Erythropoietin - Structure

Human derived erythropoietin is an acidic glycoprotein with a molecular mass of about 30.4 kDa, wherein half of its molecular mass is sugar groups (Fisher, 2010). The polypeptide backbone is estimated to be approximately 18 kDa (Ng et. al, 2003). The human EPO gene encodes for five exons and four introns and is situated at chromosome 7, q11-22 (Ng et. al, 2003). Translation produces an initial prohormone product, which requires further posttranslational processing for activation (Ng et. al, 2003).The acidic nature of the molecule is due to the presence of acidic residues.The mature hormone is composed of 165 amino acids with two disulfide bonds between Cys⁷-Cys¹⁶¹ and Cys²⁹-Cys³³ (Hadley and Levine, 2007).

 

Human EPO mRNA

        1 cccggagccg gaccggggcc accgcgcccg ctctgctccg acaccgcgcc ccctggacag

       61 ccgccctctc ctccaggccc gtggggctgg ccctgcaccg ccgagcttcc cgggatgagg

      121 gcccccggtg tggtcacccg gcgcgcccca ggtcgctgag ggaccccggc caggcgcgga

      181 gatgggggtg cacgaatgtc ctgcctggct gtggcttctc ctgtccctgc tgtcgctccc

      241 tctgggcctc ccagtcctgg gcgccccacc acgcctcatc tgtgacagcc gagtcctgga

      301 gaggtacctc ttggaggcca aggaggccga gaatatcacg acgggctgtg ctgaacactg

      361 cagcttgaat gagaatatca ctgtcccaga caccaaagtt aatttctatg cctggaagag

      421 gatggaggtc gggcagcagg ccgtagaagt ctggcagggc ctggccctgc tgtcggaagc

      481 tgtcctgcgg ggccaggccc tgttggtcaa ctcttcccag ccgtgggagc ccctgcagct

      541 gcatgtggat aaagccgtca gtggccttcg cagcctcacc actctgcttc gggctctggg

      601 agcccagaag gaagccatct cccctccaga tgcggcctca gctgctccac tccgaacaat

      661 cactgctgac actttccgca aactcttccg agtctactcc aatttcctcc ggggaaagct

      721 gaagctgtac acaggggagg cctgcaggac aggggacaga tgaccaggtg tgtccacctg

      781 ggcatatcca ccacctccct caccaacatt gcttgtgcca caccctcccc cgccactcct

      841 gaaccccgtc gaggggctct cagctcagcg ccagcctgtc ccatggacac tccagtgcca

      901 gcaatgacat ctcaggggcc agaggaactg tccagagagc aactctgaga tctaaggatg

      961 tcacagggcc aacttgaggg cccagagcag gaagcattca gagagcagct ttaaactcag

     1021 ggacagagcc atgctgggaa gacgcctgag ctcactcggc accctgcaaa atttgatgcc

     1081 aggacacgct ttggaggcga tttacctgtt ttcgcaccta ccatcaggga caggatgacc

     1141 tggagaactt aggtggcaag ctgtgacttc tccaggtctc acgggcatgg gcactccctt

     1201 ggtggcaaga gcccccttga caccggggtg gtgggaacca tgaagacagg atgggggctg

     1261 gcctctggct ctcatggggt ccaagttttg tgtattcttc aacctcattg acaagaactg

          1321 aaaccaccaa aaaaaaaaaa

 

Figure 1: Primary structure of human erythropoietin (mature hormone).

http://pmj.bmj.com/content/79/933/367.full

The sugar portion of the erythropoietin molecule consists of three N-linked oligosaccharide chains and one O-linked oligosaccharide chain (Fisher, 2010). The N-linked glycans are critical for the biological activity of EPO in vivo (Hadley and Levine, 2007). The terminal sialic acid residues of these chains play an essential role in protecting the erythropoietin molecule from degradation by the liver (Fisher, 2010). Studies have shown that EPO, with absent sialic acid moieties, is rapidly removed by galactose receptors of hepatocytes, as galactose is the penultimate sugar of these oligosaccharide chains. However, the introduction of these sialic acid residues into recombinant EPO via site-directed mutagenesis significantly enhances in vivo survival of the molecule (Hadley and Levine, 2007).

Figure 2: Functional unit of N-linked oligosaccharide chain from EPO.

http://pmj.bmj.com/content/79/933/367.full

According to Figure 2, the N-linked sugar unit plays a functional role in maintaining EPO structure. The main core is rich in Mannose, and serves to maintain the polypeptide conformation (Ng et. al, 2003). The branched sugar chain segment may serve some role pertaining to EPO activity, and further stabilizes the molecule in the blood (Ng et. al, 2003). Terminal sugars are composed mainly of sialic acids and are a primary source of EPO activity and interaction with other molecules (eg; receptors) (Ng et. al, 2003).

Erythropoietin is initially synthesized as a 193 amino acid prohormone (Hadley and Levine, 2007). The leader sequence consists of 27 amino acid residues which is cleaved prior to secretion (Hadley and Levine, 2007). The last amino acid in the prohormone chain at the carboxyl terminal (Arg¹⁶⁶) is also removed, by an intracellular carboxypeptidase, before the mature hormone is released (Ng et. al, 2003). The projected secondary structure of RHuEPO (recombinant human erythropoietin) consists of 50% α-helix moiety. The spatial arrangement is similar to that of growth hormone (GH), wherein two alpha helical pairs run antiparallel (Ng et. al, 2003).

Figure 3: Possible tertiary conformation of human erythropoietin (with bound N-linked sugar moieties).

http://www.sciencedirect.com/science/article/pii/S0003267011015844

EPO Prohormone Protein Sequence Alignment

Species: Canis lupus familiaris (Dog)

               Felis catus (Cat)

               Homo sapiens sapiens (Human)

               Danio rerio (Zebrafish)

               Oncorhynchus mykiss (Rainbow trout)

dogEPO            MCEPAPPKPTQSAWHSFPECPALLLLLSLLLLPLGLPVLGAPPRLICDSRVLERYILEAR

catEPO            --------------MGSCECPALLLLLSLLLLPLGLPVLGAPPRLICDSRVLERYILEAR

humanEPO          -------------MGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLQRYLLEAK

zebrafishEPO      -----------------MFHGSGLFALLLMVLEWTRPGLSSPLRPICDLRVLDHFIKEAW

troutEPO          ---------------------------------------------ICDLSVLNHFIKEAW

                                                               ***  **:::: **

dogEPO            EAENVTMGCAQGCSFSENITVPDTKVNFYTWKRMDVGQQALEVWQGLALLSEAILRGQAL

catEPO            EAENVTMGCAEGCSFSENITVPDTKVNFYTWKRMDVGQQAVEVWQGLALLSEAILRGQAL

humanEPO          EAENITTGCAEHCSLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQAL

zebrafishEPO      DAEAAMRTCKDDCSIATNVTVPLTRVDFEVWEAMNIEEQAQEVQSGLHMLNEAIGS----

troutEPO          DAEAAMRACKDACSIATNFTVPLTRVDFDVWEAMNIEERAQEVQSGLHVLNEAISS----

                  :**     * : **:  *.*** *:*:* .*: *:: ::* ** .** :*.**:     

dogEPO            LANASQPSETPQLHVDKAVSSLRSLTSLLRALGAQKEAMSLPEEASPAPLRTFTVDTLCK

catEPO            LANSSQPSETLQLHVDKAVSSLRSLTSLLRALGAQKEATSLPEATSAAPLRTFTVDTLCK

humanEPO          LVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRK

zebrafishEPO      -LQISNQTEVLQSHIDASIRNIASIRQVLRSLSIP---EYVPPTSSGEDKETQKISSISE

troutEPO          -LQASNQTDVLQSHIDASISNIASIRQVLRSLSIP---EYVPPTSGGEDKEMQIVSSISE

                    : *:  :  * *:* :: .: *:  :**:*.        *  :.    .    .:: :

 

dogEPO            LFRIYSNFLRGKLTLYTG--EACRRGDR

catEPO            LFRIYSNFLRGKLTLYTG--EACRRGDR

humanEPO          LFRVYSNFLRGKLKLYTG--EACRTGDR

zebrafishEPO      LFQVHVNFLRGKARLLLANAPVCRQGVS

troutEPO          LFQVHINFL-------------------

                                **: : :  ***

Key:  * = identical amino acid,   . = weak similarity, and  : = structural similarity.

- Prepared using ClustalW software program

EPO Prohormone Alignment Score

 CLUSTAL 2.1 Multiple Sequence Alignments

Sequence type explicitly set to Protein

Sequence format is Pearson

Sequence 1: humanEPO       193 aa

Sequence 2: dogEPO         206 aa

Sequence 3: catEPO         192 aa

Sequence 4: zebrafishEPO   183 aa

Sequence 5: troutEPO       136 aa

Start of Pairwise alignments

Sequences (1:2) Aligned. Score: 78.2383

Sequences (1:3) Aligned. Score: 82.2917

Sequences (1:4) Aligned. Score: 31.1475

Sequences (1:5) Aligned. Score: 30.8824

Sequences (2:3) Aligned. Score: 93.75

Sequences (2:4) Aligned. Score: 31.694

Sequences (2:5) Aligned. Score: 31.6176

Sequences (3:4) Aligned. Score: 32.2404

Sequences (3:5) Aligned. Score: 32.3529

Sequences (4:5) Aligned. Score: 87.5

Guide tree file created:   

There are 4 groups

Start of Multiple Alignment

Aligning...

Group 1: Sequences:   2      Score:3004

Group 2: Sequences:   3      Score:2746

Group 3: Sequences:   2      Score:2079

Group 4: Sequences:   5      Score:1076

Alignment Score 5339

The alignment score indicates that EPO structure is most conserved between dog (Canis lupus familiaris) and cat (Felis catus) with a 93% sequence homology. Human EPO and trout EPO displayed the lowest structural homology with a score of 30.8%. Across all species, especially in mammals, the polypeptide backbone remains highly conserved and changes in structure are attributed mainly to single amino acids and the associated carbohydrate groups (Ng et. al, 2003).

References

-  Hadley, M. E. & Levine, J. E. (2007). Endocrinology (6^th ed.). Prentice Hall, Pearson Education: Upper Saddle River, NJ

- Fisher, J. W. (2010) Landmark advances in the development of erythropoietin. Experimental Biology and Medicine. 235 (12): 1398-1411

- Ng, T., Marx, G., Littlewood, T., & Macdougall, I. (2003) Recombinant erythropoietin in clinical practice, Postgraduate Medical Journal, 79: 367-376

Erythropoietin - Origin

Erythropoietin (EP [16]), also known as erythropoietin factor or EPO, is a humoral peptide hormone that is synthesized by cells of the kidney and is involved in regulating erythropoiesis, or red blood cell production (Silverthorn, 2009).

Figure 1: Molecular structure of erythropoietin. http://upload.wikimedia.org/wikipedia/commons/thumb/a/a1/Erythropoietin.png/250px-Erythropoietin.png

Hormonal regulation of erythropoiesis was first suggested in 1906 by Paul Carnot, a professor of medicine in Paris, and his co-worker Clotilde Deflandre (Jelkmann, 2007). In their experiment, the investigators subjected rabbits to a bloodletting wherein 30 mL of blood was obtained on the first day, a second blood sample was obtained the following day, after which the serum was injected into normal rabbits. The results showed that the concentration of erythrocytes increased up to 40% within two days (Jelkmann, 2007). The investigators concluded that the serum must have contained a hematopoietic factor, a chemical substance which stimulates the formation of blood cell components.

In 1948, researchers of red blood cell production, Eva Bonsdorff and Eeva Jalavisto from Helsinki, named this factor “erythropoietin” (Jelkmann, 2007).

 

 

Figure 2: Paul Carnot (left), and Allan Erslev(right). http://en.wikipedia.org/wiki/File:Paul_Carnot.jpg

http://lifeinlegacy.com/2003/WIR20031122.html

Allan Erslev, professor of medicine at Thomas Jefferson University, confirmed this finding and provided conclusive proof of the existence of EPO. He conducted an experiment in which large volumes of plasma (100-200 mL) was transfused from anemic rabbits (hematocrit below 20%) to normal rabbits (Jelkmann, 2007). The recipient rabbits responded with a significant increase in reticulocytosis, an increase in immature erythrocytes and after several days showed a higher hematocrit value of the blood (Jelkmann, 2007).

Erslev resolved that there must be a factor within the blood serum responsible for rapidly stimulating the production of red blood cells. He further predicted the therapeutic value of the erythropoietic factor in medicine, if it could be isolated and purified, against certain kidney disorders and infections (Jelkmann, 2007).

 

 

Figure 3: Scheme depicting biogenesis of erythropoietin.

http://studydroid.com/imageCards/07/9h/card-7652883-back.jpg

 

Figure 4: Hybridization studies on hypoxic monkey kidneys (C and D). Erythropoietin production by interstitial cells, showing EPO mRNA over peritubular interstitial cells (E), and in anemic mouse kidneys (F). http://ebm.rsmjournals.com/content/235/12/1398/F2.expansion.html

 

In further studies, EPO and EPO mRNA was extracted from the kidneys of hypoxic rodents. It was discovered that the hormone was primarily present in the cortex of the kidney (outer layer) and not in the medulla (Jelkmann, 2007). An in situ hybridization study indicated a subgroup of fibroblasts in the cells lining the peritubular capillary, as the site of EPO gene expression in the kidneys. Other organs, such as the liver, spleen, lung, bone marrow, and brain were shown to express EPO mRNA (Jelkmann, 2007). Erythropoietin from the brain is thought to act as a paracrine neuroprotective factor, as it is unable to enter the general circulation of the body due to the blood brain barrier (Jelkmann, 2007). 

Erythropoietin is a glycoprotein molecule consisting of 165 amino acids and contains over 40% carbohydrate, primarily sialic acid and other sugars (Hadley & Levine, 2007). The sialic acid residues in the molecule acts as a protectant, and is necessary for biological activity in vivo, as the absence of these residues (asialo form of EPO) causes the hormone to be cleared too rapidly by the liver, preventing it from exerting its hematopoietic effects (Hadley & Levine, 2007).

The purpose of EPO is to enhance the proliferation of erythrocyte precursor cells in the hematopoietic tissues (red marrow) of the bones or the fetal liver, into erythroblasts which are cells programmed to become erythrocytes (Hadley & Levine, 2007). The final result of this action is an increased number of erythrocytes in the blood, and therefore a higher hematocrit (Silverthorn, 2009).

 

EPO release is stimulated by hypoxic (low oxygen concentration) conditions in the tissues, and is inhibited during conditions which create tissue hyperoxia (high oxygen concentration). Any activity or agent which increases oxygen consumption and metabolic rate such as growth, high altitude exposure, hormones, and pharmaceutical agents, will increase the need for oxygen and subsequently stimulate EPO (Hadley & Levine, 2007). EPO action will increase red blood cells in the system, and therefore increase the oxygen transport to the tissues (Silverthorn, 2009).

Presently, erythropoietin is manufactured artificially using recombinant DNA technology because of its potential as a therapeutic agent and has already been used in treatment of anemia and certain cancers (Fisher, 2010). Preliminary studies have shown that mutations in certain regions of the peptide structure produce a neuroprotective protein without erythropoietic effects, which may be used to treat certain neurodegenerative diseases (Fisher, 2010).

References

- Silverthorn, D. U. (2009). Human Physiology, An integrated approach (5^th ed.). Benjamin Cummings, Pearson Education : San Francisco, CA

- Jelkmann, W. (2007). Erythropoietin after a century of research: younger than ever. European Journal of Haematology. Institute of Physiology, University of Luebeck: Luebeck, Germany

-  Hadley, M. E. & Levine, J. E. (2007). Endocrinology (6^th ed.). Prentice Hall, Pearson Education: Upper Saddle River, NJ

 

- Fisher, J. W. (2010) Landmark advances in the development of erythropoietin. Experimental Biology and Medicine. 235 (12): 1398-1411

Erythropoietin....stay tuned.

This blog was designed as an assignment for the BIOL 4450 course at Memorial University.