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