16 Extracellular Hemoglobins from Annelids, and Their Potential Use in Biotechnology Franck Zal and Morgane Rousselot

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16 Extracellular Hemoglobins from Annelids, and their Potential Use in Biotechnology Franck Zal and Morgane Rousselot

Abstract products. The ﬁrst generations of blood substitutes (hemo- This chapter highlights the diversity of invertebrate hemoglobin oxygen carriers) were manufactured using intra- globin structures, and gathers together up of 100 years of cellular hemoglobin (human and bovine) in order to research data. Among invertebrates, annelid hemoglobin function outside the red blood cells. However, HEMARINA structures have been the most widely studied, probably due technologies are based on a natural extracellular hemoglo- to their typical symmetric shapes and sizes that allow direct bin. This circulatory pigment, which is present in the blood observations to be made using transmission electron vessels of A. marina, has evolved over a million years and is microscopy. Hemoglobin seems to be present in all phyla, able to function extracellularly. Currently, two main prod- which suggests a very ancient origin. Using the hemoglobin ucts are under development at HEMARINA, namely 1 of the marine annelid Arenicola marina,theFrenchbio- HEMO2life (for organ preservation) and HEMOXYCar- 1 technology startup HEMARINA is developing medical rier (as a universal oxygen carrier).

16.1 During evolution, three different categories of oxygen carriers Introduction were selected, each of which retained a characteristic color (Table 16.1). Of these pigments, hemoglobin (Hb), with a typical All aerobic cells require oxygen and nutrients to fuel their red color, is the most familiar and is present in almost all phyla, energetic and growth requirements. They ﬁnd these elements whereas chlorocruorin, which is very similar to the Hb, is in their environment for biomolecule synthesis, and discard the characterized by a green color and exists in four families of degradation products of their metabolism. However, multi- polychete annelids. Hemocyanin, a blue pigment, is currently cellular organisms are not directly in contact with the external present in certain mollusks and arthropods, while hemerythrin, environment where oxygen is available, and in this context two which is pink in color occurs more sporadically among the physical mechanisms exist for the transportation of the respira- animal kingdom (Figure 16.1). tory gases (oxygen and carbon dioxide), namely diffusion and The heme-containing pigments are the most common among convection. Diffusion mechanisms are effective only over small life kingdom as they are found in 33% of zoological classes distances (millimeters or less), and so the process plays an (Toulmond, 1992); yet, surprisingly, these pigments are also important role only in unicellular organisms. If the distance present in plants (Landsmann et al., 1986; Bogusz et al., 1988; between the external and internal media is important, however, Fuchsman, 1992). Although the molecules are characterized by then a convection process will be substituted in place of diffusion. an important diversity of molecular weight and structure (Royer, Yet, these two processes may intervene at either the same time or 1992; Terwilliger, 1992), the active site is always similar and is ¼ separately during the circulation and respiration processes. The constituted by a protoporphyrinic tetrapyrrolic ring (Mr þ primary function of the circulation process involves the require- 616.5 Da) that has an iron atom at the center (Fe2 ) (Figure 16.2). ments for oxygen and nutrients, as well as the elimination of This Fe–protoporphyrin complex constitutes the heme mole- carbon dioxide and metabolic waste. Consequently, the circulat- cule. The same active site is also found in other enzymes, such ing ﬂuids establish important ties between distant specialized as cytochromes, catalases and peroxidases. In the case of hemo- cellular populations and the external environment via the inter- globin the iron atom is only bound by an amino acid from the mediate of the exchange organs. In this context, blood has a key polypeptide chain, the proximal histidine. The complex consti- position as it contains specialized molecules known as respiratory tuted by the active site and the polypeptide chain forms the basic pigments. These exist either intracellularly or extracellularly, and functional unit that is able to bind oxygen only in a reversible are able to bind oxygen in a reversible fashion, which increases fashion. Two categories of heme-containing groups exist, but in considerably the ability of the blood to carry these gases. both cases the heme molecule is a chelate of iron associated with

Outstanding Marine Molecules: Chemistry, Biology, Analysis, First Edition. Edited by Stephane La Barre and Jean-Michel Kornprobst. Ó 2014 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2014 by Wiley-VCH Verlag GmbH & Co. KGaA. CH16 01/30/2014 0:7:29 Page 362

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Table 16.1 Classification and localization of the different respiratory pigment across the animal and plant kingdoms.

a) Pigments (localization) Phylum Mr (Da) Active sites N Intracellular Without heme Hemerythrin Sipunculida Brachiopoda 40–110 103 2 Fe atoms 2 Annelida Priapula Extracellular Hemocyanin Arthropoda 4 105 to 3 106 2 Cu atoms 6–48 Mollusca 3 106 to 9 106 70–160

Intracellular With heme Hemoglobin stricto sensu Vertebrate 65 103 1 Fe atom 4 Hemoglobin of invertebrates dissolved in their body ﬂuids Mollusca Echinodermata Annelida 17–130 103 1 Fe atom 1–8 Echiuria Hemoglobin cytoplasmic Myoglobin Vertebrate 1 Invertebrate 1 Fe atom 1 Hemoglobin Bacteria 1 Fe atom 1 Leghemoglobin Leguminous plants 1 Fe atom 1 Neurhemoglobin Annelida 1 Fe atom 1 Extracellular Hemoglobins Platyhelminthes Nematyhelminthes 17 103 Nemerta to 1 to >8 Mollusca 12 106 Arthropoda Erythrocruorin Annelida 4 105 Vestimentifera To >8 Pogonophora 4 106 Chlorocruorin Annelida 3 106 to 4 106 >8

a) N ¼ no. of active sites capable of binding an oxygen molecule.

a protoporphyrin IX which is termed ferroprotoheme and The chimeric bi-domain chains (40 kDa) are created by the contains two vinyl groups (CHCH2); this complex is the covalent association of two domain chains, one of which bears most common (Falk, 1975), but in ferrochlorocruoroporphyrin a heme group. or chlorocruoroheme the vinyl group is replaced by a formyl Linear chimeric multidomain chains created by the covalent group (CH¼O) (Fischer and Seemann, 1936) (Figure 16.2). The association of several globin domains. latter group is present only in the chlorocruorins. It is possible to The main aim of this chapter is to describe the invertebrate distinguish between ﬁve different categories of globin chains hemoglobins known to date, and brieﬂy to review the extrac- (for a review, see Vinogradov et al., 1993): ellular Hbs obtained from annelids. The details also provided of Single-domain chains are the most common in the animal two therapeutical applications from Arenicola marina Hb. kingdom. They comprise about 145 amino acids and have an active site, and their existence is documented as three different Hbs, namely intracellular, extracellular, or cytoplasmic. 16.2 These chains can associate to create monomeric, dimeric, Annelid Extracellular Hemoglobins tetrameric or polymeric Hbs. Truncated single-domain chains that contain about 116–121 With numerous reviews having been produced on annelid amino acids. extracellular hemoglobins (EHbs) and chlorocruorins, the inter- Chimeric single-domain chains have Mr 40 kDa, are cyto- est of the international “invertebrate hemoglobinist” commu- plasmic, and are characterized by an N-terminal region that is nity was directed towards this remarkable family of molecules abletobindahemegroupandaC-terminalwithseveralfunctions. (Mangum, 1976a; Mangum, 1976b; Antonini and Chiancone, CH16 01/30/2014 0:7:29 Page 363

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Figure 16.1 Occurrence of the respiratory pigments among the major phyla and classes (Toulmond and Truchot, 1993). The color dots indicate the type of respiratory pigments eventually present. Red ¼ hemoglobins; green ¼ chlorocruorins; purple ¼ hemerythrins; blue ¼ hemocyanins. The absence of a dot means that no respiratory pigment has yet been detected. However, the presence of dot does not mean that all of the group’s species possess the respiratory pigment mentioned. With kind permission by La Recherche.

1977; Weber, 1978; Chung and Ellerton, 1979a; Garlick, 1980; Linker chains, which possess very few or no heme groups and Vinogradov et al., 1980c; Vinogradov, Kapp, and Ohtsuki, 1982; play an important role in the assembly of one-twelfth of the

Vinogradov, 1985a; Vinogradov, 1985b; Gotoh and Suzuki, molecule. These chains have Mr-values of between 22 and 1990; Riggs, 1990; Terwilliger, 1992; Lamy et al., 1996). The 27 kDa. EHbs are present in the three annelids classes, namely poly- Some authors have revealed the importance of glycans on the chetes, oligochetes, and achetes. The molecules are very large assembly of EHbs from Perinerreis aibuhitensis, and have termed biopolymers with high molecular weights in the range of 3000– this mechanism “carbohydrate gluing” (Ebina et al., 1995; 4000 kDa, and corresponding sedimentation coefﬁcients of Matsubara et al., 1996; Yamaki et al., 1996). However, this 54–61 S, respectively. Each molecule consists of an assembly mechanism cannot be universal as not all of these molecules of about 200 polypeptide chains that belong to between six are glycosylated (e.g., Arenicola marina EHb; Zal et al., 1997a, and eight different types that are usually grouped into two 1997b). The EHbs of annelid are likewise characterized by an categories: acidic isoelectric point and low heme and iron contents, about Functional chains, which possess an active site that is able to two-thirds of that observed for other hemoglobins, and corre- bind oxygen reversibly, and correspond to globin chains with sponding to the presence of one mole of heme per 20000–

Mr-values of about 15 and 18 kDa. 27000 Da of protein. This result indicated that not all of the CH16 01/30/2014 0:7:32 Page 364

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(a) COO– COO– (b) COO– COO–

CH2 CH2 CH2 CH2

CH2 CH2 CH2 CH2 H H C C C C C C CH HC C C H3C C C 3 C C C C C N N C C N N C HC Fe CH HC Fe CH NC N C NC N C

H2C C C C C CH3 H2C C C C C CH3 C C C C C C C C H H H C CH C O CH3 2 CH3 H H

Figure 16.2 Heme structure (Toulmond and Truchot, 1993). (a) Ferroprotoporphyrin or ferroprotoheme (porphyrin IX) corresponding to the hemoglobin oxygen-binding site; (b) Ferrochlorocruoroporphyrin, corresponding to the chlorocruorin oxygen-binding site. One of the vinyl groups (CHCH2)of molecule (a) takes over from a formyl group (CHO). With kind permission by La Recherche.

polypeptide chains possessed a heme group (Vinogradov, Kapp, images revealed hexagonal structures with diameters of 22–26 nm, and Ohtsuki, 1982; Vinogradov, 1985a; Vinogradov, 1985b). where each molecule was constituted by two superimposed hexa- gons with a height of 11–17 nm (Levin, 1963; Roche, 1965). This structure was typically referred to as a hexagonal-bilayer (HBL). 16.3 Each hexagon was composed of an assembly of six elements with a Architecture water-drop shape (Kapp and Crewe, 1984; Van Bruggen and Weber, 1974), and were also referred to as having a hollow globular Interest in the global structure of these molecules ﬁrst emerged structure (HGS) (De Haas et al., 1996a, 1996b, 1996c, 1996d) or during the 1960s, some 30 years after the molecular weights of the one-twelfth. The molecule was built up by 12 of these subunits, pigments had been determined. The initial studies involved obser- each of molecular weight ca. 250 kDa, being association one with vations using transmission electron microscopy (TEM), and the another. Subsequently, these observations were extended to other ﬁrst micrographs were obtained from Arenicola marina (Roche, molecules belonging to other annelid species, and revealed com- Bessis,andThiery,1960a;Roche,Bessis,andThiery,1960b).The pounds of similar dimensions (Table 16.2).

Table 16.2 Dimension of several extracellular hemoglobins measured from TEM images.

Species High (nm) a (nm)a) b (nm)b) Stainingc) References

Polycheta Nephthyidae Nephtys incisa 19.8 1.2 31.6 1.1 AU Messerschmidt et al., 1983 Nereidae Tylorrynchus heterochaetus 18.2 28.4 Suzuki, Kapp, and Gotoh, 1988 19.2 1.2 7.5 0.8 30.1 0.8 Kapp et al., 1982 Perinereis aibuhitensis 20.0 1.8 29.4 0.8 Matsubara et al., 1996 Arenicolidae Arenicola marina 16.0 0.8 25.5 0.8 30.0 PT Levin, 1963 16.0 24.0 30.4 PT Roche, 1965 14.8 20 28.4 PT Breton-Gorius, 1963 19.7 27.5 30.0 AU Toulmond et al., 1990 Abarenicola afﬁnis 17.0 25.5 PT Chung and Ellerton, 1982 Opheliidae Euzonus mucronata 17.0 25.5 MA, PT Terwilliger et al., 1977 Ophelia bicornis 17.5 26.0 27.5 AU Mezzasalma et al., 1985 Travisia japonica 16.0 23.5 PT Fushitani et al., 1982 Eophilia tellinii 19.0 28.0 AU Cejka et al., 1989 Eunicidae Eunice aphroditois 17.9 0.3 26.3 0.3 AU Bannister, Bannister, and Anastasi, 1976 CH16 01/30/2014 0:7:32 Page 365

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Cirratulidae Cirriformia sp. 17.0 24.0 AU Van Gelderen, Shlom, and Vinogradov, 1981 Terrebelidae Pista pacifica 17.0 0.3 25.0 0.5 MA, PT Terwilliger and Koppenheffer, 1973 Thelepus crispus 17.0 0.3 25.0 0.5 MA, PT Terwilliger and Koppenheffer, 1973 Ampharetidae Amphitrite ornata 10.5 21.8 PT Mangum, 1976 29.0 AU Chiancone et al., 1980 Alvinellidae Alvinella pompejana 24.5 MA, PT Terwilliger and Terwilliger, 1984 19.7 27.0 30.4 AU Toulmond et al., 1990 Oligocheta Tubificidae Tubifex tubifex 16.0 26.0 Stockel,€ Mayer, and Keller, 1973 21.1 31.2 AU Kapp and Crewe, 1984 Lumbricidae Lumbricus terrestris 20.0 30.0 AU Levin, 1963 16.0 0.7 26.5 0.0 PT Levin, 1963 16.0 26.5 PT Roche, 1965 23.7 37.0 AU Ben Shaul, 1974 20.0 0.9 28.2 0.8 29.9 0.8 Kapp et al., 1982 21.5 31.3 AU Kapp and Crewe, 1984 Eisenia fetida 17.5 26.0 AU Frossard, 1982 16.5 26.0 PT Frossard, 1982 14.0 25.0 PT Ochiai and Enoki, 1981 Limnodrilus gotoi 16.0 0.7 22 1.2 AU Yamagishi et al., 1966 Maoridrilus montanus 25.0 PT Ellerton, Bearman, and Loong, 1987 Megascolecidae Pheretima communis 16.7 26.0 PT Ochiai and Enoki, 1979 Pheretima hilgendorfi 16.7 26.0 PT Ochiai and Enoki, 1979 Acheta Hirudidae Haemopis sanguisuga 15.2 1.4 24.4 2 AU Wood, Mosby, and Robinson, 1976 a) Distance between two parallel sides. b) Maximal molecule diameter. c) AU ¼ uranyl acetate; PT ¼phosphotungstate, MA ¼ ammonium molybdate.

The hexagon center is generally devoid of subunits and was mucronata (Terwilliger et al.,1977b),andArenicola marina (Zal constituted by a hole of about 7–8 nm diameter; however, some et al., 1997a, 1997b) (Figure 16.3). The existence of a central annelid EHbs were shown to possess a central subunit, such as subunit suggested that this was similar or equivalent to another Oenone fulgida (Van Bruggen and Weber, 1974), Nepthys hom- one-twelfth of the native molecule, although to date no central bergii (Wells and Dales, 1976), Nepthys incisa (Wells and Dales, subunit has been isolated and/or characterized. Ghiretti- 1976; Messerschmidt et al., 1983; Vinogradov and Kapp, 1983), Magaldi et al. (1985) also postulated that the subunit for Ophelia Ophelia bicornis (Ghiretti-Magaldi et al., 1985; Mezzasalma et al., bicornis would differ from the other one-twelfth, while Mes- 1985; Cejka et al., 1991, 1992), Maoridrilus montanus (Ellerton serschmidt et al. (1983) proposed that the central subunit for et al., 1987), Glossoscolex paulistus (El Idrissi Slitine, Torriani, Nephtys incisa was too small to correspond to one-twelfth of the and Vachette, 1990), Eophila tellinii (Cejka et al., 1989), Euzonus native molecule.

Figure 16.3 Three-dimensional cryomicroscopic structure of Arenicola marina EHb. Ludovic Jouan, 2003. CH16 01/30/2014 0:7:33 Page 366

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The use of TEM revealed a symmetric structure, and conse- one-twelfth level. The latter level of symmetry was also con- quently several quaternary structure models were proposed. firmed by Martin et al. (1996a) after crystallization and observa- However, in order to construct an accurate model, knowledge tion of the subunit at 2.9 A resolution. is required of three parameters: (i) the molecular weight of the biopolymer in its native condition; (ii) the number and relative amounts of subunits and polypeptide chains that constituted the 16.4 molecule; and (iii) the accurate molecular weight of each com- Model of Quaternary Structures ponent. Access to these parameters was gradually achieved due to the emergence of new technologies such as mass spectrome- Molecular weight determinations using the primary sequence try and multiangle light-scattering, which were first applied by permitted the realization of several models of quaternary one of the present chapter authors (F.Z.). structure for these EHbs. However, several approaches were A sixfold axis of symmetry was identified by using X-ray employed to understand the different component assemblages diffraction with a resolution of 6 A (Royer and Hendrickson, inside the biopolymers (Table 16.3). 1988), although the existence of minor components was suggested without this symmetry. The same observation was made 16.4.1 several years later by Boekema and van Heel (1989), when using Electron Microscopy electron microscopy with a resolution of 20 A. An analysis using small-angle X-ray scattering of Lumbricus terrestris EHb – the Based on TEM images which showed molecular symmetry, most extensively studied molecule – revealed the presence of a Rossi-Fanelli et al. (1970) proposed a model for Lumbricus central subunit that was not visible with TEM (Pilz, Schwarz, terrestris EHb (Figure 16.4). This model was inspired by the and Vinogradov, 1980), in contrast to Tylorrynchus heterochaetus where the subunit was visible (Pilz et al., 1988). The same conclusion was drawn in the case of Arenicola marina and Glossoscolex paulistus EHbs (Wilhelm, Pilz, and Vinogradov, 1980; El Idrissi Slitine, Torriani, and Vachette, 1990). A three- dimensional reconstruction of Lumbricus EHb confirmed these results when using scanning transmission electron microscopy (STEM), and suggested that the mass could correspond to one- twelfth at the molecule center (Crewe, Crewe, and Kapp, 1984). Alternatively, Vinogradov et al. (1986) suggested that the mass could correspond to the linker chains. The same observation was made via a three-dimensional reconstruction of the molecule by cryomicroscopy (Schatz et al., 1995; Taveau et al., 1999), but these authors suggested that only immunolabeling could confirm the exact nature of the central subunit. The major conclusions deduced by Schatz et al. (1995) were the presence Figure 16.4 First quaternary structure model proposed for Lumbricus of a sixfold axis of symmetry, in agreement with Royer and terrestris extracellular haemoglobin. Reused from Rossi-Fanelli et al., 1970 Hendrickson (1988), and a threefold axis of symmetry at the with kind permission from Elsevier.

Table 16.3 Main structural models proposed in the literature for the extracellular hemoglobins of annelids and Vestimentifera.

Species No. of globin No. of linker Methods Assembly for the References chains chains one-twelftha)

Tylorrynchus heterochaetus 192 24 HPLC (T þ M)4 þ L2 Suzuki et al., 1990a þ þ Tylorrynchus heterochaetus 144 36 ESI-MS (T M)3 L3 Green et al., 1995 Perinereis aibuhitensis 192 24 HPLC (T þ M)4 þ L2 Matsubara et al., 1996 þ þ Arenicola marina 156 42 ESI-MS (T M9) L3.5 Zal et al., 1997a Lumbricus terrestris 192 24 HPLC (T þ M)4 þ L2 Ownby et al., 1993 þ þ Lumbricus terrestris 144 36 HPLC (T M)3 L3 Vinogradov et al., 1991 Lumbricus terrestris 144 36 ESI-MS (T þ M)3 þ L3 Martin et al., 1996b þ þ Macrobdella decora 144 42 ESI-MS (D M2)3 L3.5 Weber et al., 1995 Alvinella pompejana 144 60 ESI-MS (T þ M)3 þ L5 Zal et al., 1997b þ þ Alvinella pompejana 120 72 ESI-MS (T M2)2 L6 Zal et al., 1997b Riftia pachyptila 144 36 ESI-MS (D þ M)4 þ L3 Zal et al., 1996a a) M ¼ monomeric globin chain; D ¼ covalent dimer of globin chains; T ¼covalent trimer of globin chains; L ¼ linkers chains. CH16 01/30/2014 0:7:33 Page 367

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Figure 16.5 Proposed model for Lumbricus terrestris extracellular hemoglobin using transmission electron microscopy. (a) Face view; (b) Internal bracelet constituted by D1 (central ring) and D2 (external ring) subunits, where the twelfths of the molecule were fixed. With Courtesy by J.C. Taveau, 1991.

Figure 16.6 Proposed model for all annelid hexagonal-bilayer ﬁ ﬁ hemoglobin. (a) Trion constituted by three polypeptide chains, two with ndings of Guerritore et al. (1965), who were rst to establish a the similar tertiary structure of the myoglobin (a and b) and one chain structure of chlorocruorine from Spirographis. In this model, the different (c); (b) Hexatrion built by the association of six trions; (c,d) polymer was constituted by an HBL structure with a maximum Localization of these chains on the whole hexagonal-bilayer hemoglobin diameter of 26.5 nm, where each hexagon was composed of six (c, profile view; d, face view). Reused from (Hendrickson, 1983) with kind identical triangular subunits (A in Figure 16.4), located at each permission from Elsevier. corner. Each of the subunits was built from three smaller subunits (B in Figure 16.4), assembled in a triangle. The molecular weights were estimated as 270 kDa for A and 92 kDa for B. (1971). The molecule would be characterized by the same The model proposed for the Lumbricus terrestris EHb by folding as Mb but with the third (c) chain being different. The Vinogradov et al. (1986) is known as the “bracelet model,” three chains would be associated in trimer or “trion” fashion, and is constituted by the assembly of monomers M and trimers with six trions associated to a “hexatrion,” which itself would

T with Mr-values of 16 and 50 kDa, respectively. The cohesion of be associated by 12 to create the EHb molecule (Figure 16.6). this structure was produced by a central ring constituted by two This model described a biopolymer with Mr 3880 kDa, con- other dimeric subunits that were devoid of heme, D1 and D2, structed from an association of 216 chains of which 144 would

with Mr-values of 31 and 37 kDa, respectively. The assembly of possess heme. these subunits was modeled by Taveau (1991), based on TEM The deficiency in the stoichiometry of one mole of heme per images (Figure 16.5). polypeptide chain proved to be a major result, and allowed an understanding of the quaternary structure of these huge mol- 16.4.2 ecules. However, the scientific community found this result very Estimation of Heme Number and Minimal Molecular Weight difficult to accept, and both Antonini and Chiancone (1977) and Garlick (1980) concluded that these results may have been due The minimal molecular weight of the native EHb, using either to purification artifacts. Subsequently, the absence of stoichi- the heme or the number of iron atoms, revealed a value of about ometry was admitted without real proof, although Fushitani one mole of heme per 27 000 Da of protein. This value was et al. (1982a) rejected this hypothesis using high-performance higher than for the smaller subunit found on EHb, determined liquid chromatography (HPLC). In fact, these authors showed either by electrophoresis or by using the primary sequences. that the polychete EHb from Travisia japonica was assembled fi fi These observations con rmed that not all of the polypeptide from ve components with Mr-values ranging between 14 and chains possessed a heme group, in contrast to vertebrate Hbs. 18 kDa, each of which contained one heme, although the The mean mass of 27 000 Da corresponded to one heme for minimal molecular weight was about 23 000 Da (Fushitani about 1.5–2 globin chains. et al., 1982a; Fushitani et al., 1982b; Fushitani, Morimoto, Waxman (1971) suggested a model for the EHb of Arenicola and Ochi, 1983) (Table 16.4). cristata in which two polypeptide chains of the three would be When Suzuki et al. (1983) were able to isolate (by HPLC) only able to bind a heme group. Some years later, Hendrickson and six heme-components from the EHb of Perinereis brevicirris, Royer (1986) proposed a unique model for EHb molecules, their result was in agreement with that of Fushitani et al. based on similar aspects of TEM images. In the latter model (1982a). Subsequently, all of these results were challenged, the molecule was proposed to have been built by the assembly and following the determination of the primary sequences of of three different chains, of which two (a and b)wouldcontain three globin chains involved in the trimer subunit of Lumbricus a heme group, in agreement with the findings of Waxman EHb, Fushitani and Riggs (1988) recalculated the heme CH16 01/30/2014 0:7:33 Page 368

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Table 16.4 Minimum molecular weight (Da) obtained by iron or heme percentage. Their results suggested that all three chains pos- content, or by electrophoresis (SDS–PAGE). sessed heme and a proportion of about 32.5 kDa of protein without heme per one-twelfth of the molecule that remained M Species Minimal r (Fushitani and Riggs, 1988). Using the HPLC data, Gotoh and Fe or Heme SDS–PAGE Suzuki (1990) proposed a quaternary model for Tylorrynchus heterochaetus EHb which, according to these results, would Polycheta Nephtys incise 22 400 11 000 consist of an assembly of four globin chains. This model Tylorrhynchus heterochaetus 26 500 12 000 described a molecule built by assembling 192 globin chains. Perinereis cultifera 23 700 13 000 Later, Suzuki et al. (1990a) sequenced two linker chains without Arenicola marina 23 613 14 000 heme from the Tylorrynchus EHb, and confirmed that not all Arenicola cristata 26 100 13 000 polypeptide chains in EHb would contain a heme group, thus fi Abarenicola af nis 23 525 13 700 rejecting in facto their previous model. Abarenicola pacifica 24 700 15 800 Euzonus mucronata 23 370 12 500 Travisia japonica 23 000 14 000 16.4.3 Travisia fetida 23 400 14 600 Small-Angle Light Scattering Eunice aphroditois 26 700 14 600 Marphysa sanguinea 27 589 14 000 The model proposed by El Idrissi Slitine (1991) for Arenicola Pista pacifica 24 454 15 000 marina was obtained by the assembly of 186 basic spherical Thelepus crispus 15 000 units of 21 A radius. This radius was calculated as being the Alvinella pompejana 23 400 13 900 distance between the next two spherical units, and was inferior Oligochaeta ’ Tubifex tubifex 21 000 13 000 to the units own diameter. This model was based on three Glossoscolex paulistus 25 250 ND different levels: (i) a “peripheral structure” made from 14 Lumbricus terrestris 24 500 12 400 elemental spheres set out in three stairs (4/6/4); (ii) a “central Maoridrilus montanus 23 900 13 500 structure” formed by six spheres arranged in this way (1/4/1); Eisenia fetida 24 700 14 800 and (iii) a “contact structure” constituted by two spheres, Pheretima communissima 25 430 14 500 which occupied the free space between the peripheral and Acheta central structures (Figure 16.7). This model described a mol- Haemopis grandis 24 000 13 000 “ ” Haemopis sanguisuga 24 800 12 600 ecule built by the assembly of 12 peripheral structures linked Dina dubia 21 200 12 000 together by 12 “contact structures”;thisassemblagesur- ND ¼ not determined. rounded a protein material, the density of which could not be attributed exactly to a complete one-twelfth (El Idrissi Slitine, Torriani, and Vachette, 1990). This model described

Figure 16.7 Model proposed for the extracellular hemoglobin from Arenicola marina, based on small-angle X-ray scattering data. P ¼ peripheral structure; C ¼ central structure; L ¼ contact structure. Courtesy of Fouzia El Idrissi Slitine, 1991. CH16 01/30/2014 0:7:34 Page 369

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a molecule of 168 chains, of which 144 globin chains and 24 global Mr of 3800 kDa. In addition, the model could explain linker chains were devoid of heme. the heterogeneity of the results observed with SDS–PAGE, as the molecule could be cleaved randomly at different levels 16.4.4 (Figure 16.8). Low- and High-Pressure Liquid Chromatography and The second model proposed for Lumbricus terrestris was SDS–PAGE built by assembling 192 globin chains (i.e., a, b, c, d)and24 linker chains (i.e., three majors L1, L2andL3 and one minor To date, the details of two major quaternary models for L4) (Fushitani and Riggs, 1988; Ownby et al., 1993; Zhu et al., Lumbricus terrestris EHb have been reported. For both models, 1996). For this model, each twelfth would be constituted by a

each twelfth is constituted only by the globin chain, and the dimer of tetramer [(a,b,c,d)2] that would be able to dimerize to twelfths were tied together by the intermediate of linker create a tetramer of [(a,b,c,d)4].Themoleculewouldbe ﬁ chains that possessed no or little heme. The rst of these constituted in the following way: [(a,b,c,d)4,L2] 12, where models was proposed by Vinogradov et al. (1986), and a, b, c and d corresponded to globin chains, and L to linker described a molecule assembled from 12 subunits (“dodeca- chains. This conceptual model would have a Mr of 3800 kDa, mers”) and constituted 12 globin chains (Vinogradov et al., as for the bracelet model (Fushitani and Riggs, 1988; Riggs, 1991; Sharma et al., 1996). The molecule was composed of 1990). 144 globin chains, associated with 36 linker chains. Although, the names of the different components have 16.4.5 changed with time, at this point the ﬁrst nomenclature Electrospray Ionization-Mass Spectrometry will be used. For Vinogradov’smodel,amonomerM

(i.e., d)withaMr of 16 750 Da possessing a heme group A more accurate technique, electrospray ionization-mass spec- was associated with a trimer subunit T (i.e., a, b, c) with a Mr trometry (ESI-MS), was successfully applied to some HBL Hbs, of about 50 000 Da; together, this complex constituted a allowing the determination of their complete polypeptide chain tetramer. The trimer complex constituted the covalent assem- composition. The EHbs examined were from Macrobdella decora bly of three globin chains with Mr about 16 000 Da, and hence (Weber et al., 1995), Tylorrynchus heterochaetus (Green et al., each twelfth or dodecamer was constituted by the assembly of 1995), Lumbricus terrestris (Martin et al., 1996b), Riftia pachyptila three tetramers (3 (M þ T)). These twelfths were tied (Zal et al., 1996a), and Arenicola marina (Zal et al., 1997a, 1997b). together in the native molecule by two different monomers The models of these molecules, obtained by ESI-MS, were D1andD2ofMr about 31 and 32 kDa, respectively. D1and compared to those obtained with HPLC in Table 16.3. The D2 were suspected of being devoid of heme, and to have a model obtained for Arenicola marina EHb HBL, using ESI- protein bracelet at the molecule centers. MS data, is shown in Figure 16.9. The one-twelfth protomer

The bracelet model was based on TEM images as well as would be constituted by the following assembly [(3a1)(3a2)2T], dissociation–reassociation experiments (Kapp et al., 1984, where T corresponds to either T3, T4, or T5. T1 and T2 were 1987; Mainwaring et al., 1986; Vinogradov, 1986; Vinogradov probably located at the molecule center, as noted in previous et al., 1986), and contained 200 polypeptide chains with a studies.

(a) (b)

T3 a1 a1 a2 a2 T5 T4 a1 a2 T T T T4 T5 a2 a2 T3 a2

Figure 16.9 Model of Arenicola marina HBL Hb, adapted from Waxman Figure 16.8 Cleavage possibility of the extracellular hemoglobin from (1971). (a) Drawing of typical HBL Hb, top view. Each one-twelfth is Lumbricus terrestris, according to the bracelet model proposed by constituted by one trimer (T2, T3 and T4), as noted by Waxman (1971), Vinogradov et al. (1986). This scheme explains the heterogeneity of SDS– with nine monomeric chains. The two trimers (T), for example, T1 and T1 PAGE results. (a) Face view (T ¼ trimer; M ¼ monomer; D1(V) and D2 or T1 and T2 at the center of the HBL structure, are represented as viewed (VI) ¼ linker chains; (b) Profile view. V and VI correspond with the first on the TEM image; (b) Detail of a one-twelfth, showing the arrangement of nomenclature used to name these chains. Courtesy of S. Vinogradov. monomeric chains (Zal et al., 1997a). CH16 01/30/2014 0:7:34 Page 370

370 j 16 Extracellular Hemoglobins from Annelids, and their Potential Use in Biotechnology

16.5 16.6 Biotechnology Applications Organ Preservation

As noted brieflyabove,manyresearchgroupsworldwidehave In 2005, almost 93 000 organ transplants were conducted investigated the annelid EHb, demonstrating the great interest worldwide, and the number of transplants is growing each in this molecule among the international scientific commu- year by 5%. For example, 25 000 transplants were conducted nity. Among these groups, the more active in the field in in the US alone in 2003. In France, the annual number of France was led by Prof. Andre Toulmond and was located transplants is constantly growing, having risen from 3115 in initially in Paris on the University Paris VI campus (Pierre-et- 1998 to 4946 in 2011. During the latter year about 300 people Marie-Curie University). In 1993, this laboratory moved to the died in France due to a lack of transplants, whilst in 2012 some Marine Biological Station Roscoff (Finistere, France), and 10 400 patients were awaiting transplant. The success of organ Prof. Toulmond became the Director of this very old and transplantation has resulted in a rapid increase in the discrep- famous center (founded in 1872 by Prof. Henri de Lacaze- ancy between the number of patients waiting to be transplanted Duthiers, at the Sorbonne University), between 1993 and and the number of transplants available. The available solutions 2003. Franck Zal, a PhD student of Andre Toulmond, began include splitting an organ between several receivers (e.g., liver), to work on Arenicola marina in 1993, and described in detail xenotransplantation (using organs from animals, mostly cardiac the structure of several EHbs. Zal discovered that the Hb valves or islets of Langerhans from pigs), or the culture of molecule of Arenicola marina (HbAm) had all the character- organs from stem cells. Unfortunately, as these solutions are istics of a “universal oxygen carrier” that medics had been only at the exploratory stages, a much better solution might be to seeking for several decades for therapeutic applications. extend the duration of organ preservation after collection from a HbAm has exceptional properties: cadaver, by using hypothermic continuous reperfusion so as to minimize organ loss. It is naturally extracellular and polymerized. The main objective of organ preservation is to extend the Its molecular weight is 50-fold that of human Hb. period of ex vivo viability of an organ, allowing for its transfer It has functional O -binding and -liberating properties similar 2 from the removal center to the transplant center. Under classical to those of human Hb (HbA inside the red blood cell). static organ conservation conditions, the conservation times Each HbAm molecule can bind 156 molecules of oxygen, possible before ischemia sets in differ widely and depend on compared to four in the case of human HbA the organ concerned. For example, the kidney, heart, liver, lungs It has naturally antioxidative properties. and pancreas are very sensitive to cold ischemia. Historically, and before this discovery, two approaches had The limited life span of transplants is due to deleterious been investigated to develop universal oxygen carriers: (i) a effects induced by interruption of the blood circulation (ische- chemical method using perfluorocarbons; and (ii) biological mia) which, at 37 C, causes a rapid cellular necrosis of the methods using human or bovine Hb (Ketcham and Cairns, removed organ. Ischemia can be retarded by refrigerating the 1999; Gulati, Barve, and Sen, 1999; Winslow, 2000. In March organ, as a decrease in temperature causes a decline in cellular 2007, Dr Franck Zal and Dr Morgane Rousselot created the energy requirements (the O2 requirement at 5 C is only 5% of French biotechnology company, HEMARINA, in order to that at 37 C). Ischemia in hypothermia (cold ischemia) starts as develop and promote the molecule HbAm, which was seen soon as the washed organ is refrigerated, and continues until it to possess all of the characteristics necessary to become the is reperfused in the recipient. Cooling does not completely stop leading third-generation blood substitute. In addition to the cellular metabolism, but does slow down the speed of enzymatic data outlined above, the principal characteristics of HbAm are reactions and delays cellular death. However, the use of cold that: ischemia is time-limited, as the absence of a sufficient supply of O causes a metabolic shift towards anaerobic glycolysis. This The functional properties are totally independent of 2 leads to the generation of lactic acid with cellular acidification secondary molecules, such as 2,3-DPG. from the rupture of lysosomal membranes and cellular destruc- The molecule functions without any chemical modification, tion by the liberated proteolytic enzymes. The results is an and with no additional treatment. accumulation of various toxins, including cytotoxic free radicals, The molecule has a functional capability at temperatures that are passed to the receiver during organ reperfusion. between 4 C and >30 C, which is a major and essential advantage. 16.6.1 The absence of any vasoconstrictor effects is in contrast to all Preservation Solutions other products developed to date and referred to as hemoglobin oxygen carriers (HBOCs) (Tsai et al., 2012). The deleterious effects of cold ischemia can be attenuated Although several major applications for HbAm have already through the use of preservation solutions, which allow the been identified, attention here will be focused on only two, reduction of cellular edema by maintaining the extracellular namely organ preservation pending transplantation and the osmotic pressure, thus preventing intracellular acidosis and development of an innovative oxygen carrier. reducing reperfusion injuries due to free radical accumulation. CH16 01/30/2014 0:7:34 Page 371

16.7 Anemia j 371

Some years ago, the ANSM (Agence National de Securitedu 16.7 Medicament) questioned the efficiency of organ preservation Anemia solutions. According to a World Health Organization reports, one in ten 16.6.2 people admitted to hospital requires a blood transfusion. The Hypothermic Continuous Reperfusion majority of these cases are anemia, due to a decrease in Hb levels following the loss or reduced production of red blood cells In general, following its removal a transplant can be conserved by (RBCs). A loss of RBCs may be due to hemorrhage during a simple immersion in a preservation solution at 4 C. This static trauma (accidents, injuries, burns) or during surgery (coronary technique is simple and inexpensive; however, due to the urgency artery bypass, organ transplants, hip replacement, etc.). Anemia of the situation related to lack of organs and to the degree of can also be caused by hemolytic anemia (e.g., drepanocytosis). A ischemia of organs currently being transplanted, a hypothermic reduced RBC production may occur in cases of global medullary continuous reperfusion technique has been developed. This insufficiency and bone marrow cancer, as well as being a side dynamic technique, which is presently being applied to kidney effect of radiotherapy or chemotherapy. The aim of transfusing transplants, requires sophisticated and expensive equipment. RBC concentrates is to maintain or to restore a sufficient level of Currently, two main dynamic systems have been developed, Hb. Typically, 50% of all RBCs are transfused to patients aged the performances of which are similar. Unfortunately, the use >65 years, and demographics show that, in developed countries, of perfusion equipment is limited to a small number of centers in this age category will double over the next 30 years. Certain types Europe, and the preliminary results do not indicate a significantly of surgery that are becoming increasingly common require an better performance than static methods. essential number of RBCs (e.g., up to 100 RBC units for one Because it is functional in the natural environment at low liver transplantation). Moreover, these trends are also observed temperature, HbAm was manufactured by HEMARINA as the in emerging countries (India, China, Brazil), and it is agreed 1 product HEMO2life which, when added to several clinical that worldwide RBC demands to treat anemia will continue to storage media, insured oxygenation of the organs. The recovery increase. 1 of organ grafts maintained in media with HEMO2life and then As an example, in 2009 the number of RBCs transfused was transplanted into pig models was shown to be excellent (Fig- about 93 million units, which represents an increase of 33% 1 fi ure 16.10). HEMO2life is currently at the nal stage of compared to 2003. Moreover, this situation is not expected to registration, with efficiency having already been demonstrated improve with demands continuing to increase, especially when 1 for kidney, lung, and heart. HEMO2life also possesses natu- associated with a stagnation or even regression in donations. rally an intrinsic superoxide dismutase (SOD) activity on Today, the exclusion criteria for donors are expanding due to HbAm, which allows it to protect the cells against oxidative emergent risks relating to transfusions (Nile virus, severe acute damage caused by an accumulation of free radicals liberated respiratory syndrome, prions, chikungunya virus, etc.), and this 1 during cold ischemia. HEMO2life could also increase the is causing a regression in the number of regular donors. Experts efficiency of dynamic systems for the conservation of kidneys, in the field maintain that the shortage in blood products world- and probably also for other organs with shorter preservation wide is 100 million liters per year, and these requirements are times (e.g., heart, liver, pancreas, lung). constantly increasing.

Figure 16.10 Kidney function following reperfusion. Porcine kidneys were cold-flushed and preserved for 24 h with UW or HTK supplemented with M101 at a concentration of 0 g l 1 (UW and HTK groups) or 5 g l 1 (UW þ M101 and HTK þ M101 groups). Follow-up was performed on the day before transplantation (D-1) and after transplantation (at 1 h ¼ R60; from day 1 to day 14 ¼ D1 to D14; and 1 month ¼ M1). Values shown are mean SEM. , p <0.05 versus UW; y,p<0.05 versus HTK; n ¼ 5. UW ¼ University of Wisconsin organ preservation solution, patented by the university (also called VIASPAN); HTK ¼ Histidine-tryptophan-ketoglutarate organ preservation solution (also called Custodiol); M101 ¼ Molecule 101, the development name given by HEMARINA to the hemoglobin of Arenicola marina. CH16 01/30/2014 0:7:34 Page 372

372 j 16 Extracellular Hemoglobins from Annelids, and their Potential Use in Biotechnology

16.7.1 to obtain a higher efﬁciency of this practice. HEMOXYCar- 1 Hemoglobin Oxygen Carriers rier , which is highly soluble and stable when diluted, should temporarily mediate the lack of oxygen inherent in the simple Initially, HBOCs were developed in response to this blood use of a ﬁlling solution. shortage. The short half-life of HBOCs (ranging from several Anemia can be caused by intense and brutal bleeding hours to 2–3 days) compared to RBCs (120 days) means that after a trauma, typically accidents, gunshot wounds or they are only suitable for use in acute anemia (programmed rupture of the vascular system (aneurysm). The tolerance surgery, traumatology), though these represent 60% of current of certain vital organs (brain, heart) to a massive reduction

prescriptions for RBCs. Today, experts estimate that oxygen in O2 supply is weak. Trauma situations generally occur transporters will not replace RBCs from donors for the treat- outside hospitals and are managed by mobile emergency ment of anemia. In the case of HBOCs, this is due to the very units, and often the blood group of the victim is not known. large amounts of active material that must be produced, the The stocks of O blood (universal donor) carried by emer- limited sources of raw materials, and the costs related to gency vehicles are insufficient to compensate for important chemical modifications. Perfluorinated compounds (PFCs) losses and, in the worst cases, only filling solutions are are limited in their therapeutic applications, as only small used. In this situation, emergency units require a product quantities can be used because of their high retention, and the that is compatible with all blood groups, and easy to store need for concomitant use of an oxygen mask. Nevertheless, and use. This type of product would be of special interest HBOCs can represent an additional therapy for certain appli- for the treatment of victims of natural catastrophes and of cations, notably normovolemic hemodilution and emergen- war casualties. The intravenous administration of HEMOX- 1 cies relating to hemorrhagic shocks. YCarrier permitted a rapid, efficient and simple restora- tion of the oxygenation capacity affected during massive blood loss in an animal model, without any side effects 16.7.2 (Rousselot et al., 2006; Tsai et al., 2012). This molecule is Normovolemic Hemodilution stable at ambient temperature and can also be lyophilized. Moreover, its properties would allow mobile emergency Normovolemic hemodilution has been recommended for units to dispose of sufficient products to meet important decades. The principle is simple: blood is taken from the and unpredictable demands. patient who will undergo surgery and replaced by filling Preclinical studies performed on rodents have shown that the liquids (colloids and crystalloids) in order to maintain the systemic injection of this molecule does not generate immuno- intravascular volume. After surgery, the blood that had been genicity and allergic reactions, and does not provoke any vaso- removed is re-transfused to the patient. Dilution of the circu- constrictive effects. It has been possible to directly visualize the lating blood reduces the number of RBCs lost by hemorrhage efficiency of the molecule on hypoxic tissues immediately after during the intervention, which in turn reduces the need for systemic perfusion (Rousselot et al., 2006; Tsai et al., 2012; T. Le homologous transfusions postoperatively. This has a double Gall et al., unpublished results). advantage, in that homologous RBC stocks are saved, and there is an avoidance of exposure to homologous blood, which carries a sizeable residual risk. In France today, three accidents occur for every 1000 homologous transfusions. These prob- 16.8 lems occur mainly due to blood typing errors (donor or Conclusion receiver) or to sanitary issues (bacteria or viruses below detection levels during tests). Available data on normovolemic To summarize, as opposed to the development of first- and hemodilution show, however, that the objective targeted by second-generation HBOCs that modify intracellular hemoglo- this strategy (a reduction in homologous blood use) is rarely bin (human or bovine) in order to function extracellularly, a attained under most surgical conditions. The mathematical natural extracellular hemoglobin from the invertebrate Areni- modeling of normovolemic hemodilution indicates that the cola marina (HbAm) was used. This circulatory pigment, chances of reaching this goal are much higher if the volume of whichispresentinthebloodvesselsoftheinvertebrate, blood removed is high before preoperative blood losses. How- hasevolvedovermillionsofyearsandisabletofunction ever, in practice this theoretically optimal hemodilution is extracellularly. It was shown, using vertebrate animal models, rarely achieved because of issues relating to individual toler- that this molecule does not induce vasoconstriction, which ance levels of patients. was the major secondary effect of the last generation of HBOCs. As a consequence, HbAm can be considered a ® 16.7.2.1 HEMOXYCarrier lead molecule for the future development of an oxygen carrier HEMARINA is currently developing a product called for ischemia or anemia applications. In this respect, two 1 HEMOXYCarrier , which contains HbAm, for such applica- products are currently under development by the HEMARINA 1 fi 1 tions. The coadministration of HEMOXYCarrier with lling company, namely HEMO2life for organ preservation, and 1 liquids would allow a greater degree of hemodilution in order HEMOXYCarrier as a universal oxygen carrier. CH16 01/30/2014 0:7:34 Page 373

References j 373

Acknowledgments Nelly Rolland for its help to get on time all journal author- isations for this review. Conﬂict of interest: It should be noted Dr Franck Zal is very grateful to Prof. A. Toulmond, who was his that the authors are the founders of HEMARINA and hold advisor during his PhD studies and introduced him to the company stock. hemoglobin research ﬁeld. The authors would also like to thank

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About the Authors Franck Zal is the cofounder of HEMARINA and recognized on the structure–function relationships of the annelid extrac- internationally for his studies in the ﬁeld of the respiratory ellular hemoglobin. He then spent three years abroad as part of pigments of invertebrates. He received his PhD degree with a post-doctoral program, two years at the University of Santa honors in oceanography in 1996 from the University of Paris Barbara in California (USA), and one year at the University of VI (Pierre-et-Marie-Curie), where his doctoral research focused Antwerp (Belgium). For his work, he received in 2001 the CH16 01/30/2014 0:7:35 Page 376

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bronze medal of CNRS (French National Research Center). the Research and Development Analytical Chemistry Depart- Dr Zal is the author of over 70 publications in international ment of GlaxoSmithKline in the UK. In 2002, she obtained a peer-reviewed scientific journals, the author of ten patents, and degree in Chemical Engineering (Diplome^ d’Ingenieur) at has made presentations at over 100 international scientific ENSCMu (Ecole Nationale Superieure de Chimie de Mulhouse) meetings. He was also the head of an academic research group with qualifications in Chemistry-Biology-Healthcare. During the working on marine ecophysiology at a marine station in same year she obtained her master’s degree in Interface Chem- Brittany, CNRS-UPMC. In 2007, he decided to create HEMAR- istry-Biology and molecular modeling. She received her PhD INA, a marine biotechnology start-up located in Morlaix with honors in biochemistry in 2006 from the University of Paris (Finistere, France). The Capital Magazine identified this com- VI (performed at the Station Biologique of Roscoff), where her pany in France as the most promising biotechnology company. doctoral research focused on the properties of Arenicola marina HEMARINA won almost all prizes for innovative enterprise hemoglobin (HbAm) and its potential use as a blood substitute. in France, and especially the great prize of the Senat in France In 2006, she obtained a grant “Jeune Createur d’Entreprise” in 2007. Franck is also the cofounder of the Enterprise club delivered by Bretagne Innovation in order to create the Bio- Bretagne Biosciences and was vice-president of France Biotech. technology society Hemarina. She possesses strong expertise in analytical chemistry and structural biology). Dr Rousselot is the Morgane Rousselot PhD is cofounder of HEMARINA, and in author of eight publications in international peer-reviewed sci- charge of the operational aspects of Hemarina research and entific journals, the author of four patents, and has made development programs. In 2001, she was employed for a year in presentations at over 10 international meetings.