Molecular mass of macromolecules and subunits and the quaternary structure of hemoglobin from the microcrustacean Daphnia magna Tobias Lamkemeyer1,2, Bettina Zeis1, Heinz Decker3, Elmar Jaenicke3, Dieter Waschbusch3, ă Wolfgang Gebauer4, Jurgen Markl4, Ulrich Meissner4, Morgane Rousselot5, Franck Zal5, ă Graeme J Nicholson6 and Rudiger J Paul1 ă Institut fur Zoophysiologie, Westfalische Wilhelms-Universita Munster, Germany ¨ ¨t, ¨ ¨ Proteom Centrum Tubingen, Eberhard-Karls-Universitat, Tubingen, Germany ¨ ¨ ¨ Institut fur Molekulare Biophysik, Johannes Gutenberg Universitat, Mainz, Germany ă ă Institut fur Zoologie, Johannes Gutenberg Universita Mainz, Germany ăt, ă Equipe Ecophysiologie: Adaptation et Evolution Moleculaires, UPMC–CNRS UMR 7144, Station Biologique, BP 74, Roscoff, France Institut fur Organische Chemie, Eberhard-Karls-Universitat,Tubingen, Germany ă ă ă Keywords glycosylation; hemoglobin; macromolecule; molecular mass; quaternary structure Correspondence T Lamkemeyer, Interfakultares Institut fur ă ă Zellbiologie, Proteom Centrum Tubingen, ă Eberhard-Karls-Universitat, Auf der ă Morgenstelle 15, D-72076 Tubingen, ă Germany Fax: +7071 295359 Tel: +7071 2970556 E-mail: Tobias.Lamkemeyer@unituebingen.de (Received 23 March 2006, revised 17 May 2006, accepted 30 May 2006) doi:10.1111/j.1742-4658.2006.05346.x 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, hemoglobinpoor 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 hemoglobinrich 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 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 nm Abbreviations AUC, analytical ultracentrifugation; BN-PAGE, blue native polyacrylamide gel electrophoresis; ESI-MS, electrospray ionization mass spectrometry; Hb, hemoglobin; MALLS, multiangle laser light scattering; MRA, multireference alignment; MSA, multivariate statistical analysis; RuBPs, ruthenium II tris(bathophenanthroline disulfonate) FEBS Journal 273 (2006) 3393–3410 ª 2006 The Authors Journal compilation ª 2006 FEBS 3393 Structure of Daphnia magna hemoglobin T Lamkemeyer et al Invertebrate hemoglobins show very high structural diversity in comparison with the uniform tetrameric structure in vertebrates They range from the 17 kDa single-chain globins found in bacteria, algae, protozoa and plants to the large multisubunit, multidomain hemoglobins found in nematodes, molluscs and crustaceans up to the giant annelid and vestimentiferan hemoglobins of about 3600 kDa, which are composed of globin and nonglobin subunits [1] Specifically, the structure and function of the hemoglobin (Hb) of the microcrustacean Daphnia magna have been addressed by many studies over the last decades Daphnia magna Hb is freely dissolved in the extracellular fluid It is a multisubunit Hb composed of didomain globin chains The synthesis of this Hb is regulated by ambient oxygen concentration, temperature and juvenoid hormones [2–4] Environmental hypoxia, for example, may cause an increase of Hb concentration in D magna by a factor of 16, corresponding to Hb concentrations between 55 and 888 lmol hemL)1 [5,6] Concomitant changes of oxygen affinity between 1.02 and 0.15 kPa (P50) have been reported [7], which are related to Hb multiplicity (isohemoglobins composed of different Hb subunits) [1,8,9] The biological advantages of increased Hb concentration and oxygen affinity under hypoxia are manifold [1,9] Accordingly, Daphnia is currently the focus of investigations aimed at the structure and evolution of its globin genes as well as of its physiologic adaptations [1] Hb synthesis takes place in the fat cells and epipodite epithelial cells of D magna [10], which are the only known sites of Hb synthesis in crustaceans [1] The seven known Hb subunits (DmHb) [3,8,11] are encoded by at least six Hb genes (dmhb) [12] At least four of them are organized in a cluster in the order dmhb4, dmhb3, dmhb1 and dmhb2 [11] On the basis of nucleotide and derived amino acid sequences, the molecular masses of Hb subunits are 36.228 kDa (dmhb1), 36.177 kDa (dmhb2), 36.217 kDa (dmhb3) (sequence data from [11]) and 35.921 kDa (dmhb4) (sequence data from [12]) However, the molecular masses of the Hb subunits experimentally determined by gel electrophoresis were 36.2 (DmHbA–DmHbD), 37.9 (DmHbF, DmHbG) and 40.6 kDa (DmHbE) [8], raising the question of the reason for this difference For the molecular mass of the native D magna Hb complex, reported values vary between 494 and 670 kDa, depending on the methods used (gel filtration, ultracentrifugation, gel electrophoresis [13–15]) Suggesting very low molecular masses of about 31– 33 kDa for the Hb subunits and 494 kDa for the native multimer, Ilan et al [14] concluded that 16 3394 polypeptide chains, each carrying two heme-binding domains, form one D magna Hb macromolecule From electron micrographs, two models of the threedimensional structure have been suggested [14]: (a) a cyclic structure composed of all 16 subunits; and (b) a dihedral structure, in which the subunits are grouped in two layers stacked in an eclipsed orientation Accordingly, the present data on the molecular mass of the native Hb complex as well as the Hb subunits are inconsistent In addition, there are only two hypothetical models concerning the structure of the multisubunit assembly, which has stimulated further studies on the structure of D magna HB Three-dimensional reconstruction from transmission electron microscopy promises to be a satisfactory way to describe the quaternary structure of high molecular mass invertebrate respiratory proteins [16–18] To elucidate the important structural characteristics of the extracellular Hb of D magna, the molecular mass of the native Hb complex as well as those of denatured Hb subunits were determined by analytical ultracentrifugation and multiangle laser-light scattering (MALLS) or by MS and gel electrophoresis, respectively, in normoxically (pale) and hypoxically (red) raised D magna In addition, possible post-translational modifications were investigated To investigate the quaternary structure of D magna Hb, transmission electron microscopy and three-dimensional reconstruction of the macromolecule were carried out Results The native Hb complex from the hemolymph of red D magna purified by chromatofocusing and diluted in 10 mm ammonium acetate buffer was subjected to ultracentrifugation to determine the sedimentation velocity (Fig 1A) Use of the van Holde–Weischet analysis on the data (Fig 1B) showed that approximately 75% of the native protein sedimented with a sedimentation coefficient of 17.4 ± 0.1 S (Fig 1C) The remaining proteins sedimented at 16.3–17.1 S Subsequent sedimentation equilibrium experiments (Fig 2) resulted in a molecular mass of 583 kDa for the native Hb complex from the hemolymph of red D magna Owing to their small quantity, the molecular mass of slower-sedimenting proteins could not be analyzed with this method MALLS analyses of pale and red D magna Hb obtained by purification of crude animal extracts via gel filtration or chromatofocusing provided molecular mass determinations during the elution of peaks (Fig 3) On average, the molecular mass was 597.5 ± 49 kDa (n ¼ samples: 562.3, 576.4 and FEBS Journal 273 (2006) 3393–3410 ª 2006 The Authors Journal compilation ª 2006 FEBS time 0.6 A 0.5 0.4 0.3 0.2 0.1 center 0.0 6.0 6.1 6.2 bottom 6.3 6.4 24 22 B 20 18 16 14 12 0.00 6.5 6.6 radius (cm) % of total concentration sedimentation coefficient (S) Fig Sedimentation velocity analysis of the hemoglobin (Hb) of red (Hb-rich) Daphnia magna A preparation of Hb purified by chromatofocusing was centrifuged at 116 480 g at 20 °C; the protein concentration in the cell was 1.0 mgỈmL)1 in 10 mM ammonium acetate buffer, pH 6.7 (A) The absorbance at 280 nm along the length of the cell was recorded every (B) The sedimentation coefficient was determined with the van Holde–Weischet extrapolation plot (C) The distribution plot shows that approximately 75% of the protein sedimented at 17.4 ± 0.1 S, whereas only a small fraction sedimented more slowly (16.3–17.1 S) Structure of Daphnia magna hemoglobin absorbance at 280 nm T Lamkemeyer et al 0.01 0.02 0.03 time-0.5 (min-0.5) 653.9 kDa; for each sample, the molecular mass is averaged over 130 measured points) for pale D magna Hb and 590.4 ± 11.1 kDa (n ¼ samples: 576.2, 582.2, 593.2, 596.0 and 604.2 kDa; for each sample, the molecular mass is averaged over 130 measured points) for red D magna Hb A second peak in case of pale D magna (Fig 3A) was heterogeneously composed (polydispersity), as indicated by the inclination of individual measuring points The molecular mass of these proteins (Hb dissociation products or other proteins) was 385 ± kDa Using a multiphasic buffer system according to Laemmli [19], gel electrophoresis (SDS ⁄ PAGE) resulted in the separation of D magna Hb subunits into three different bands (Fig 4A) with molecular masses of 40.0, 38.1 and 35.1 kDa (pale D magna) and 40.0, 36.9 and 35.1 kDa (red D magna) As such separations may be due to a specific buffer system instead of reflecting actual mass differences [20], the protocol of Weber and Osborn [21] was employed: in this case, only one band for both Hb from the hemolymph of pale D magna and Hb from red D magna appeared (Fig 4B), corresponding to a molecular mass of approximately 39.2 kDa Bands with molecular masses above 66 kDa may originate from undissociated Hb molecules or impurities in the sample For an exact determination of the molecular mass of D magna Hb subunits, electrospray ionization mass spectrometry (ESI-MS) analyses were performed For red D magna Hb purified by gel filtration, an ion series in the mass ⁄ charge ratio (m ⁄ z) range of 1500–3000 0.04 6.7 6.8 6.9 7.0 100 80 C 60 40 20 16.0 16.5 17.0 17.5 18.0 sedimentation coefficient (S) occurred under denaturing conditions (Fig 5A) Deconvolution of the spectrum resulted in a single significant peak which corresponded to a molecular mass of 36.482 ± 0.0015 kDa (Fig 5B) The acidic conditions used for ESI-MS analysis led to the dissociation of the heme group (616.5 Da) from the polypeptide chains Thus, the final mass for the didomain subunit is 37.715 kDa, including two heme groups To test for post-translational modifications of red D magna Hb subunits, a staining technique specific for glycosylated proteins was used (Fig 6A), followed by staining with ruthenium II tris(bathophenanthroline disulfonate) (RuBPs) (Fig 6B) All Hb subunit spots and the glycosylated proteins of the CandyCane marker (Fig 6C) were specifically labeled after staining for glycoproteins The remaining marker proteins became visible only after silver staining for total protein Obviously, D magna Hb subunits are glycosylated These staining results are corroborated by enzymatic deglycosylation of Hb subunits After two-dimensional gel electrophoresis, the Hb subunits A–D appear as a train of spots, whereas the subunits E–G also differ in molecular mass [3,8] (Fig 7A) To test whether this separation is influenced by subunit glycosylation, Hb (purified by gel filtration) was incubated with different sets of enzymes removing only N-linked sugars (Fig 7B), only O-linked carbohydrates (Fig 7C) or both types of glycans (Fig 7D), respectively The untreated sample (Fig 7A, control) represents the typical spot pattern of red D magna Hb consisting of the subunits A–D and F The Hb pattern after incubation FEBS Journal 273 (2006) 3393–3410 ª 2006 The Authors Journal compilation ª 2006 FEBS 3395 Structure of Daphnia magna hemoglobin A T Lamkemeyer et al 1.0 I II 0.6 absorbance (280 nm) 0.8 III 0.4 0.2 center bottom 0.0 B residual s 0.05 0.00 -0.05 I 0.05 II 0.00 -0.05 III 0.05 0.00 -0.05 0.0 0.5 1.0 1.5 position, rmp - rmc 2.0 2.5 (cm2) Fig Sedimentation equilibrium analysis of the hemoglobin (Hb) of red Daphnia magna A preparation of Hb purified by chromatofocusing was centrifuged at 4660 (I), 5900 (II) and 7280 (III) g at °C, until equilibrium was achieved (A); the protein concentration was 1.0 mgỈmL)1 in 10 mM ammonium acetate buffer, pH 6.7 The results at all speeds were globally fitted, resulting in a molecular mass of 583 kDa The residuals (B: I–III) reflect the deviation of individual measuring points from the calculated fit, indicating the quality of the fit and the calculated molecular mass mp, measuring point; mc, meniscus with N-glycosidase F (Fig 7B) is obviously identical to that of the untreated sample Incubation with enzymes specific for the removal of O-linked glycans led to the occurrence of additional spots (marked with white circles in Fig 7C) with a higher electrophoretic mobility Consequentially, these spots also emerged, when the Hb sample was treated with the whole set of enzymes removing N-linked and O-linked glycans (Fig 7D) Potential N-linked glycosylation sites (NXT ⁄ S) were not found in the amino acid sequences of D magna Hb [22] Prediction of O-linked glycosylation was performed using the recently released NetOGlyc 3.1 server [23], and the results indicated the pre A-segments of 3396 Fig MALLS analysis of the hemoglobin (Hb) of (A) pale (Hb-poor) and (B) red (Hb-rich) Daphnia magna purified by gel filtration or chromatofocusing, during the elution on a gel filtration column (Superose 6-C) The solid curve represents the refractive index signal profile versus the elution volume, and the distribution of the molecular weight values is represented by crosses The latter information yielded molecular masses of 597.5 ± 49 kDa (n ¼ samples from different animal groups) for pale and 590.4 ± 11.1 kDa (n ¼ samples from different animal groups) for red D magna Hb The samples from pale D magna additionally showed a second protein component of a lower molecular mass (384.5 kDa) domain as sites for glycosylation events in all Hb sequences The numbers of possible glycosylation sites exceeding the threshold (G-score ¼ 0.5) were 11 for DHb and DHb2 and 12 for DHb3 and DHb4, respectively For identification of the carbohydrates bound to D magna Hb subunits, saccharides were released by methanolysis and separated by GC followed by MS Chromatograms of the carbohydrates of D magna Hb, of the corresponding blank and of a mannose standard are shown in Fig The dominant carbohydrate in D magna Hb is mannose, which was unambiguously identified by the analysis of a mannose standard (grayshaded curve in Fig 8) A lower, but still highly significant, increase (compared to the blank; Fig 8, inset) in the intensity of galactose and glucose was also observed (the dashed line in Fig represents the FEBS Journal 273 (2006) 3393–3410 ª 2006 The Authors Journal compilation ª 2006 FEBS T Lamkemeyer et al A Marker (kDa) pale Structure of Daphnia magna hemoglobin B red Marker (kDa) pale red 66 45 36 66 45 29 24 36 20.1 29 24 14.2 20.1 Tris buffer phosphate buffer Fig The influence of the buffer system used for gel electrophoresis on the band pattern of pale and red Daphnia magna hemoglobin (Hb) The hemolymph of pale (2.0 lg of Hb) and red (2.0 lg of Hb) D magna was subjected to electrophoresis according to Laemmli [19], using either Tris buffer (A) or sodium phosphate buffer (according to Weber and Osborn [21]) (B) In the first case, Hb was separated into three bands (pale D magna, 40.0, 38.1 and 35.1 kDa; red D magna, 40.0, 36.9 and 35.1 kDa), whereas in the second case, only a single band at 39.2 kDa was detected maximum total ion current of the blank: glucose, about 2500) The results of all experiments to demonstrate phosphorylation of Hb subunits, however, were negative Neither specific staining of phosphorylated proteins in gels (Pro Q Diamond stain) nor detection by western blotting using antibodies specific for phosphoserine and phosphotyrosine residues or different MS methods showed any indication of phosphorylation of D magna Hb subunits (data not shown) To assess the organization of the native Hb complex, electron microscopy and three-dimensional reconstructions were employed After staining with 2% uranyl acetate, the highly concentrated Hb macromolecules in the hemolymph of red D magna (Fig 9C,D) frequently showed specific clover-leaf structures (marked by arrows) These characteristic clover-leaf structures were scarcely found in the hemolymph of pale D magna (Fig 9A,B) In addition to Hb molecules, another type of protein was detected in the hemolymph of pale and red D magna (probably ferritin, marked by asterisks in Fig 9A,C,D) Because of its higher hemolymph concentration and higher degree of structural detail, red D magna Hb was considered to be more promising for single-particle image processing As purification by gel filtration did not completely remove the ferritin, which would have interfered with image analysis, Hb was purified by chromatofocusing, resulting in a small quantity of ferritin in comparison to the large number of Hb molecules in the samples In addition, structures clearly representing ferritin molecules were omitted from subsequent image analysis The macromolecules within a sample were spread across the holes in a perforated carbon support film, causing the molecules to have many different axial orientations relative to the electron beam As the three-dimensional organization of subunits in a native Hb complex of D magna is as yet unknown, different symmetry features had to be tested and verified Therefore, the simplest symmetry with minimum assumptions (C2; two-fold symmetry, bottom and top of the molecule different) was chosen for an initial estimation From the established threedimensional model, it could be expected that the bottom and the top of the molecule would be identical (D symmetry) Manual and automated (anchor set) Euler searches were then systematically performed for twofold to eight-fold D symmetries (D2–D8) For the Hb of red D magna, the class averages calculated from electron micrographs seemed to suggest a tetrameric (D4) symmetry (Fig 9E) Moreover, the best correspondence between class averages and reprojections was found for this symmetry, so that D4 symmetry was finally selected and used for three-dimensional reconstructions The preliminary three-dimensional model of ˚ red D magna Hb has a sub-30 A resolution (Fig 9F) In top view (Fig 9F, center), the molecule has the overall shape of a square Each side of the square has a length of approximately 12 nm A single mass in the center of the molecule was observed However, this apparent mass may be an artefact of the negative staining procedure (see Discussion) In side views (Fig 9F, left and right), the molecule appears as a compressed sphere with a height of about nm Without additional information such as X-ray data, the molecule’s handedness cannot be defined, meaning that it cannot be determined whether the real structure or its mirror image was reconstructed Discussion Molecular mass and sedimentation coefficient of Hb macromolecules Crustacean hemoglobins are large polymers with molecular masses between 240 and 800 kDa, and sedimentation coefficients between 11 and 19 S Reported FEBS Journal 273 (2006) 3393–3410 ª 2006 The Authors Journal compilation ª 2006 FEBS 3397 Structure of Daphnia magna hemoglobin A T Lamkemeyer et al 1921.1 2147.0 intensity (%) 2281.2 1825.1 100 2433.2 1659.3 2606.9 50 1500 2000 m/z 3000 2500 B Intensity (%) 100 36482 Fig Mass spectra of red Daphnia magna hemoglobin (Hb) purified by chromatofocusing (A) Charge state ESI-MS spectrum (B) MaxEnt-deconvoluted spectrum Prior to MS, the Hb samples had been desalted and denatured by adding acetonitrile ⁄ water containing 0.2% formic acid In (A), seven of the 10 peaks representing the subunit monomer are labeled In (B), the molecular mass (Da) of the Hb subunit (without heme) is given above the peak 50 36000 36500 37000 37500 38000 molecular mass (Da) C A 180 A F C B 82 97 66 D glycoprotein stain 42 B F C A B 29 D RuBPs stain 18 values for the molecular mass of D magna Hb vary considerably (494–670 kDa) Molecular mass determinations by gel filtration and gel electrophoresis yielded values between 500 kDa [24] and 670 kDa [15] However, interactions between 3398 Fig Staining for glycosylation of red Daphnia magna hemoglobin (Hb) Hb purified by gel filtration (100 lg) was subjected to two-dimensional gel electrophoresis and a staining procedure specific for glycosylated proteins (A) Hb spots became visible after staining for glycosylated proteins Subsequently, the gel was stained with ruthenium II tris(bathophenanthroline disulfonate) (RuBPs) for total protein analysis (B) To prove the specificity of the staining technique, the CandyCane molecular mass marker (C) consisting of glycosylated and nonglycosylated proteins in alternating order was stained for glycoproteins (left lane, bold italicized digits: after staining with Pro Q Emerald 488) and total protein (right lane: after silver staining) protein molecules and matrix, whether beads (gel filtration) or polyacrylamide ⁄ agarose (gel electrophoresis), may cause false estimations of molecular mass Actually, it is not the molecular mass, but the effective molecular radius, that determines the mobility of FEBS Journal 273 (2006) 3393–3410 ª 2006 The Authors Journal compilation ª 2006 FEBS T Lamkemeyer et al Structure of Daphnia magna hemoglobin A C1 C2 A1 A2 B1 B2 F1 F2 D1 D2 * control B C1 C2 A1 A2 B1 B2 * F1 F2 D1 D2 de-N C * de-O D * de-N/O Fig Mobility shift assays after enzymatic deglycosylation of Daphnia magna hemoglobin (Hb) To determine the type of glycosylation (N- or O-linked), Hb was deglycosylated using different sets of enzymes, and this was followed by two-dimensional gel electrophoresis (A) Control: addition of water instead of enzymes to the reaction mixture (B) Hb incubated with N-glycosidase F (cleavage of N-linked sugars) (C) Release of O-linked sugars (D) Release of N- and O-linked sugars New spots occurring due to the removal of O-linked or O- and N-linked glycans are marked with white circles One spot, which is usually not found in Hb subunit patterns, is labeled with an asterisk a macromolecule during gel filtration [25] The accuracy of SDS ⁄ PAGE is also limited to about 10– 40%, because of, for example, unusual amino acid composition, glycosylation or phosphorylation [21,26,27] Covering the charge of native proteins by Coomassie Brilliant Blue during gel electrophoresis (blue native PAGE; BN-PAGE) resulted in a molecular mass of about 600 kDa in the case of native D magna Hb (data not shown) During analytical ultracentrifugation, protein molecules are freely dissolved and no interactions with a matrix take place Provided that the partial specific volume of a protein analyzed is exactly known, the error of measurement is below 3% [20] Using the partial specific volume of D magna Hb (0.749 mLỈg)1; measured by Ilan et al [14]), sedimentation equilibrium experiments on red D magna Hb (Figs and 2) revealed a molecular mass of 583 kDa The value used for the partial specific volume is similar to those of other invertebrate hemoglobins such as those of Caenestheria inopinata (0.747 mLỈg)1 [28]), Lepidurus apus lubbocki (0.745 mLỈg)1 [29]), Lumbricus terrestris (0.740 mLỈg)1 [30]), Planorbis corneus (0.745 mLỈg)1 [31]), and Triops longicaudatus (0.743 mLỈg)1 [32]) The measured sedimentation coefficients were comparable (between 17.4 and 17.8 S) in all studies on D magna Hb [13,14] (this study) However, the molecular mass suggested by Sugano and Hoshi [13] for D magna Hb (670 kDa) was not determined by sedimentation equilibrium experiments, but was deduced from the measured sedimentation coefficient (17.8 S) As they found identical sedimentation coefficients for D magna and Moina Hb, they concluded that the molecular masses of D magna and Moina Hb (molecular mass: 660–670 kDa, determined by ultracentrifugation [33]) were identical Previous sedimentation equilibrium experiments on D magna Hb [14] gave a molecular mass of 505 ± 35 kDa in a first experiment and one of 483 ± 27 kDa in a second experiment, resulting in an overall molecular mass of 494 ± 33 kDa For the subunits of D magna Hb, the reported molecular mass (about 31 kDa; also determined by ultracentrifugation) was distinctly lower than that from the ESI-MS data of our study (37.715 kDa) or the value deduced from amino acid sequence (36.2 kDa) Accordingly, molecular masses seemed to be generally underestimated in that previous ultracentrifugation study, which may be due to the buffer system used (0.1 m sodium phosphate, pH 6.8) To additionally verify the results from the ultracentrifugation experiments, the molecular mass of the D magna Hb complex was determined by MALLS (Fig 3) Zhu et al [34] have shown that the determination of molecular mass by MALLS could have an error of measurement below 2% The determined values (590.4 ± 11.1 kDa for red D magna and 597.5 ± 49 kDa for pale D magna) agree well with the data from ultracentrifugation (583 kDa for red D magna) The second peak in case of pale D magna Hb could be a dissociation product of Hb, as partial dissociation of D magna Hb at pH 9–10 yielded a 353 kDa fragment determined by gel filtration [35] FEBS Journal 273 (2006) 3393–3410 ª 2006 The Authors Journal compilation ª 2006 FEBS 3399 Structure of Daphnia magna hemoglobin T Lamkemeyer et al Fig GC mass analysis of carbohydrate moieties of Daphnia magna hemoglobin (Hb) Saccharides were released by methanolysis and separated by GC Comparison with a mannose standard (gray-shaded curve) identifies this saccharide as the dominant glycan in D magna Hb A significant increase of galactose and glucose in comparison to the background level [dashed line ¼ maximum total ion current of the blank (inset)] was also observed One peak marked with an asterisk could not be identified Molecular mass of Hb subunits The reported molecular mass of Daphnia Hb didomain subunits varies between 31 and 40 kDa [14,24] Hb proteins of twice this molecular mass have also been reported for crustaceans (Lepidurus [36]; Daphnia pulex [37,38]; Daphnia magna [15]), presumably resulting from subunit dimerization For Triops longicaudatus and Cyzicus Hb subunits, molecular masses between 15 and 21 kDa have been found [32,39] To determine the molecular mass of protein subunits, SDS ⁄ PAGE is often used because of its advantageous properties (rapid and sample-saving) However, a frequently neglected problem arises from the buffer system used A common protocol makes use of a Tris buffer system [19] Weber and Osborn [21] introduced another buffer system (sodium phosphate buffer) For Hb, the number of separated subunits is lower with the Weber and Osborn method than with a Laemmli SDS ⁄ PAGE protocol For the subunits of Daphnia pulex Hb, Dangott and Terwilliger [37] determined five bands using the Laemmli protocol, but only two prominent bands employing the Weber and Osborn protocol In this study on D magna Hb subunits, three bands were found using the Laemmli protocol and only a single band with the Weber and Osborn protocol (Fig 4) Gielens et al [40] have reported that hemocyanin and some other proteins bind only 0.7 g sodium dodecyl sulfate (SDS) per g protein in Tris ⁄ HCl and Tris ⁄ glycine buffers, whereas 1.4 g SDS per g protein is bound in phosphate buffers Rochu and Fine [20] concluded that Tris ions may be attracted by amino acid residues, which are negatively charged at the pH values used in SDS ⁄ PAGE, and accordingly, proteins may not be fully saturated with the detergent, with the consequence that the lower 3400 density of negative charges may not lead to a strictly mass-dependent electrophoretic mobility Moreover, Tris ions may decrease the number of SDS monomers in solution by promoting the formation of micelles Consequently, during Laemmli SDS ⁄ PAGE the separation of polypeptides is influenced not only by the molecular mass but also by the degree of coverage of surface charges, which depends on the amino acid composition of proteins Phosphate buffers, however, not specifically interfere with the binding of SDS to polypeptide chains, permitting their saturation with SDS [20] Accordingly, a Weber and Osborn SDS ⁄ PAGE protocol may better reflect molecular masses The deviating electrophoretic mobility of Hb subunits in Tris-buffered gels despite similar molecular masses may also be caused by differences in their primary structure Actually, it has been reported for a variant of serum prealbumin that a single-point mutation, which results in methionine instead of threonine, leads to a different migration behavior and an apparently lower mass of the variant prealbumin form during SDS ⁄ PAGE [41] In addition, glycosylated proteins are known to show unusual migration behavior during gel electrophoresis [27] The observed difference in migration behavior of D magna Hb subunits in buffer systems according to Laemmli [19] compared to Weber and Osborn gels [21] may therefore indicate a posttranslational modification of Hb subunits (see below) To determine the molecular mass of D magna Hb subunits exactly, ESI-MS experiments were performed After deconvolution of the raw spectrum, a single peak was high above background level The molecular mass of this major component was calculated to be 37.715 kDa, including two heme groups The difference between the detected mass and the molecular masses calculated from amino acid sequences may be caused by FEBS Journal 273 (2006) 3393–3410 ª 2006 The Authors Journal compilation ª 2006 FEBS T Lamkemeyer et al Structure of Daphnia magna hemoglobin A B * C D * * E F Fig Electron micrographs (A–D) and three-dimensional reconstruction (E–F) of Daphnia magna hemoglobin (Hb), which was purified by gel filtration and negatively stained with 2% uranyl acetate for electron microscopy or was purified by chromatofocusing and negatively stained with 5% ammonium molybdate containing 0.1% trehalose on holey carbon grids for three-dimensional reconstructions (A) Hb of pale animals (B) Magnification of a section in (A) (C) Hb of red animals (D) Magnification of a section in (C) (Hb molecules showing the clover-leaf structure are indicated by arrows Ferritin molecules are indicated by asterisks Bar, 50 nm.) (E) After a multireference alignment, matchable images were treated by multivariate statistical analysis Three examples of the resulting class averages are shown in (E) which correspond approximately to the views of the three-dimensional reconstruction (F), showing the characteristic clover-leaf structure of red D magna Hb: ˚ presumed top (F, center) and two side views (F, left and right) of the three-dimensional reconstruction of D magna Hb at sub-30 A resolution [Note different scaling of molecules in (E) and (F).] In the center of the Hb molecule, a single mass was observed (F, center) (White areas represent molecular masses.) glycosylation (see below) Considering that the relationship between Hb genes and Hb subunits is not yet completely determined, this post-translational modification may also be the reason for the discrepancy between the result of one dominating component in ESI-MS meas- urements and the expected number of six gene products (i.e the maximum number assuming expression of all known Hb genes [12]) or at least four different subunit types that can be detected as main components by twodimensional electrophoresis in Hb from red animals FEBS Journal 273 (2006) 3393–3410 ª 2006 The Authors Journal compilation ª 2006 FEBS 3401 Structure of Daphnia magna hemoglobin T Lamkemeyer et al [42], respectively The subunits contributing to the Hb of red D magna are mainly A, B, D and F, and accordingly, more than one main subunit type would have been expected in the ESI-MS spectrum Although three of these subunits (A, B and D), comprising almost 70% of the total subunits present in Hb from red Daphnia [42], seem to be of similar size in both gel electrophoresis systems used, their expected size differences should have been resolved by MS Again, glycosylation may contribute to the observed discrepancy Different glycan structures seem to generate a complex mixture of masses leading to a variety of charge states, which are difficult to resolve by ESI-MS Actually, under the experimental conditions used for ESI-MS, only a single subunit type could be detected reliably It is most likely that this phenomenon may also be affected by electrospray ionization suppression, which is typically observed in ESI-MS of extracts from biological samples [43] However, the value measured was used to calculate the number of subunits in the native aggregate Based on the molecular mass of macromolecule and subunits, the number of subunits per macromolecule can be calculated For red D magna, the molecular mass of the native Hb complex (ultracentrifugation, 583 kDa; MALLS, 590.4 kDa, red, and 597.5 kDa, pale) divided by the subunit mass (ESI-MS: 37.715 kDa) results in 16 subunits per Hb macromolecule, independent of the method applied for the determination of the Hb complex mass Because of an underestimation of both macromolecule and subunit mass (see above), Ilan et al [14] came to the same conclusion of 16 subunits per D magna Hb macromolecule Hb glycosylation The mean molecular mass of D magna Hb subunits calculated from the nucleotide and the derived amino acid sequences (Hb genes dmhb1–dmhb4 [11,12]) is 36.207 ± 0.027 kDa Accordingly, the experimentally determined value for the predominant peak found in ESI-MS is 275 Da higher (red D magna Hb subunits) than the calculated value Actually, the subunits of red D magna Hb were found to be glycosylated (Fig 6) using the Pro-Q Emerald 488 stain Although Pro-Q Emerald 300 dye is capable of detecting proteins with a higher sensitivity (300–1 ng) and a broader dynamic range (500–1000-fold), the Pro-Q Emerald 488 dye is the most sensitive dye for detection of glycoproteins in gels, when a laser-based gel scanner such as the FLA-2000 is used for imaging [44] For proteins with a high carbohydrate content (9–42%), the detection sensitivity is reported to be between and ng with a linear dynamic range of 128–255-fold Even glycoproteins with lower 3402 carbohydrate content (3–7%) were successfully detected (sensitivity 19 ng, linear dynamic range 64-fold) [44] Protein isoforms that have the same amino acid sequence, but different glycosylation profiles, often appear as trains of spots on two-dimensional separations, which can differ in pI and ⁄ or molecular mass [45] Hence, in a next step, enzymatic deglycosylation was performed using different sets of N- and O-glycosidases to remove only one type of glycan or completely remove all common glycans (Fig 7) Additional spots occurred when the Hb samples were treated with enzymes releasing specifically O-linked glycans or with all enzymes, respectively After treatment with N-glycosidase F, which cleaves only N-linked glycans, additional spots were not found These results indicate that the carbohydrates of Hb are O-linked This is in accordance with amino acid sequence analyses for determination of the glycosylation type Whereas no N-linked glycosylation sites are present in D magna Hb [22], analyses using the NetOGlyc 3.1 server [23] revealed 11–12 potential O-linked glycosylation sites in Hb subunits exclusively in the pre-A segments This server is reported to correctly predict 76% of glycosylated residues and 93% of nonglycosylated residues It is intended for extracellular proteins and can predict sites for completely new proteins without losing its performance [23] In order to identify the carbohydrates bound to Hb subunits, saccharides were released by methanolysis and analyzed by GC followed by MS (Fig 8) Mannose was identified as the dominant sugar, whereas galactose and glucose were found in smaller quantities Presently, directly O-linked mannose cannot be removed enzymatically However, the occurrence of additional spots in two-dimensional gel electrophoresis (Fig 7) indicates (a) that some mannose is indirectly bound to the protein, allowing a cleavage, and ⁄ or (b) that the mobility shifts originate from the removal of galactose and glucose The fact that the spots A–D and F were still present after h of incubation with deglycosylating enzymes can be explained by incomplete deglycosylation or the presence of mannose directly bound to the proteins Although glycosylation is a common post-translational modification of the respiratory pigments of invertebrates [46], Hb subunits of Daphnia pulex were reported to be not glycosylated [38] However, the analysis of distribution of glycosylation among taxonomic groups showed that even closely related species may not necessarily share close similarities in their glycan diversity [47] In conclusion, all experimental results concerning the molecular mass of Hb subunits can be explained FEBS Journal 273 (2006) 3393–3410 ª 2006 The Authors Journal compilation ª 2006 FEBS T Lamkemeyer et al satisfactorily in the light of glycosylation It can be assumed that the molecular mass found by ESI-MS, which was higher than the values calculated from the amino acid sequences, is due to addition of a glycan moiety Although O-linked glycans are relatively small compared to N-linked glycans, they can effectively cover charges at the surface of a protein Therefore, this modification is suggested to influence the migration behavior of Hb subunits during gel electrophoresis by interfering with binding of SDS, leading to three bands or a single band during one-dimensional separations, depending on the buffer system used (Tris versus phosphate buffer) As indicated by the alteration of Hb subunit patterns after two-dimensional gel electrophoresis due to enzymatic deglycosylation, the presence of glycans influences isoelectric focusing of subunits as well, supporting the idea that glycans affect the net charge of Hb subunits Quaternary structure Owing to its large size, the gross structure of a crustacean Hb molecule can be assessed by electron microscopy Ilan et al [14] performed electron microscopy of D magna hemolymph proteins and detected a small number of ring-like projections (diameter: approximately 14 nm), which they considered to be Hb On the basis of these electron micrographs and assuming 16 subunits per macromolecule, they discussed structural models for the multisubunit assembly, including a cyclic and two dihedral structures containing either both heterologous and isologous bonds or two types of isologous bond Based on pH dissociation experiments, they favored a dihedral symmetry, D8, containing both heterologous and isologous bonds This model consisted of 16 subunits grouped in two layers stacked in an eclipsed orientation, the eight subunits of each layer occupying the vertices of a regular eightsided polygon As this model was based on the small number of ring-like projections, it may, however, not be valid Reporting difficulties in the electron microscopy of Daphnia Hb, the authors applied a contrast enhancement technique (rotational photography) to improve the resolution of objects with a rotational symmetry However, the major fraction of other macromolecules visible in the preparation [14] was not resolved by this technique All Hb-utilizing animals possess ferritin as iron-storage protein in the hemolymph In Nereis virens hemolymph, for example, a small number of ring-like molecules (molecular mass about 500 kDa; diameter approximately 10 nm diameter) were also detected apart from a major fraction of Hb [48] This minor fraction was identified as ferritin Structure of Daphnia magna hemoglobin particles, due to their electron-dense iron cores Accordingly, the minor fraction of ring-like particles within the hemolymph of D magna (Fig 9A,C,D) may also represent ferritin molecules and not Hb Electron micrographs of the hemolymph of red D magna revealed predominant structures of tetrameric symmetry (clover-leaf structures; see Fig 9C,D, arrows), which are supposed to represent red D magna Hb macromolecules In pale D magna hemolymph (Fig 9A,B), these specific structures were present only to a much lower degree As red D magna Hb is composed of a different set of subunits than pale D magna Hb [8,42], it is possible that both Hb isoforms also display a different quaternary structure After purification of red D magna hemolymph by chromatofocusing [removal of ring-like molecules (‘ferritin’), but retention of high Hb quantities], numerous different views of the Hb molecules were obtained by negative staining of these molecules across holey carbon grids, allowing a three-dimensional reconstruction by single-particle analysis Negative staining techniques have been widely used to obtain images of macromolecules with high contrast Although the stain may invade aqueous channels, structural information is basically limited to the shape of a macromolecule, which may also be distorted due to air drying [49] Despite these restrictions, this method is used in highresolution electron microscopy of macromolecules as an important first step in identifying characteristic views and has been used with great success in numerous computer reconstructions of viruses and other large macromolecular assemblies [49] The gross structure of D magna Hb turned out to be much more complex than hitherto suggested (Fig 9E,F) Consisting of 16 subunits with a molecular mass of 37.715 kDa, the macromolecule has the overall shape of a square Three-dimensional reconstructions of subunits revealed by homology modeling [42] were fitted into that of the macromolecule, but the resolution of the quaternary structure model made it difficult to determine the exact positions of individual subunits in the multimer However, tight packing of subunits was evident (data not shown) Each side of the square is approximately 12 nm in length, and a single mass in the center of the macromolecule was observed (Fig 9F, center) This apparent mass, however, may be an artefact, because in conventional negative staining, the contrast medium may localize preferentially on the surface of a molecule, leaving its center more or less free of stain In negative staining, protein masses are visualized because of their lower electron density compared to the contrast medium Therefore, the incomplete filling of the molecule’s center with FEBS Journal 273 (2006) 3393–3410 ª 2006 The Authors Journal compilation ª 2006 FEBS 3403 Structure of Daphnia magna hemoglobin T Lamkemeyer et al ammonium molybdate ⁄ trehalose leads to a low electron density, which thus may generate the impression of a mass In side views (Fig 9F, left and right), the macromolecule appears as a compressed sphere with a height of about nm However, flattening of the macromolecule may have originated from negative staining and, in reality, it may be more spherical A unique feature conserved in D magna Hb chains is the presence of an unusual N-terminal extension This extension is reminiscent of the 18-residue C-terminal extension found in the two-domain Hb chain of a parasitic nematode, Ascaris suum, the so-called polar zipper sequence Initially, the extensions of individual subunits were suggested to act as cement between the subunits in the center of the octamer [50], and an equivalent function of the pre-A segments was discussed for D magna Hb subunits [11] Detailed analysis of A suum Hb finally revealed that these extensions not play a role in stabilization of the quaternary structure once formed, but rather function as intramolecular chaperones, aiding assembly of the nascent Ascaris Hb octamer [51] In fact, the relevance of the pre-A segments in D magna Hb subunits seems to be determined by glycosylation Amino acid sequence analyses using the NetOGlyc 3.1 server revealed possible O-glycosylation sites only in the pre-A segments Since O-glycosylation is mainly a posttranslational and postfolding event, which affects the structure of a protein by determining the secondary, tertiary and quaternary structure (aggregation, multimerization) and occurs only at surface-exposed serine and threonine residues [52,53], it can be assumed that the pre-A segments are located on the surface of the molecule Concerning its biological impact, glycosylation is suggested to be important for the folding, oligomerization and transport of proteins and is reported to confer protease and heat resistance to glycoproteins [53] as well as to increase the solubility of proteins [52], as in the case of vitellogenin from the decapod crustacean Cherax quadricarinatus [54] A localization of the pre-A segments in the center of the Hb macromolecule is also not supported by the three-dimensional reconstruction, as the apparent central mass is possibly an artefact Instead, the existing data indicate that these segments may be involved in adjusting important biophysical properties of the native molecule For a definite elucidation of the fine structure of D magna Hb, however, three-dimensional reconstructions based on macromolecules prepared by cryo-electron microscopy have to be established The exceptional characteristic of D magna of pursuing a regulatory strategy to cope with changing oxygen and temperature conditions by strong variations in the quantity (concentration) and quality (subunit composi3404 tion and oxygen affinity) of its respiratory protein [55,56] has made D magna an example of molecular adaptation or acclimatization The information available on D magna Hb gene promoters and open reading frames as well as protein subunits and subunit composition [2,4,8,11,12,42] offers the chance to relate the structure and function of this interesting molecule The present study has contributed to this approach: (a) the determination of the molecular mass of the native Hb complex and its subunits as well as the number of subunits in the macromolecule; (b) evidence for glycosylation of their pre-A segments, which may support macromolecule assembly and provide additional functional qualities; and (c) a first reconstruction of the native Hb complex Experimental procedures Animals and Hb samples Female D magna Straus organisms were initially obtained from the Staatliches Umweltamt, Munster, Nordrheină Westfalen, Germany and kept in laboratory culture for many years They were raised in iron-enriched (1 mgỈL)1 ferrous iron [57]), standard culture medium [58] at 20 °C ‘Pale’ D magna, i.e those with a low Hb concentration, were raised under normoxic conditions (oxygen partial pressure: 20.7 kPa) in a 40 L aquarium by gentle ventilation with room air ‘Red’ D magna, i.e those with an elevated Hb concentration, were obtained by reducing the air pressure inside closed L glass vessels to 15% of atmospheric pressure (oxygen partial pressure: 3.1 kPa) Animals were fed daily with algae (Desmodesmus subspicatus) The media were changed every weeks Hemolymph was obtained by cutting off the animals’ second antenna and collecting it with a lL capillary tube (minicaps; Hirschmann, Eberstadt, Germany) Determination of Hb concentration was carried out as described previously [5,6] Gel filtration was performed on a Superdex 200 column (10 mm · 300 mm) equilibrated with 50 mm Tris ⁄ HCl, pH 8.0 (for details, see [24]) Separation of Hb isoforms by chromatofocusing was performed on a MonoQ HR10 ⁄ 10 column (Pharmacia, Uppsala, Sweden) using a pH gradient between pH 8.1 and 4.2 (for details, see [42]) Analytical ultracentrifugation Sedimentation velocity and sedimentation equilibrium experiments were carried out in a Beckman Optima XL-I analytical ultracentrifuge (Palo Alto, CA) using an An-50Ti rotor Velocity runs were conducted at 20 °C at 116 480 g using 0.2 mgỈmL)1 Hb of red D magna in 10 mm ammonium acetate buffer (pH 6.7) Sample cells with 12 mm double-sector charcoal-filled epon centerpieces and quartz FEBS Journal 273 (2006) 3393–3410 ª 2006 The Authors Journal compilation ª 2006 FEBS T Lamkemeyer et al windows were used for all experiments The absorbance of the cells was scanned at 280 nm every during the run Velocity data were analyzed using the method of van Holde and Weischet [59], as implemented in the program ultrascan 6.0 Sedimentation coefficients (s20,w) were corrected for 20 °C and water Sedimentation equilibrium runs were conducted at °C at 4660 g, 5900 g and 7280 g using protein concentrations of 0.2 mgỈmL)1 Hb of red D magna in ammonium acetate buffer Sample cells with six-channel charcoal-filled epon centerpieces and quartz windows were used for all experiments Samples were run for at least 24 h at each speed The absorbance of the cells was scanned at 280 nm, and it was assumed that equilibrium was achieved when scans taken at intervals of h showed no significant change Equilibrium data were analyzed with the program ultrascan 6.0, employing the global fit routine to integrate data taken at different speeds For all calculations, a partial specific volume of 0.749 cm3Ỉg)1 was used, as published for D magna Hb by Ilan et al [14] MALLS MALLS measurements were performed with a DAWN EOS system (Wyatt Technology Corp., Santa Barbara, CA) directly on-line with an HPLC system (Waters, Milford, MA) Gel filtration was performed on a · 30 cm Superose 6-C column (fractionation range from to 5000 kDa; Pharmacia) equilibrated with 0.1 m Tris ⁄ HCl buffer, pH 7.0 The eluate (flow rate 0.5 mLỈmin)1) was simultaneously monitored with a photodiode array detector (Waters 2996) and a refractive index detector (Waters 2414) The MALLS instrument was placed directly before the refractometer to avoid back pressure on the instrument’s cell Chromatographic data were collected and processed with the astra software (Wyatt Technology Corp.) The Zimm fit method was used for molecular mass determinations [60] In this method, the variation rate of the refractive index as a function of concentration, dn ⁄ dc, was set to 0.193 mLỈg)1 (typical for human Hb [61]) BSA monomer (Sigma, St Louis, MO) was used for normalizing various detectors’ signals relative to the 90° detector signal Hb of red and pale D magna (concentration 0.14– 0.93 mgỈmL)1; dissolved in 10 mm ammonium acetate buffer, pH 6.7) purified by gel filtration or chromatofocusing was used The sample was kept at °C until the elution, which was performed at ambient temperature Analysis of subunit molecular mass by gel electrophoresis Two methods of molecular mass analysis by SDS ⁄ PAGE were employed The multiphasic buffer system according to Laemmli [19] was used with 3.6% stacking gels (0.125 m Tris, 0.1% SDS, pH 6.8) and 12% separating gels (0.559 m Tris, 0.1% SDS, pH 8.8) Electrophoresis buffer consisted Structure of Daphnia magna hemoglobin of 25 mm Tris, 250 mm glycine, and 0.1% SDS The continuous buffer system according to Weber and Osborn [21] was used with a 12% separating gel (0.1 m sodium phosphate, 0.1% SDS, pH 7.1) without a stacking gel Electrophoresis buffer consisted of 0.1 m sodium phosphate and 0.1% SDS (pH 7.1) Two micrograms of pale and red D magna Hb dissolved in sample buffer [0.5 m Tris ⁄ HCl, 3.5% SDS, 10% glycerin, pH 6.8, containing bromophenol blue, and 0.1 m sodium phosphate, 1% SDS, 1% dithiothreitol, 30% glycerin, pH 7.1, containing bromophenol blue] were incubated at 95 °C for 10 and loaded onto the gel Minigels (about 85 · 60 · mm) were run at a current of 15 mgel)1 for 70 min, 20 mgel)1 for 30 and 30 mgel)1 for 150 (Tris gel) and 105 (sodium phosphate gel), respectively Gels were fixed in 20% trichloroacetic acid for 20 and stained in a solution containing final concentrations of 0.1% Coomassie R250, 30% methanol, and 10% acetic acid for 30 Destaining was performed by diffusion in a solution of 25% methanol and 10% acetic acid on an orbital shaker, until protein bands were clearly visible ESI-MS ESI-MS was performed under denaturing conditions to determine the molecular masses of the subunits of D magna Hb Electrospray data were acquired on an ESI-Q-TOF (QTOF II; Micromass, Altrincham, UK) mass spectrometer scanning over the m ⁄ z range 600–5000 at 1.5 sỈscan)1 Data were accumulated over to produce the final spectrum Spectra were obtained on an Ouest-genopoleÒ sequencing ⁄ genotyping platform setup at Roscoff Samples at a concentration of 0.5 lgỈlL)1 in acetonitrile ⁄ water (1 : 1, v ⁄ v) containing 0.2% formic acid were introduced into the electrospray source at lLỈmin)1 The cone voltage (counter electrode to skimmer voltage) was set to 60 V Mass scale calibration employed the multiply charged series from horse heart myoglobin (16951.5 Da; Sigma Cat No M-1882) The raw ESI-MS spectra were deconvoluted using the maximum entropy based software (maxent I) supplied with the instrument [62], in order to find the approximate mass of each subassembly (monomer and polymer) Hb fractions obtained by chromatofocusing of crude extracts of red and pale D magna were desalted by washing with Milli-Q water and centrifugation in Amicon kDa filter devices (Millipore, Bellerica, MA) 10 times at °C, before ESI-MS analysis Molecular masses are based on the atomic weights of the elements given by IUPAC Analysis of Hb glycosylation and phosphorylation Two-dimensional gel electrophoresis of D magna Hb Hb of red D magna purified by gel filtration [24] was diluted in rehydration solution containing m urea, m FEBS Journal 273 (2006) 3393–3410 ª 2006 The Authors Journal compilation ª 2006 FEBS 3405 Structure of Daphnia magna hemoglobin T Lamkemeyer et al thiourea, 2% CHAPS, 0.5% pharmalytes ⁄ 10, and 65 mm dithiothreitol to a final volume of 125 lL Immobilized pH gradient (IPG) strips (7 cm, pH 3–10, Biorad, Munich, Germany) were actively rehydrated (50 V) for 12 h After focusing (maximum 4000 V, 22 000 Volthours), the IPG strips were equilibrated for 15 in m urea, 2% (w ⁄ v) SDS, 30% (w ⁄ v) glycerol, 50 mm Tris, pH 8.8, containing 1% dithiothreitol; this was followed by incubation in the same solution, but with dithiothreitol replaced by 4% iodoacetamide, for an additional 15 The IPG strips were then placed on 12% separating gels (thickness mm) in the second dimension (15 mgel)1 for 15 min, 30 mgel)1 for 70 min) The gels were silver stained or stained for glycoproteins, followed by staining with RuBPs (see below) Glycoprotein staining Hb of red D magna (100 lg, Hb purified by gel filtration) was subjected to two-dimensional gel electrophoresis For demonstration of glycosylated proteins, the Pro-Q Emerald 488 Glycoprotein Gel and Blot Stain kit (Molecular Probes, Gottingen, Germany) was used ă according to the manufacturers protocol Glycoprotein staining was followed by total protein staining using the fluorescence stain RuBPs, synthesized according to [63], and staining was performed according to [64] or via silver staining To prove the specificity of the staining technique, the CandyCane glycoprotein molecular mass standard (Molecular Probes) ranging from 14 to 180 kDa was used When separated by electrophoresis, the CandyCane standards appear as alternating bands corresponding to glycosylated and nonglycosylated proteins Fluorescent signals were detected using an FLA-2000 laser scanner (Fuji Photo, Tokyo, Japan) Enzymatic deglycosylation For removal of carbohydrate moieties, the Glycoprotein Deglycosylation Kit (Merck Biosciences, Schwalbach, Germany), which contains all enzymes and reagents needed to remove all N-linked, all simple O-linked, and virtually all complex O-linked oligosaccharides from glycoproteins, was used according to the manufacturer’s protocol To analyze the type of glycosylation (N- or Olinked sugars), 50 lg of Hb purified by gel filtration [24] was incubated either with N-glycosidase F (removal of N-linked sugars), or a mix of endo-a-N-acetylgalactosaminidase, a2–3,6,8,9-neuraminidase, b-1,4-galactosidase, and b-N-acetylglucosaminidase (removal of O-linked sugars), or a mixture of all five enzymes (removal of N- and O-linked sugars), for h at 37 °C A sample in which water was added instead of enzymes served as a control Deglycosylation was monitored by mobility shift assays using separation of proteins by two-dimensional gel electrophoresis 3406 GC ⁄ MS For identification of carbohydrates, crude extracts of D magna were purified by gel filtration [24] Eluates containing Hb were desalted by washing with water and centrifuging using Nanosep spin columns (MWCO 10 kDa, Pall Life Sciences, Ann Arbor, MI) and concentrated to dryness in a rotational vacuum concentrator (RVC 2-25, Christ, Osterode am Harz, Germany) An eluate of the gel filtration column eluting at the same time as the Hb was lyophilized and analyzed by GC for the presence of saccharides originating from the used buffer and ⁄ or from the column material (blank) Carbohydrates were cleaved by methanolysis (0.6 m HCl in absolute methanol for 18 h at 70 °C), and the methyl glycosides were trimethylsilylated [100 lL of : BSTFA (bis(trimethylsilyl) trifluoroacetamide) ⁄ pyridine for h at 60 °C] The derivatization solution was directly analyzed by GC ⁄ MS (Agilent 6890 ⁄ 5973, Agilent, Waldbronn, Germany) on a DB-5 (J+W, Folsom, CA) capillary [25 m · 0.25 mm, film thickness (df) ¼ 0.25 lm] Individual saccharides were identified on the basis of their mass spectra, their retention times and characteristic ratio of the a- and b-anomers Analysis of phosphorylation For the detection of possible phosphorylation, Hb of red D magna (purified by gel filtration [24]) was subjected to one-dimensional gel electrophoresis Staining of phosphorylated proteins was performed using the Pro Q Diamond Phosphoprotein Stain Kit (Molecular Probes) according to the manufacturer’s protocol, followed by total protein staining using the fluorescence stain RuBPs In addition, proteins were transferred onto PVDF membranes (Schleicher & Schuell, Dassel, Germany) after one-dimensional gel electrophoresis using a semidry western blot apparatus (Peqlab, Erlangen, Germany) and incubated separately with antibodies directed against phosphotyrosine (monoclonal anti-phosphotyrosine, clone PY20, Sigma, Steinheim, Germany) or phosphoserine residues (phosphoserine antibody Q5, Qiagen, Hilden, Germany) Antibody binding was detected using alkaline phosphatase For MS, Hb of red animals was digested with trypsin and phosphorylated peptides were enriched using immobilized metal affinity chromatography with Fe3+, Ga3+ and Zr4+ as well as affinity chromatography with TiO2 Nano-LC-MS ⁄ MS-measurements were accomplished on a nano-HPLC (Ultimate system, Dionex GmbH, Idstein, Germany) coupled to a linear ion trap (QTrap 4000, Applied Biosystems, Framingham, MA) operating in precursor ion scanning mode The reporter ion mass of 79 Da for the phosphate group in negative mode was used for detection of Ser- ⁄ Thr-phosphorylated peptides, and the reporter ion mass of 216.043 Da was used for the immonium ion of phosphotyrosine in positive scanning mode FEBS Journal 273 (2006) 3393–3410 ª 2006 The Authors Journal compilation ª 2006 FEBS T Lamkemeyer et al Electron microscopy and three-dimensional reconstruction Specimen preparation Conventional negative staining with 2% uranyl acetate of D magna Hb purified by gel filtration [24] was performed by the single-droplet procedure as described by Harris and Horne [65] For single-particle image processing, D magna Hb purified by chromatofocusing (for details, see [42]) was prepared using the holey carbon negative staining method, as described by Harris and Scheffler [66] The Hb concentration was optimized by direct electron microscopic assessment of the distribution of particles embedded within the thin film of negative stain (5% ammonium molybdate + 0.1% trehalose) across the holes of the grid Structure of Daphnia magna hemoglobin thank Professor Maier, University of Tubingen, for the ă synthesis of ruthenium II tris(bathophenanthroline disulfonate) The excellent help of Dr Gnau and Dr Buckenmaier, Proteom Centrum Tubingen (PCT), ă concerning molecular weight determinations by MS is gratefully acknowledged We also thank Dr Sickmann and Jorg Reinders from the Protein Mass Spectromeă try and Functional Proteomics Group (Rudolf Virchow Centre, University of Wurzburg) for their ă expertise in analysis of phosphorylation by MS Franck Zal and Morgane Rousselot would like to ´ thank the Conseil Regional de Bretagne and CNRS for their financial supports The Proteom Centrum Tubingen is supported by the Ministerium fur Wisă ă senschaft und Kunst, Landesregierung Baden-Wurtă temberg Electron microscopy A Tecnai 12 transmission electron microscope (with LaB6 filament) and a Zeiss EM900 were used Electron micrographs were recorded on Kodak 4489 electron image film The instrumental magnification was 49 000-fold Digitization and image processing Electron micrographs were digitized using a Primescan drum scanner (Heidelberger Druckmaschinen, Heidelberg, Germany) at a resolution of 2700 d.p.i The resulting reso˚ lution ratio was 2.855 pixel)1 in the digitized micrograph Image processing was performed within the imagic software package (Image Science GmbH, Berlin, Germany) Single-particle analysis was started with about 7500 single particles from 10 different electron micrographs Because of an unequal background, a bandpass filter was used to buffer noise and remove brightness shifts from the single-particle images For equalization of transverse and rotational distortions, a multireference alignment (MRA) was performed [67] The aligned and matchable images could be treated by multivariate statistical analysis (MSA) [67,68] The resulting class averages, each based on about 12 single particles, showed randomly orientated molecules with much better quality than the original images For three-dimensional reconstruction, a Euler search was performed using different symmetry features, to calculate the relative orientation (Euler angles) of the class images From determined relative spatial angles, the three-dimensional reconstruction was conducted The process was repeatedly performed by iterative improvement of the reconstruction by quality estimation during every single step of the process Acknowledgements The technical assistance of Marita Koch, Ina Buchen and Nicole Sessler is gratefully acknowledged We References Weber RE & Vinogradov SN (2001) Nonvertebrate hemoglobins: functions and molecular adaptations Physiol Rev 81, 569–628 Gorr TA, Cahn JD, Yamagata H & Bunn HF (2004) Hypoxia-induced synthesis of hemoglobin in the crustacean Daphnia magna is hypoxia-inducible factor-dependent J Biol Chem 279, 36038–36047 Lamkemeyer T, Zeis B & Paul RJ (2003) Temperature acclimation influences temperature-related behaviour as well as oxygen-transport physiology and 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Fig The influence of the buffer system used for gel electrophoresis on the band pattern of pale and red Daphnia magna hemoglobin (Hb) The hemolymph of pale (2.0 lg of Hb) and red (2.0 lg of Hb)... experiments (Fig 2) resulted in a molecular mass of 583 kDa for the native Hb complex from the hemolymph of red D magna Owing to their small quantity, the molecular mass of slower-sedimenting proteins