Miyake et al.
2008), and ribosome biogenesis (Flygare et al. 2007; Badhai et al. 2009). In addition, haploinsufficiency of RPS19 is associated with cooperative downregulation of other ribosomal proteins as well as genes responsible for translation in DBA affected cells (Gazda et al. 2006;
Badhai et al. 2009).
Hypothesis for how a defect in ribosome synthesis could result in DBA
One hypothesis (Ellis & Massey 2006) suggests that ribosomal proteins are expressed in amounts that differ from one tissue to the other and that haploinsufficiency for a particular ribosomal protein may be rate limiting for ribosome assembly in that specific tissue. According
7 to this hypothesis, erythroid progenitor cells are the initial site where haploinsufficiency for at least one ribosomal protein, RPS19, becomes rate limiting for ribosome biogenesis. The hypothesis also states that: "in CD34+ cells from normal control, RPS19 expression is relatively low but not limiting for 40S subunit assembly" (Ellis & Massey 2006). On the other hand, patients with DBA display a block in erythropoiesis, more precisely at the BFUe and CFUe stage (figure 1). This indicates that haploinsufficiency for a ribosomal protein has severe effects on erythroid progenitor cells in the bone marrow (Ellis & Lipton 2008).
Western Blotting
Western blot (alternatively called protein immunoblot or electroblot) is an analytical technique that allows for detection of proteins in a given sample consisting of homogenized tissue or cell extract. The technique is based on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) in which proteins migrate through a polyacrylamide gel and are separated upon size. In principle smaller proteins migrate faster than larger proteins through the gel. The technique can separate both native and denatured proteins. The rate of migration of native proteins is strongly affected by their three-dimensional structure. Denaturation straightens the polypeptide chains so that the sodium dodecyl sulphate gives the same negative charge to each amino acid residue. Denatured proteins therefore migrate through SDS gel at a rate proportional to the number of amino acids they contain. Efficiently separated proteins are transferred onto a membrane, either Polyvinylidene Fluoride (PVDF) or nitrocellulose. This process is done in a chamber in which an applied electric current forces all proteins to be transferred into the membrane at the position they had after separation on the gel (figure 2).
Transferred proteins are now detectable by hybridization with specific antibodies. Primary antibodies are specifically designed to interact with target proteins. The bound primary antibodies then are visualized by fluorescently labeled secondary antibodies. These reactions result in visible bands on the membrane when exposed to appropriate wave length in an imaging system. One advantage using this method is that one can analyze several samples at the same time by cutting the membrane into strips and by probing them with one or more antibodies labeled with different fluorophores.
Figure 2. Western blotting.
Anode (+)
Filter papers
Polyacrylamide gel
PVDF membrane Filter papers
Cathode (-) Blotting papers
Blotting papers
Protein movement
8 Aim of the study
The aim of this study was to investigate the levels of different ribosomal proteins in erythroid progenitor cells as well as in more differentiated erythroid cells in four healthy individuals. To test the above hypothesis I planned to measure the levels of five selected ribosomal proteins in different erythroid cell types from four healthy individuals. Estimation of the level of ribosomal proteins in three cell fractions from early, intermediate and late stages in erythropoiesis, could be the initial step in providing a ribosomal protein profile. The profile is then compatible with the one from DBA patients and/or any other ribosomal profile from both healthy individuals and DBA patients. Any ribosomal protein profile in DBA could then be evaluated by such a control profile to see if there is any rate limiting manner of one or more particular ribosomal proteins. To study the role of ribosomal protein levels in ribosome assembly, it is required to know if there is any common pattern of expression in healthy cells to be compared to DBA-affected cells.
Analysis of the relative amounts of ribosomal proteins in two ribosomal subunits was another aspect of this study. dfgsgge vffsdvfvfvfssdfsfsfwsfswgrsgrghhhhhhhhhhhhhhhhhhhhhhhhhhhh
9
Results
Optimization of antibodies was needed prior to hybridizing specific antibodies with target proteins.
Optimization of antibodies
The aim at this step was to select at least two antibodies against small subunit proteins and two against large subunit proteins. To optimize the experimental procedure, I extracted proteins from three different human cell lines (LCLs, K562 and HEK293T). For each cell line, protein samples were analyzed by western blotting (data not shown). Of more than 12 tested antibodies against different ribosomal proteins (RPs), anti-RPS12, anti-RPS19, anti-RPS20, anti-RPL9 and anti- RPL30, five antibodies were selected for further use. The selected antibodies were the ones that gave positive results when they were tested on proteins extracts from cell lines.
Analysis of protein expression levels in four healthy individuals
Selection of cells: Bone marrow samples from four healthy individuals were obtained and three different cell types (GpA-, CD34- and GpA+) were sorted out using immunomagnetics at the Karolinska Institute, Stockholm.
Protein preparation: I extracted total proteins from the cells using Trizol reagent. Negatively charged proteins were then separated according to their sizes on an SDS-NuPAGE polyacryl- amide gel. Separated proteins were transferred to a PVDF membrane and hybridized with primary polyclonal antibodies against the five ribosomal proteins. Since the secondary antibodies were fluorescently labeled, their interaction with target proteins could be detected and visualized when the membranes were scanned with an imaging system. I used infrared Odyssey imaging device to scan western blot membranes. Relative amounts of ribosomal proteins in two studied cell types (GpA- and GpA+) are illustrated in figure 2. Results from CD34- cells were excluded because of limiting materials which did not allow duplication of the experiments. GpA- and GpA+ cells were selected as they represent very early and very late stages in erythropoiesis, respectively.
The relative level of each of the five tested ribosomal proteins was normalized to β–actin in four individuals. The mean values from two measurements were calculated for three out of four samples (individual 1, 3 and 4). Individual 2 values were based on one measurement (figure 2). Although one individual out of four (Indv. 3) did not show the same pattern for three small subunit ribo- somal proteins (RPS12, RPS19 and RSP20), when it comes to the mean value from all four indi- viduals, the tendency is observed to be increased when erythroid cells pass through erythropoiesis or more clearly when they are more differentiated (Appendix 1). Some western blot pictures are shown in appendix 2.
.
10
0 0,2 0,4 0,6 0,8 1
GPA- GPA+
RPS12
0 1 2 3 4
GPA- GPA+
RPS19 -0,5
0 0,5 1 1,5 2 2,5 3
GPA- GPA+
RPS20
-1 0 1 2 3 4
GPA- GPA+
RPL9
0 0,5 1 1,5 2
GPA- GPA+
RPL30
Small subunit proteins vs. large subunit proteins
In order to compare the levels of ribosomal proteins in the two ribosomal subunits, the average amount of both small (RPS12, RPS19 and RPS20) and large (RPL9 and RPL30) subunit proteins were calculated for different cell types (Figure 3). The amount of large subunit proteins was higher compared to small subunit proteins. To analyze the relative amount of the two subunits protein levels in two different cell phases, mean values of the three SSU proteins and two LSU proteins,
A D
C
E B
Fig 2. Relative amounts of five ribosomal proteins in GPA- and GPA+ sorted bone marrow cells. The average level of two small subunit ribosomal proteins (RPS12: p= 0,86; RPS19:
p=0,20 and RPS20: p=0,23) and two large subunit ribosomal proteins (RPL9: p= 0,04and RPL30:
p=0,05), all normalized to β-actin, were calculated after collecting data from four healthy individuals. Values in this graph are based on two independent measurements from individual 1,3 and 4 and only one measurement for individual 2.
Relative proteinexpression levels Relative proteinexpression levels
Relative proteinexpression levels Relative proteinexpression levels
Relative proteinexpression levels
11
Figure 3. Comparison of ribosomal proteins from A) small subunit and B) large subunit ribosomal proteins. Mean values were calculated between three small subnit ribosomal proteins RPS12, RPS19 and RPS20) and two large subunit ribosomal proteins (RPL9 and RPL30) in both GpA+ and GpA- cells. Expression levels of both small subunit (p= 0,094) and large subunit (p= 0,0043 ) proteins show an increase in GpA+ cells when it compared to GpA- cells.
both normalized to β-actin, were calculated in both GpA+ and GpA- cells. Figure 4 illustrates the SSU/LSU ratio which was significantly higher in GpA- cells. Although the ratio was based on only five ribosomal proteins, it potentially suggests differences throughout erythropoiesis with respect to the stoichiometry of ribosomal proteins. The SSU/LSU ratio showed a reduction to almost 1/2 in GpA+ cells compared to GpA- cells (figure 4). This observation also suggests that in normal tissue, the expression of LSU proteins is increased in later phases of erythropoiesis relative to expression of SSU proteins.
Figure 4. Ratio of small subunit ribosomal protein levels (RPS12,RPS19 and RPS20) to large subunit ribosomal protein levels (RPL9 and RPL30) in GpA+ and GpA- cells. The mean value for each ribosomal protein group (SSU and LSU) was calculated after normalizing against β-actin. The ratio shows a significant decrease in GpA+
cells (P= 0,0042). The ratio in GpA- cells is set to one.
.
A B
Relative proteinexpression levels Relative proteinexpression levels
Relative proteinexpression levels
GpA+
GpA+
GpA- GpA- GpA+
12
Discussion
Analysis of expression level of ribosomal proteins could shed light on unknown molecular mechanisms underlying DBA as well as other diseases linked to ribosomal biogenesis. The present study showed an overall tendency toward elevation of expression levels of five tested ribosomal proteins through red blood cell differentiation.
Ribosomal proteins are highly expressed in more differentiated erythroids
On one hand, higher expression of ribosomal proteins in GpA+ cells compared to GpA- cells could support the idea that BFU-E is the stage of proliferation as the result of erythropoietin stimulation.
Rapidly growing cells need high production of cell components including proteins. Since ribosomes are the main protein synthetic machinery, ribosomal proteins are required to be in appropriate amount to meet the cellular demand for protein synthesis. In fact DBA patients do not respond properly to erythropoietin stimulation (Flygare & Karlsson 2007). On the other hand, this expression level difference could confirm the idea that there is a higher ribosome biosynthesis in respond to high hemoglobin demand after CFU-E stage. As demonstrated in figure 2, the tendency for elevation of ribosomalprotein expression was observed for at least in five ribosomal proteins. In normal bone marrow, erythroid progenitor cells need to synthesize huge amounts of hemoglobin before losing their nucleus while in DBA bone marrow a block is defined in BFU-E stage as the main failure (Flygare & Karlsson 2007).
Ribosomal proteins in four different individuals
RPS19 is one of the ribosomal proteins required for ribosome assembly because of its role in the ribosomal 40S subunit structure (Flygare et al. 2007; Badhai et al. 2009). Besides, gene therapy with introducing RPS19-containing vectors into CD34+ bone marrow cells improved the colony- forming ability in erythroids in RPS19-deficient DBA patients (Hamaguchi et al. 2002) which means DBA patients with mutations in the RPS19 gene show a defect in proliferation of multipotent progenitor cells. My data showed that RPS19 expression levels, at least in three out of four tested individuals, increased through the erythropoiesis lineage. This observation was in agreement with the conclusion from studies showing correction in erythroid proliferation in bone marrow progenitor cells from DBA after transferring RPS19 gene (Flygare et al. 2008). Another SSU protein, RPS20, shows the same pattern in one individual as its expression decreased slightly in GpA+ cells. The other tested SSU protein, RPS12, appeared to show a different expression profile among four individuals as a tendency to be increased is observed in only one individual out of four (Appendix 1). The increased tendency is observed for the two tested LSU proteins (Fig 2C
& appendix 1).
Small subunit ribosomal proteins vs. large subunit ribosomal proteins
Comparison of three SSU and four LSU proteins in four healthy individuals in this study showed a tendency toward increased levels in later erythropoisesis stages (Figure 3), although a significant difference was observed only for LSU proteins. These data could suggest that at least some SSU
13 proteins including RPS19 were expressed in lower amounts during erythropoiesis when compared to LSU proteins. The observed lower expression levels for small subunit proteins could possibly supports the hypothesis that suggested ribosomal proteins are expressed in amounts that differ from each other (Ellis & Massey 2006).
One study on ribosomal proteins suggested a significant reduction for three SSU proteins (RPS20, RPS21 and RPS24) and not for LSU proteins in cells induced by siRNA against RPS19 mRNA (Badhai et al 2009) which may suggest that RPS19 has a co-downregulation effect on SSU proteins and not on LSU components. As illustrated in figure 3, the LSU proteins levels were increased 3- fold in GpA+ cells but this increase was only 1.5-fold for the SSU proteins levels.This significant- ly different (p<0.05) expression level in two studied cell types could indicate that there might be a higher expression of LSU components as progenitor cells transition into mature red blood cells in bone marrow. In the other word, the biosynthesis of the LSU proteins might proceed independent- ly of that of the SSU proteins. No mechanism is yet known though which adjust levels of LSU and SSU proteins. In addition, a reduction of almost half in GpA+ cells was observed in the SSU/
LSU ratio (Figure 4). This observation may suggest an assumption that in normal bone marrow, the effect of differentiation may cause LSU proteins to be expressed in higher amounts while SSU proteins are expressed in lower amount or remain at the same level as they were in the early stage. If so, then there is a possibility that cooperative significantly reduction of at least three SSU proteins, including RPS19 and RPS20, in DBA-like cells (Badhai et al 2009) is because of a relatively lower expression of SSU proteins. It can also suggest that the biosynthesis of the LSU proteins proceeds independently of the SSU ones and no mechanism is yet known which adjusts LSU proteins to SSU proteins. Although my data showed a possible tendency for some ribosomal levels of proteins to be increased in more differentiated erythroid cells, many questions still remain concerning varieties in expression levels. Not only one ribosomal protein amount but any extra-ribosomal factors could affect other ribosomal protein expression patterns.
Future perspective
This work started with testing 12 different ribosomal proteins in variety of cell lines and ended up with only five ribosomal proteins in two bone marrow cell types. This fact indicates that there is still a lot to be done in order to indentify a consensus profile of ribosomal proteins expression level in different stages of erythropoiesis both in healthy and DBA-affected cells. The observed increase in ribosomal proteins during erythroid differentiation on one hand could correspond to an increase in functional ribosomes and on the other hand could be a result of other extra-ribosomal factors.
Any of 70 ribosomal proteins might be rate limiting in different subsets of tissues which have not been found yet. Investigation of expression levels of other ribosomal proteins in as many healthy individuals as possible as well as DBA patients would be a high potential follow up study.
Effects of other factors like protein-protein interactions or polymorphic factors, as reported before, could affect ribosomal protein genes and alter their expression (Ellis & Massey 2006; Gazda et al.
2006 and Badhai et al. 2009).
Many questions are still remaining to be answered: Is there a significant difference between SSU and LSU in terms of expression levels in more differentiated cells? Do other ribosomal proteins levels increase throughout erythropoiesis? Does the expression pattern of RPS19 differ from that of
14 expression pattern differ from the other ribosomal proteins? If so, does it induce any up- regulatation and/or down-regulation effects on other ribosomal proteins? Is any of the ribosomal proteins rate limiting for ribosome assembly in bone marrow? A ribosomal profile could reveal many mysteries behind mechanism causing DBA. Thousands of unknown facts is remain to be discovered including alteration in expression and relative amounts of ribosomal proteins in healthy individuals as well as DBA patents.
15 Materials and Methods
Sample preparation
Cell separation: Three different cell types were isolated from bone marrow samples of four healthy individuals. Cell separation was performed using immunmagnetic cell separation technique at the Karolinska hospital, Stockholm. Magnetic beads coated with monoclonal antibodies against target proteins were applied to detect the target protein. Target proteins in this study were hematopoietic cell surface markers such as CD34 and Glycophorin A which are recognizable in bone marrow progenitor cells and mature eryhtroid cells, respectively. CD34- and GpA- cells ware isolated after washing the column containing CD34+ and GpA+ cells attached to the beads. This study was based on ~1.5 ×106 GpA+ cells and ~12×106GpA- cells from two different stages.
Protein extraction: Protein extracts were prepared from three bone marrow cell types of four healthy individuals using TRIzol® reagent according to Total RNA/Protein Isolation Reagent (Life Technologies™ ) according to the manufacture’s protocol. Protein pellets were then dissolved in 1% Sodium Dodecyl Sulphate at 55°C.
Western blotting
Protein separation and transfer: Dissolved proteins were prepared for Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophorsis (SDS-PAGE). 20 µl of each sample were mixed with 4 µl NuPAGE Sample Buffer (Invitrogen ™) and 2 µl of dithiothreitol (SIGMA) as reducing agent.
Samples then were heated at 70°C for 10 min to break all remaining oxidized bounds in proteins in advance to loading on the polyacrylamide gel (10% Bis-Tris SDS-PAGE, Invitrogen ™). The gel ran in running buffer (1x NuPAGE Running Buffer and 1 ml antioxidant/1000 ml Running Buffer, Invitrogen ™) in a Novex Mini-Cell (Invitrogen ™) for 40 min at 200V. 2 µl Page Ruler™ protein Prestained Ladder was used as a protein size marker. After electrophoresis proteins were electroblotted on to PVDF Immobilon-FL membrane (Millipore) in transfer buffer (1x Nupage Transfer Buffer and 10% methanol) according to manufacturer's protocol. All filter papers and blotting pads were soaked in transfer buffer before use. The PVDF membrane was soaked with methanol for few seconds and was rinsed with transfer buffer just before using. The membrane transfer was run in Novex Mini-cell (invitrogen ™) for 90 min at 40V.
Blocking the membrane: Protein containing PVDF membranes were blocked over night at 4°C with 2%BSA solution in 1× Phosphate Buffered Saline containing 8 gr NaCl, 0.2 gr KCl, 1.44 gr Na2HPO4 and 0.24 gr KH2PO4 in 100 ml ddH2O (1X PBS). This was required to avoid any false positive results due to non-specific interactions between antibody and the membrane as the solid membrane binds to proteins.
Primary antibodies: The membranes were hybridized with primary antibodies against ribosomal proteins of interest (Table 2). All primary antibodies but one were produced by Swedish Human Proteome Resource (HPR) against ribosomal proteins. An anti-RPS19 monoclonal antibody was kindly provided by Professor Farrizio Loreni in Rom, Italy. All membranes were probed with anti β-actin antibody (Abcam) as internal control to normalize values. Usually one primary antibody
16 was added at a time. The membrane was then rinsed to remove unbound antibodies and bound primary antibodies were picked up by secondary fluorescent antibodies. Membranes were incubated overnight in 2% BSA solution in 1× PBS at 4°C before incubation with primary antibodies. All primary antibodies were incubated overnight at 4°C except for anti-RPS19 which was probed for 1hr at room temperature. Table 2 contains all information about primary and secondary antibodies I used.
Secondary antibodies: Secondary antibodies were added after rinsing membranes three times of 10 minutes in room-temperature washing buffer (1% Tween-20 in 1× PBS ) to remove all remaining primary antibodies with non specific binding. All secondary antibodies were incubated at room temperature for 1hr.
Membrane scanning: Membranes were then rinsed again three times 10 minutes with the washing buffer and two times of 10 minutes in 1× PBS at room temperature to remove all residual of secondary antibodies. Western blots were analyzed in Odyssey infrared imaging system (Li-Core Bioscience) according to the manufacture manual. After exposure to infrared light, mouse and rabbit antibodies turned red (absorbance at 800 nm) and green (absorbance at 700 nm) respectively. The relative amount of proteins was calculated following normalization to β-actin integrated intensity.
Table 2. Primary and secondary antibodies used in this study.
Primary antibody Dilutions Host Source Secondary antibody Source Dilutions*
Anti-RPS12 1:50 Goat HPR Anti-rabbit IgG** LiCor Bioscience 1:10000 Anti-RPS19 1:250 Mouse HPR Anti-rabbit IgG Molecular probes® 1:10000
Anti-RPS20 1:250 Goat HPR Anti-mouse IgG *** LiCore Bioscience 1:10000 Anti -RPL9 1:500 Goat HPR Anti-rabbit IgG LiCore Biocience 1:10000
Anti -RPL30 1:500 Goat HPR Anti-rabbit IgG LiCore Bioscience 1:10000
Anti -Β- Actin 1:10000 Goat HPR Anti-mouse IgG Molecular probes® 1:10000
*All antibodies were diluted in 2% BSA in 1% PBS.
** Alexa Fluor 680 rabbit anti-human IgG
***IRDye 800-labeled mouse anti-human IgG
17
Acknowledgments
This project was done in a research group supervised by Professor Niklas Dahl at the Medical Genetic section of Department of Genetics and Pathology of Uppsala University.
I am thankful to HPR group for preparing antibodies, Dr. Farbrizio Loreni in Italy for preparing monoclonal antibody, Dr. Eva Hellberg-Lindstrom at Karolinska Institute for preparing bone marrow cells as well as all bone marrow donors and Lothar Dieterich for helping me out with imaging equipment.
I am grateful to all presents and former members of Dahl group: Jitendra, Jimmy, Joakim, Dana, Miriam, Chikari, Aysha and Johanna for being nice, helpful and friendly during last ten months.
I am also thankful to my friends Sonia, Sara, Hamid, Mehdi and Reza and all other department members who were helpful and supportive during the time of my stay in the department.
Thank you Niklas for having me in your group and being so nice to me, your kindness and consideration is deeply appreciated. Jens Schuster and Anne-Sophie Fröjmark, you are two of the best supervisors I have ever had. Your knowledge, skill and patience will be remaining unforgettable.
18 References
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20 Appendix 1
Relative expression level of five ribosomal proteins in four individuals
21
22
23 Appendix 2
Sample pictures of western blots for five tested proteins and beta actin
β-actin(46kDa) RPS12(13kDa) RPS19(16kDa)
RPS20(13kDa) RPL9(22kDa) RPL30(13kDa)
GpA+ CD34-
GpA-
M
