Morgane Rousselot

Novel dissociation mechanism of a polychaetous annelid extracellular haemoglobin

The extracellular haemoglobin of the marine polychaete, Arenicola marina, is a hexagonal bilayer haemoglobin of ≈ 3600 kDa, formed by the covalent and noncovalent association of many copies of both globin subunits (monomer and trimer) and nonglobin or ‘linker’ subunits. In order to analyse the interactions between globin and linker subunits, dissociation and reassociation experiments were carried out under whereby Arenicola hexagonal bilayer haemoglobin was exposed to urea and alkaline pH and the effect was followed by gel filtration, SDS/PAGE, UV‐visible spectrophotometry, electrospray‐ionization MS, multiangle laser light scattering and transmission electron microscopy. The analysis of Arenicola haemoglobin dissociation indicates a novel and complex mechanism of dissociation compared with other annelid extracellular haemoglobins studied to date. Even though the chemically induced dissociation triggers partial degradation of some subunits, spontaneous reassociation was observed, to some extent. Parallel dissociation of Lumbricus haemoglobin under similar conditions shows striking differences that allow us to propose a hypothesis on the nature of the intersubunit contacts that are essential to form and to hold such a complex quaternary structure.

High-level Production of Recombinant Arenicola Marina Globin Chains in Escherichia Coli: A New Generation of Blood Substitute

This work reports for the first time the expression of a soluble B2 globin chain that is part of the extracellular hexagonal-bilayer haemoglobin from Arenicola marina. Two recombinant B2 globins were produced, one fused with gluthatione S-tranferase (B2-GST) and the other without a fusion tag (RecB2) and requiring a different purification procedure. We also describe a new method for the expression of globin that uses Studiers auto-induction medium together with the heme precursor δ-aminolevulinic acid. Media supplementation with the heme precursor δ-aminolevulinic acid in the culture increased heme synthesis by E. coli leading to the expression of the recombinant B2 globins in their active form. RecB2 and B2-GST were expressed with a yield of up to 105 mg/l of E. coli culture. Our approach is rapid and requires only one chromatographic purification step for B2-GST and three purification steps for RecB2. The overall results on RecB2 and B2-GST show that the recombinant globins exhibit similar properties to those of Arenicola marina native HBL-Hb with a great stability and a strong oxygen binding. The results and methodologies described in this paper are the beginning of a work aiming at reconstituting a recombinant HBL-Hb by genetic engineering in order to produce an innovative oxygen carrier for therapeutic applications.

Microcirculation and NO-CO Studies of a Natural Extracellular Hemoglobin Developed for an Oxygen Therapeutic Carrier

Extracellular soluble hemoglobins (Hbs) have long been studied for their possible use as safe and effective alternatives to blood transfusion. While remarkable progress has been made in the use of cell-free Hb as artificial oxygen carrier, significant problems remain, including susceptibility to oxidative inactivation and propensity to induce vasoconstriction. Hemarina-M101 is a natural giant extracellular hemoglobin (3600 kDa) derived from marine invertebrate (polychaete annelid). Hemarina-M101 is a biopolymer composed of 156 globins and 44 non-globin linker chains and formulated in a product called HEMOXYCarrier®. Prior work has shown Hemarina-M101 to possess unique anti-oxidant activity and a high oxygen affinity. Topload experiment with this product into rats did not revealed any effect on heart rate (HR) and mean arterial pressure (MAP). A pilot study with the hamster dorsal skinfold window chamber model showed absence of microvascular vasoconstriction and no significant impact on mean arterial blood pressure. In vitro nitric oxide (NO) and carbon monoxide (CO) reaction kinetics measurements show that Hemarina-M101 has different binding rates as compared to human Hb. These results revealed for the first time that the presence of this marine hemoglobin appears to have no vasoactivity at the microvascular level in comparison to others hemoglobin based oxygen carriers (HBOCs) developed so far and merits further investigation.

The Structural Analysis of Large Noncovalent Oxygen Binding Proteins by MALLS and ESI-MS: A Review on Annelid Hexagonal Bilayer Hemoglobin and Crustacean Hemocyanin

Understanding the function of macromolecular complexes is related to a precise knowledge of their structure. These large complexes are often fragile high molecular mass noncovalent multimeric proteins. Classical biochemical methods for determination of their native mass and subunit composition were used to resolve their quaternary structure, sometimes leading to different models. Recently, the development of mass spectrometry and multi-angle laser light scattering (MALLS) has enabled absolute determination of native masses and subunit masses. Electrospray ionization mass spectrometry (ESI-MS) was used in denaturing and native conditions to probe subunit composition and noncovalent assemblies masses up to 2.25 MDa. In a complementary way, MALLS provides mass and size estimation in various aqueous solvents. ESI-MS method can also give insights into post-translational modifications (glycosylation, disulfide bridges ). By combining native mass and subunit composition data, structural models can be proposed for large edifices such as annelid extracellular hexagonal bilayer hemoglobins (HBL Hb) and crustacean hemocyanins (Hc). Association/dissociation mechanisms, protein-protein interactions, structural diversity among species and environmental adaptations can also be addressed with these methods. With their absolute mass determination, the very high precision of spectrometry and the versatile nature of light scattering, ESI-MS and MALLS have provided a wealth of data helping to resolve parts of controversies for HBL-Hb models and opening access to new fields of investigation in structural diversity and molecular adaptation. In this review we will focus on annelid HBL-Hb and on crustacean Hc and on the original contributions of ESI-MS and MALLS in this field.

Native and subunit molecular mass and quarternary structure of the hemoglobin from the primitive branchiopod crustacean Triops cancriformis

Many branchiopod crustaceans are endowed with extracellular, high‐molecular‐weight hemoglobins whose exact structural characteristics have remained a matter of conjecture. By using a broad spectrum of techniques, we provide precise and coherent information on the hemoglobin of one of the phylogenetically ‘oldest’ extant branchiopods, the tadpole shrimp Triops cancriformis. The hemoglobin dissociated under reducing conditions into two subunits, designated TcHbA and TcHbB, with masses of 35 775 ± 4 and 36 055 ± 4 Da, respectively, determined by ESI‐MS. Nonreducing conditions showed only two disulfide‐bridged dimers, a homodimer of TcHbA, designated D1 (71 548 ± 5 Da), and the heterodimer D2 (71 828 ± 5 Da). Carbamidomethylation of free SH groups revealed the presence of three cysteines per subunit and indicated one intrasubunit and one intersubunit disulfide bridge. Ultracentrifugation and light‐scattering experiments under nondenaturating conditions yielded mass estimates that suggested an uneven number of 17 subunits forming the native hemoglobin. This unrealistic number resulted from the presence of two size classes (16‐mer and 18‐mer), which were recognized by native PAGE and Ferguson plot analysis. ESI‐MS revealed three hemoglobin isoforms with masses of 588.1 kDa, 662.0 kDa, and 665.0 kDa. The 16‐mer and the smaller 18‐mer species are supposed to be composed of TcHbA only, given the dominance of this subunit type in SDS/PAGE. Transmission electron microscopy of negatively stained specimens showed a population of compact molecules with geometrical extensions of 14, 16 and 9 nm. The proposed stoichiometric model of quarternary structure provides the missing link to achieve a mechanistic understanding of the structure–function relationships among the multimeric arthropodan hemoglobins.

Molecular mass of macromolecules and subunits and the quaternary structure of hemoglobin from the microcrustacean Daphnia magna

The molecular masses of macromolecules and subunits of the extracellular hemoglobin from the fresh‐water crustacean Daphnia magna were determined by analytical ultracentrifugation, multiangle laser light scattering and electrospray ionization mass spectrometry. The hemoglobins from hypoxia‐incubated, hemoglobin‐rich and normoxia‐incubated, hemoglobin‐poor Daphnia magna were analyzed separately. The sedimentation coefficient of the macromolecule was 17.4 ± 0.1 S, and its molecular mass was 583 kDa (hemoglobin‐rich animals) determined by AUC and 590.4 ± 11.1 kDa (hemoglobin‐rich animals) and 597.5 ± 49 kDa (hemoglobin‐poor animals), respectively, determined by multiangle laser light scattering. Measurements of the hemoglobin subunit mass of hemoglobin‐rich animals by electrospray ionization mass spectrometry revealed a significant peak at 36.482 ± 0.0015 kDa, i.e. 37.715 kDa including two heme groups. The hemoglobin subunits are modified by O‐linked glycosylation in the pre‐A segments of domains 1. No evidence for phosphorylation of hemoglobin subunits was found. The subunit migration behavior during SDS/PAGE was shown to be influenced by the buffer system used (Tris versus phosphate). The subunit mass heterogeneity found using Tris buffering can be explained by glycosylation of hemoglobin subunits. Based on molecular mass information, Daphnia magna hemoglobin is demonstrated to consist of 16 subunits. The quaternary structure of the Daphnia magna hemoglobin macromolecule was assessed by three‐dimensional reconstructions via single‐particle analysis based on negatively stained electron microscopic specimens. It turned out to be much more complex than hitherto proposed: it displays D4 symmetry with a diameter of approximately 12 nm and a height of about 8 nm.

Gene Structure and Molecular Phylogeny of the Linker Chains from the Giant Annelid Hexagonal Bilayer Hemoglobins

Giant extracellular hexagonal bilayer hemoglobin (HBL-Hb), found only in annelids, is an ∼3500-kDa heteropolymeric structure involved in oxygen transport. The HBL-Hbs are comprised of globin and linker chains, the latter being required for the assembly of the quaternary structure. The linker chains, varying in size from 225 to 283 amino acids, have a conserved cysteine-rich domain within their N-terminal moiety that is homologous to the cysteine-rich modules constituting the ligand binding domain of the low-density lipoprotein receptor (LDLR) protein family found in many metazoans. We have investigated the gene structure of linkers from Arenicola marina, Alvinella pompejana, Nereis diversicolor, Lumbricus terrestris, and Riftia pachyptila. We found, contrary to the results obtained earlier with linker genes from N. diversicolor and L. terrestris, that in all of the foregoing cases, the linker LDL-A module is flanked by two phase 1 introns, as in the human LDLR gene, with two more introns in the 3′ side whose positions varied with the species. In addition, we obtained 13 linker cDNAs that have been determined experimentally or found in the EST database LumbriBASE. A molecular phylogenetic analysis of the linker primary sequences demonstrated that they cluster into two distinct families of linker proteins. We propose that the common gene ancestor to annelid linker genes exhibited a four-intron and five-exon structure and gave rise to the two families subsequent to a duplication event.

In vivo biodistribution and oxygenation potential of a new generation of oxygen carrier

Natural giant extracellular hemoglobins (Hbs) from polychaete annelids are currently actively investigated as promising oxygen carriers. Their powerful oxygenating ability and their safety have been demonstrated in preclinical studies, motivating their development for therapeutic and industrial applications. HEMARINA-M101 (M101) is derived from the marine invertebrate Arenicola marina. It is formulated as a manufactured product designated HEMOXYCarrier(®) (HEMARINA SA, France). The aim of the present study was to unveil the fate of M101 after a single intravenous (i.v.) injection in mice. For this purpose, M101 was tagged with a far-red fluorescent dye. Repeated non-invasive fluorescent imaging revealed a rapid diffusion of M101 in the whole body of animals, reaching all the examined organs such as brain, liver, lungs and ovaries. Functional M101 was circulating in bloodstream for several hours, without inducing any obvious side-effects. Last, a single i.v. injection of M101 in mice bearing human-derived subcutaneous tumors demonstrated the ability of this Hb to reduce hypoxia in poorly vascularized tissues, thus supporting the biological relevance of M101 oxygen release to vertebrate tissues. Altogether, these results further encourage the development of M101 as an oxygen carrying therapeutic.

Advancement in recombinant protein production using a marine oxygen carrier to enhance oxygen transfer in a CHO-S cell line

Abstract Recombinant proteins, particularly proteins used as therapeutics, are widely expressed for bioprocessing manufacturing processes. Mammalian cell lines represent the major host cells for bioproduction, according to their capacities of post-translational modifications and folding of secreted proteins. Many parameters can affect cell productivity, especially the rate of oxygen transfer. Dissolved oxygen, in high or low proportions, is a crucial parameter which can affect cell viability and thus productivity. HEMARINA has developed a new technology, commercially proposed as HEMOXCell®, to improve cell culture at a large production scale. HEMOXCell® is a marine oxygen carrier having properties of high oxygen sensitivity, to be used as an oxygen additive during cell culture manufacturing. In this study, we investigated the effects of HEMOXCell® on the culture of the commonly used CHO-S cell line. Two main objectives were pursued: 1) cell growth rate and viability during a batch mode process, and 2) the determination of the effect of this oxygen carrier on recombinant protein production from a CHO-transfected cell line. Our results show an increase of CHO-S cellular growth at a rate of more than four-fold in culture with HEMOXCell®. Moreover, an extension of the growth exponential phase and high cell viability were observed. All of these benefits seem to contribute to the improvement of recombinant protein production. This work underlines several applications using this marine-type oxygen carrier for large biomanufacturing. It is a promising cell culture additive according to the increasing demand for therapeutic products such as monoclonal antibodies.