ERYTHROPOIETIN STRUCTURE AND FUNCTION
Erythropoietins structure plays a crucial role in the production of red blood cells. It is bound to its cognate receptor on erythroid progenitors in the bone marrow to rescue these cells from apoptosis, which allows them to proliferate and differentiate into circulating erythrocytes. A mutagenesis experiment is discussed in brief to understand the method of determining structure-function relationship of erythropoietin molecule. In the end, the effect of carbohydrate on the structure and stability of erythropoietin is discussed.
INTRODUCTION
Protein and its types
In an era of scientific and medical breakthroughs our fascination with knowing more about the formation and function of certain cells have become great from scientific and medical points of view. Researchers believe that since Proteins are crucial organic components to life, and therefore we should endeavour to know more about the structure and function of proteins. It is well known that proteins in general have several fundamental functions in all organisms processes. For instance, proteins distributed between building blocks combine with other substances to create the cell from which we are created (Lesk, 2010). Moreover, the functions of proteins vary, some proteins act as catalysts such as enzymes which accelerate the biochemical reactions to maintain the cell to be active and alive yet others assist cells to communicate or to transfer or store important substances from macromolecule to electrons such as oxygen in haemoglobin, and to construct the complex merge of tissues to build the creatures bodies. However, the focus of this analysis is on erythropoietin proteins, understanding the function and structures of erythropoietins is important because they have the ability to modulate unfolding process for development (Lesk, 2010). They consist of the same basic building block, twenty different amino acids joined together with covalently by peptide bond.
Researchers who study this type of proteins classify them into two major classes the first being simple proteins, which constitute amino acids only. The second group is conjugated proteins, which consist of amino acids and other chemical groups, whether organic or inorganic groups. Conjugated proteins include glycoprotein, which contain carbohydrates lipoproteins, which contain lipids and nucleoproteins, which contain nucleic acids. An active protein has four different structures. This means proteins not only have primary, unstructured, chain structures but also they have a highly organized three dimensional structure which make it easier to predict their functions (Nelson Cox, 2008). Thus, the aim of this paper is two-fold firstly, the paper examines both the structure and function of erythropoietin (EPO), which is deemed to be one of the most important glycoproteins. In addition, some of mutagenesis studies and the affect of carbohydrate on the erythropoietin structure will be discussed in this paper.
Erythropoietin (EPO)
Erythropoietin is known as the major hormone that controls erythrocyte differentiation and it also plays an essential role in protection of the physiological level of erythrocyte circulating mass. In addition, erythropoietin is formed by peri tubular capillary endothelial cell in the kidney (Wen et al. 1994). It is produced by peritubular cells in adult renal cortex as well, with a tiny quantity in the liver in foetus. Moreover, there is an extensive utilization of the recombinant human erythropoietin in treat certain diseases such as anaemia, which has registered over 2 billion through the sale of this drug. The molecular mass of erythropoietin is 30.4 kDa which is determined by sedimentation equilibrium, a technique used to find out macromolecule molecular mass (Lappin, et al., 2000). (Wen, et al. 1994. To understand the study of Erythropoietin, it is important that we first the history of the protein since it was discovered.
History
In 1906, a French professor of medicine in Paris who suggested that, hormones are responsible for the production of red blood cells. Eva Bonsdorff and other researchers invented the name of erythropoietin in 1948. Additional experiments showed that erythropoietin was a substance that circulates in the blood which was able to stimulate red blood cell production and increase in hematocrit (the volume of blood that the red blood cells occupy) (Medical Dictionary, 2010).
Furthermore, in 1970s the isolation of this substance was proved as erythropoietin, which opened doors for the application of erythropoietin in treatment purposes for diseases like anaemia. Finally, native human erythropoietin purified and its gene cloned in 1977 and 1985 respectively (Jelkmann, 2007).
Since the middle of 1980s the primary structure of human Erythropoietin, old-world Monkey, and mouse has been detected followed by rat erythropoietin gene sequence. It was found that, human Erythropoietin consists of 193 amino acid and 27 amino acid leader sequence which is cleaved to produce 166 amino acids. Glycoslyation arises in N-linked at 24, 38, and 83 Aspargin residue, and the glycoslyation of O-linked occurs at serine 126. A 166 Arginine residue is eliminated in C-terminal by an intercellular Carboxypeptidase (Lappin, et al., 2000). Human EPO consists of four cysteine associated together with disulfide bonds, that link Cysteine29 with Cysteine 33 and Cysteine 7 with Cysteine 161. Both these bonds are significant for biological activity, which will be covered below (Lappin Winter, et al., 2000).
Berndt and other researches detected the secondary structure of human EPO, they found that the human erythropoietin secondary structure has a four helical bundle in the company of intron and exon boundaries positioned in non-helical region (Lappin Winter, et al., 2000). In addition, it is predicted that this protein has folding class( folding is a scientific classification of amino acids) (Boissel, et al., 1993). Lin et al. in 1985 isolated and characterized the human erythropoietin gene from a genomic phage library. They demonstrated that the gene for EPO encoded the production of erythropoietin in mammalian cells that is biologically active in vivo (vivo means experimenting a live organism or animal) and in vitro (vitro means experimenting with a partially dead organism or a completely dead organism). Thereafter, their research opened up the door for the industrial production of recombinant erythropoietin for treatment of anaemia patients. More recently, a protein called Novel Erythropoiesis-Stimulating Protein (NESP) has been produced which has shown anti-anaemic capabilities similar to erythropoietin but has a longer terminal half-life than erythropoietin. To maintain normal haemoglobin levels, a lower dose of NESP is needed by chronic renal failure patients. The next section will discuss how the Erythropoietin functions in biological processes.
Biological functions of Erythropoietin
Erythropoietin is the most important hormone that has a main function in supporting and regulating the formation of red blood cells (RBC) in that it promotes the survival of the red blood cells by protection of the cells through apoptosis (Youssoufian, et al., 1991). It differs from the other growth factors how Because it is mainly made in a single organ, which is the kidney, and in small amounts in the liver (Boissel, 1993). In addition, it is involved in the feedback control system. It also influences the maturity of erythroid cells, and affects the increase and the activities of other cells. Because of hypoxia (lack of enough oxygen supply), raising the hormone quantity increases the production level of red blood cells. As a result of hyperoxia (in excess supply of oxygen), the amount of erythropoietin will decrease which leads to a decline of the formation of red blood cells (Youssoufian, et al. 1991). Furthermore, there is another biological purpose of erythropoietin, which includes its role in the brains response to any neuronal injury and in the wound healing process.
The Erythropoietin Pathway Retrieved from HYPERLINK httpwww.sabiosciences.compathway.phpsnErythropoietin_Pathway httpwww.sabiosciences.compathway.phpsnErythropoietin_Pathway
The erythropoietin starts its function from the Epo Receptor where there is a binding to its receptor. The receptor then forms homodimers and eventually undergoes phosphorylation through the association and interaction with tyrosine kinase JAK2. the tyrosine residues then activate some adaptors that include the STAT5(Signal Transducers and Activators of Transcription factor-5), PI3K (Phosphoinositide-3 Kinase), SHIP (SH2-containing Inositol Phosphatase), the tyrosine phosphatase SHP1 and SHP2 and the ERKs (Extracellular Signal Regulated Kinases), JNK (Jun N-terminal Kinases) and p38 MAPK (Mitogen Activated Protein Kinase) (SA Biosciences 2010).
STAT5 act as a transcription factor, binds to nuclear DNA carrying the signal from the membrane to the nucleus (Ref.3).
DISCUSSION
Determination of three-dimensional structure of Erythropoietin
The structure of Erythropoietin can be best determined by computer-based predictions. Computer based prediction of three dimensional structure can be divided into three stages. At first stage, primary amino acid sequence of erythropoietin is determined. From this sequence, secondary structure is predicted using optical measurements. With the knowledge of disulfide bonds, these secondary structural elements are then packed into a set of alternative tertiary structures (Johnson, et al., 1998). An analysis of Erythropoietin by circular dichroism method revealed around 50 alpha-helix but no detectable beta-sheet in the tertiary structure. Thus, the number of possible arrangements can be reduced using knowledge of preferred helix-helix packed geometries and the need for globular structure to form a hydrophobic core. The presumed tertiary structure is then distinguished by standard force field calculations. (Johnson, et al., 1998).
(Wen et al., 1994) Fig2 Primary and secondary structure of human Erythropoietin.
In this diagram, the single letters that is A,B,C and D show the predicted alpha-helices. The single letter codes show the sequence of amino acid of an exported protein. An asteric represents the N-linked glycosylation sites. The shaded parts show the nonhelical parts of the EPO protein and can be deleted with no significant loss of specific bioactivity (Wen et al., 1994).
Description of Erythropoietin structure
(Wen et al. 1994) Fig3 Model of the three-dimensional structure of erythropoietin.
The three diagrams (A, B and C) presented by Boissel and other researchers in 1993, clearly demonstrate the structures of EPO. Although, these models are based on computer model predictions they are useful representations in understanding how EPO is structured.
Diagram A is a Ribbon diagram which represents predicted erythropoietin tertiary structure. There are four alpha-helices in the structure, which are labeled from A to D (in magenta color) (Boissel et al., 1993). Loops interconnect the helices and they are named accordingly. There are two regions with an extended structure, which could form hydrogen bonds between Loop AB and Loop CD (in cyan color) (Boissel et al., 1993). The two loops are short fragments of amino acids, which interact with each other to shape antiparallel -sheet, the first -sheet (residues 39-41) and the second -sheet (residues 133-135) (Rashid, et al. 1998). Additionally, there are N- and O-glycosylation sites that are indicated in green and blue, respectively (see Fig 3). Disulfide bonds bridge residues 29-33 in Loop AB, and 7-161 on the N-terminal side of Helix A and the C-terminal side of Helix D (see Fig 4). The loop tracing is shown which does not represent predicted coordinates. (Boissel, et al., 1993).
Moreover, diagram B, consists of a schematic representation of erythropoietins primary structure which depicts predicted up-up-down-down orientation of the four anti-parallel alpha-helices (boxes with arrowhead in Fig 3). The large size of the two interconnecting loops AB and CD strongly suggests this folding pattern. There is a predicted short region of beta-sheet, which is delineated by the dashed rectangle. There are N-glycosylation sites, which are represented by the dotted diamonds, and also the O-glycosylation sites are present which are represented by the dashed oval. The locations of the two-disulfide bridges are also shown between the two cysteines (see in Fig 3) (Boissel et al., 1993).
Finally, diagram C, shows a cross-section of the erythropoietin molecule at the level of four alpha-helices. There are helical wheel projections, which are viewed from the NH end of each helix. In addition, there are hydrophobic residues, which are localized inside the globular structure, are indicated by filled circles. Furthermore, there are charged and neutral residues (open and gray circles, respectively) that are exposed at the surface of the molecule (see Fig 3)(Boissel et al., 1993).
(Wen et al., 1994) Fig4 Model of EPO three-dimensional structure
Structural and functional relationship of Erythropoietin
Understanding the structural and functional relationship of erythropoietin is a crucial factor. Erythropoietins structure participates crucially in its function of red blood cells production. Erythropoietin binds to its cognate receptor on erythroid progenitors in the bone marrow to rescue these cells from apoptosis, which allows them to proliferate and differentiate into circulating erythrocytes. It stimulates cells by joining and orientating two cell-surface erythropoietin receptors (EPORs), which activate an intracellular phosphorylation cascade (Rashid, et al., 1998).
The study of the structure-function relation of the erythropoietin
Results from various experiments have shown that a restricted number of residues on helices A, C, and D are involved in erythropoietins biological activity. It has been proposed that one functional domain encompassing Arg 14, Val 11 and Tyr 15 of helix A, Arg 103 and Ser 104 and Ser 100 of helix C, also Asn 147, Arg 150, Gly 151, and Leu 155 of helix D. Moreover, Leu 108 perhaps exists in the predicted C-D loop (Wen, et al. 1994). Arg 14 is predicted to be within close proximity of Ser 104 and Arg 103 (Wen, et al. 1994).
Another study was carried out to determined the crystal structure of erythropoietin- (EPObp)2 complex at 1.9 from two crystals forms, it showed that the crystal structure imposes erythropoietin a unique 120 angular relationship and orientation to obtain an optimal signalling through intracellular kinase pathways (Rashid, et al. 1998).
Besides that, erythropoietin-EPObp structure consists of a short amino-terminal helix (approximately 15 residues) followed by two - sandwich domains, D1 (N-terminal) and D2 (carboxy-terminal), both containing of 100 residues approximately. Residues 9-22 in N-terminal helix of EPObp structures is pushed into the elbow produced by the D1 and D2 domains (Sasaki, et al., 2000). Furthermore, motif positioned close to the transmembrane domain, Trp-Ser-X-Trp-Ser (WSXWS) motif (residues 209-213XGlu) in D2 (Sasaki, et al., 2000). Leu 18 makes hydrophobic contact with Phe 29, Leu 27, Leu 120, and Phe 208, in the side chin of helix residue of EPObp. If there is any modification in WSXWS sequence that will disrupt erythropoietin binding and receptor signalling. It is detected that WSXWS motif is essential for the folding and transport of receptor to the cell surface. Moreover, the efficiency of the function is improved by introducing an A211E mutation. The Glu 211 side chin of WSXWS motif of erythropoietin-(EPObp)2 complex is closest to Leu 17 of the N-terminal receptor helix. In addition, Trp 209 and Trp 212 side chains residues sandwich the hydrophobic Arg 197 side chin in the receptor fold, Ser 210 and Ser 213 within hydrogen-bonding distance of Ala 198 and Val 196 respectively. It is concluded that from the interaction of WSXWS motif with the N-terminal helix and -sheet residues Val 196, Arg 197 and Ala 198 in erythropoietin- (EPObp) 2 complex the N-terminal helix is vital in the biological stabilization of EPOR folding. Because of Gly 151 in D-helix which is important in the structural role of erythropoietin in forming a kink, the side chain of Ly 152 produce hydrophobic contact with Val 63, Trp 51 and Phe 148 in the protein core (Rashid, et al. 1998).
Further studies found that a two mutation causes loss bioactivity of erythropoietin which included the replacement of Ala at 151 or 152 positions and the other mutation to acidic residues. However, there is no influenced on the function by substitute Lys 20 (basic residues) with Ala (Rashid, et al. 1998).
FIG Schematic presentation of receptors for single cysteine-scanning mutagenesis
fig
Mutagenesis study
Mutagenesis studies have been performed to identify functionally important domains on the surface of erythropoietin molecule. One of studies prepared a number of amino acids to replace them at 51 conserved sites. This study preserved that -helical structure was permitted by Ala substitution. In COS1 and COS7 cells a high level of natural and mutant erythropoietin cDNAs were rapidly expressed (Boissel, et al., 1993). In addition, three of erythropoietin-responsive cells were utilized to examine the biological activity of wild and mutant types of erythropoietin, which are the murine HCD57 erythroleukemia cell line primary murine, erythroid spleen cells and the human UT7-EPO leukaemia cell line (Boissel, et al., 1993). A mutation applied on helix A by replacing Arg 14 with Ala, this results a significantly decrease in the biological activity, however by substituting with carbohydrates produced a loss of the specific bioactivity totally. In the same way, in helix C, the substitution of Arg 103 with Ala was absenting the biological function completely. On one hand, contrast, by replacing Ser 104 and Leu 108 with Ala also produce a decline in the bioactivity (Boissel, et al., 1993). On the other hand, on helix D the replacement of Ala at three close positions introduced a rose of erythropoietin bioactivity. Such mutagenesis studies have indicated functionally essential domains on the surface of the erythropoietin molecule (Boissel, et al., 1993).
The effect of carbohydrate on structure and stability of Erythropoietin
Erythropoietin has three N-linked and one O-linked carbohydrate, with average carbohydrate content about 40. The carbohydrate plays a significant role in the biological activities of erythropoietin. Carbohydrate may also play a role in the structure and stability of erythropoietin (Owers et al., 1991). Studies on a number of different glycosylated proteins have been done in an attempt to explain the role of carbohydrate in maintaining the structure and stability of these proteins. Results from these studies suggest that the specific effect of carbohydrate depends on the protein involved (Owers et al., 1991).
Conclusion
With this report, we are able to know that EPOs structure has a role in its function and therefore we have learned that any protein in the human body is always influenced by its structure. The sequence of the amino acids and the helices that are found in the protein describe its main function which is the production of red blood cells. Some factors also influence its functions such as the presence of oxygen in the system. The study of structure-function of elements is an important aspect of scientific research as through the information gathered, we are able to understand how certain body elements work and how their structures affect their functions.
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