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Molecular adaptation of Daphnia magna hemoglobin
Micron 35 (2004) 47–49 www.elsevier.com/locate/micron
Molecular adaptation of Daphnia magna hemoglobin Bettina Zeis*, Tobias Lamkemeyer, Ru¨diger J. Paul Institute of Zoophysiology, Westfa¨lische Wilhelms-Universita¨t, Hindenburgplatz 55, Mu¨nster 48143, Germany
Abstract Daphnia magna responds to changing environmental conditions impeding aerobic metabolism by synthesizing hemoglobin of adequate quantity and quality to maintain oxygen supply of the tissues. Hemoglobin subunit composition and its oxygen affinity were analyzed as a function of temperature as well as depending on the oxygen partial pressure of the medium. Additionally, the time course of acclimation to hypoxia was studied. Correlating structural and functional changes, the role of individual subunits for the increase in oxygen affinity is discussed. q 2003 Elsevier Ltd. All rights reserved. Keywords: Dalphnia magna; Hemoglobin; Subunit composition; Two-dimensional electrophoresis; Oxygen affinity; Oxygen supply
The ability of Daphnia magna to increase hemoglobin concentration in the hemolymph at low oxygen concentration is a well-known phenomenon (Fox et al., 1951; Kobayashi and Hoshi, 1982). The di-domain multi-subunit protein of approximately 500 kDa (Weber and Vinogradov, 2001) is coded by at least four Hb genes (Kimura et al., 1999) and synthesized in the fat cells and the epipodite epithelia (Goldmann et al., 1999). The oxygen-dependent Hb induction was studied at the protein level by analyzing the alterations of functional properties and the structural changes they are based upon. Hemoglobin concentration and its oxygen affinity were studied using spectrophotometric analysis of ml-samples (Pirow et al., 2001) and its composition of different subunits was studied by two-dimensional electrophoresis of individual hemolymph samples (Zeis et al., 2003a,b). For studies of Hb induction as a function of oxygen concentration in the medium, animals were kept in long-term cultures at normoxic, hyperoxic and six different hypoxic conditions. Hb concentration rises exponentially with decreasing oxygen concentration (Fig. 1, bottom). Along with this quantitative change, the functional quality is also altered resulting in enhanced oxygen affinity (Fig. 1, top). Analysis of subunit composition reveals a complex expression pattern. Subunit DmHbB is present in high amounts at all oxygen concentrations studied. Subunits DmHbE and DmHbG are present only at high oxygen concentrations and decrease under hypoxia. * Corresponding author. Tel.: þ49-251832-3851; fax: þ49-2518323876. E-mail address: [email protected] (B. Zeis). 0968-4328/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2003.10.015
Among the subunits induced by hypoxia, subunits DmHbC and DmHbF already increase at moderate hypoxia. Increased amounts of subunits DmHbA and DmHbD are observed only at severe hypoxia of 4.1 kPa and below (Fig. 1, bottom). Hb oxygen affinity is not changed within the wide range of 73.6– 7.2 kPa, half-maximal saturation being achieved at an oxygen partial pressure of about 1 kPa. At severe hypoxia, rising amounts of subunits DmHbA, DmHbD and DmHbF coincide with elevated oxygen affinity of the hemoglobin (P50 values of 0.5 kPa were measured in Hb from animals kept at a Po2 of 2.1 kPa; Zeis et al., 2003b). For analysis of the time-course of hemoglobin expression animals from normoxic culture were transferred to hypoxic medium (Po2 of 3.1 kPa). Within an 11-day period Hb concentration, its affinity and subunit composition were studied daily. Newly synthesized hemoglobin was detected within eighteen hours. A pronounced increase of hemoglobin concentration occurred around the third and fourth day and after 6 days, a constantly elevated level of hemoglobin concentration was reached. Increases in affinity can be aligned with these quantitative changes. Twodimensional electrophoresis reveals the domination of subunits DmHbB, DmHbE and DmHbG in Hb of normoxically raised animals. Upon the transfer to hypoxic medium, the newly expressed Hb subunits show a transient increase of subunit DmHbC and after 11 days the Hb pool is dominated by subunits DmHbD and DmHbF. The dynamics of Hb oxygen affinity elevation coincides with the increase of the latter subunits. Subunit DmHbA is not present at any time (Zeis et al., 2003a). Acclimation of D. magna to different temperatures (10, 20, 30 8C) also results in an increase of Hb concentration and
48
B. Zeis et al. / Micron 35 (2004) 47–49
Fig. 1. Functional and structural characteristics of D. magna hemoglobin as a function of oxygen tension in the culture medium. The subunit composition of D. magna hemoglobin (bottom) changes along with an increase in concentration at lower oxygen availability. The oxygen affinity is unchanged in a wide Po2 range and only at severe hypoxia P50 decreases. The elevated oxygen affinity is correlated with an induction of Hb subunits DmHbA, DmHbD and DmHbF.
affinity along with increased temperature. Since oxygen solubility is inversely correlated to temperature, changes of these parameters can be compared to the alterations as a function of oxygen concentration. The effect of temperature on Hb concentration and affinity is only partly explained by the change in oxygen availability. Subunit composition of the expressed hemoglobin species reveals an increase of subunit DmHbA at elevated temperature, its presence being correlated with the observed increase in oxygen affinity (Lamkemeyer et al., 2003).
The effects of oxygen and temperature on each individual subunit are summarized in Table 1 along with the apparent values of molecular mass and pI determined from 2D gels. Analysis of similarities and differences in subunit composition and resulting functional characteristics in these three sets of experiments can be used to correlate the presence of individual subunits with effects on oxygen affinity. From subunit composition changes as a function of oxygen, the three potential candidates responsible for increased oxygen affinity are DmHbA, DmHbD and DmHbF. It seems
Table 1 Effect of environmental changes on the contribution of individual subunits of D. magna hemoglobin to the Hb pool Subunit
Molecular weight (kDa)
pI
Oxygen conditions for induction
Hypoxic induction
Dynamics
Induction at high temperature
DmHbA DmHbB DmHbC DmHbD DmHbE DmHbF DmHbG
36.2 ^ 0.9
8.1 ^ 0.2 7.5 ^ 0.2 7.1 ^ 0.1 6.8 ^ 0.2 6.5 ^ 0.1 6.7 ^ 0.2 7.2 ^ 0.1
Severe hypoxia All oxygen conc Moderate hypoxia Severe hypoxia Normoxia, hyperoxia Moderate hypoxia Normoxia, hyperoxia
þþ þ þ þ þþ þ – þþ –
Slow
þ þþ þ þ
40.6 ^ 1.6 37.9 ^ 1.2
Transient Fast Fast
2 þ 2
B. Zeis et al. / Micron 35 (2004) 47–49
that in the case of elevated temperature, the change in affinity is brought about by the increase in the share of subunit DmHbA alone, since only minor changes in DmHbD and DmHbF contribution to the Hb pool are observed. However, this subunit (DmHbA) is not induced within the time-course studied, although a considerable increase in affinity takes place. Here, subunits DmHbD and DmHbF are increased most, so a higher oxygen affinity of these subunits compared to subunits DmHbB, DmHbE and DmHbG can also be postulated. Thus adaptation to different oxygen partial pressures leads to the expression of different D. magna hemoglobin subunits increasing the oxygen affinity. The oxygen threshold for the induction of each Hb subunit is assumed to be constant on the cellular level. However, the thresholds for the expression of the different subunits vary over a broad range of oxygen concentrations in the medium, some subunits already being present at high oxygen concentrations, some being expressed at moderate hypoxia and some being induced only at severe hypoxia. A discussion of these observed variations in subunit expression with oxygen supply must keep in mind the different positions of the two Hb synthesizing tissues (Goldmann et al., 1999). The centrally located fat cells are influenced by oxygen consuming neighbouring tissues (gut, ovaries) and their oxygen supply depends on the animals’ circulation. So these cells could experience oxygen deficiency at moderate medium hypoxia, they are the possible origin for subunits expressed already under these conditions. In contrast, the epithelia of the epipodites (which are leg appendices) face the medium directly, lacking oxygen only at low medium concentrations. These cells might be the origin of Hb subunits rising in concentration only at severe hypoxia. Thus, the acclimatisation to environmental changes is achieved by differential synthesis of hemoglobin of adequate functional properties (functional isoform multiplicity), the high heterogeneity of Daphnia hemoglobin being the result of a complex expression pattern of seven
49
subunit types in synthesizing cells sensing different oxygen concentrations. Adaptation at the macromolecular level provides Hb of adequate oxygen affinity, rather than requiring modulation of oxygen affinity by low-molecular weight effectors. This seems to be the regulatory strategy to optimize quantity and quality of the respiratory protein for altered oxygen transport requirements in D. magna due to its restricted homoiostasis capabilities.
References Fox, H.M., Gilchrist, B.M., Phear, E.A., 1951. Functions of haemoglobin in Daphnia. Proc. R. Soc. Lond. B138, 514–528. Goldmann, T., Becher, B., Wiedorn, K.H., Pirow, R., Deutschbein, M.E., Vollmer, E., Paul, R.J., 1999. Epipodite and fat cells as sites of hemoglobin synthesis in the branchiopod crustacean Daphnia magna. Histochem. Cell Biol. 112, 335–339. Kobayashi, M., Hoshi, T., 1982. Relationship between the haemoglobin concentration of Daphnia magna and the ambient oxygen concentration. Comp. Biochem. Physiol. 72A, 247–249. Kimura, S., Tokishita, S., Ohta, T., Kobayashi, M., 1999. Heterogeneity and differential expression under hypoxia of two-domain hemoglobin chains in the water flea Daphnia magna. J. Biol. Chem. 274, 10649–10653. Lamkemeyer, T., Zeis, B., Paul, R.J., 2003. Temperature acclimation to moderate influences temperature-related behaviour as well as oxygen transport physiology and biochemistry in the water flea Daphnia magna. Can. J. Zool. 81, 237–241. Pirow, R., Ba¨umer, C., Paul, R.J., 2001. Benefits of hemoglobin in the cladoceran crustacean Daphnia magna. J. Exp. Biol. 204, 3425–3441. Weber, R.E., Vinogradov, S.N., 2001. Nonvertebrate hemoglobins, functions and molecular adaptations. Physiol. Rev. 81, 569–628. Zeis, B., Becher, B., Lamkemeyer, T., Rolf, S., Pirow, R., Paul, R.J., 2003a. The process of hypoxic induction of Daphnia magna hemoglobin: subunit composition and functional properties. Comp. Biochem. Physiol. B 134, 243–252. Zeis, B., Becher, B., Goldmann, T., Clark, R., Vollmer, E., Bo¨lke, B., Bredebusch, I., Lamkemeyer, T., Pinkhaus, O., Pirow, R., Paul, R.J., 2003b. Differential haemoglobin gene expression in the crustacean Daphnia magna exposed to different oxygen partial pressures. Biol. Chem. 384, 1133–1145.
Molecular adaptation of Daphnia magna hemoglobin Bettina Zeis*, Tobias Lamkemeyer, Ru¨diger J. Paul Institute of Zoophysiology, Westfa¨lische Wilhelms-Universita¨t, Hindenburgplatz 55, Mu¨nster 48143, Germany
Abstract Daphnia magna responds to changing environmental conditions impeding aerobic metabolism by synthesizing hemoglobin of adequate quantity and quality to maintain oxygen supply of the tissues. Hemoglobin subunit composition and its oxygen affinity were analyzed as a function of temperature as well as depending on the oxygen partial pressure of the medium. Additionally, the time course of acclimation to hypoxia was studied. Correlating structural and functional changes, the role of individual subunits for the increase in oxygen affinity is discussed. q 2003 Elsevier Ltd. All rights reserved. Keywords: Dalphnia magna; Hemoglobin; Subunit composition; Two-dimensional electrophoresis; Oxygen affinity; Oxygen supply
The ability of Daphnia magna to increase hemoglobin concentration in the hemolymph at low oxygen concentration is a well-known phenomenon (Fox et al., 1951; Kobayashi and Hoshi, 1982). The di-domain multi-subunit protein of approximately 500 kDa (Weber and Vinogradov, 2001) is coded by at least four Hb genes (Kimura et al., 1999) and synthesized in the fat cells and the epipodite epithelia (Goldmann et al., 1999). The oxygen-dependent Hb induction was studied at the protein level by analyzing the alterations of functional properties and the structural changes they are based upon. Hemoglobin concentration and its oxygen affinity were studied using spectrophotometric analysis of ml-samples (Pirow et al., 2001) and its composition of different subunits was studied by two-dimensional electrophoresis of individual hemolymph samples (Zeis et al., 2003a,b). For studies of Hb induction as a function of oxygen concentration in the medium, animals were kept in long-term cultures at normoxic, hyperoxic and six different hypoxic conditions. Hb concentration rises exponentially with decreasing oxygen concentration (Fig. 1, bottom). Along with this quantitative change, the functional quality is also altered resulting in enhanced oxygen affinity (Fig. 1, top). Analysis of subunit composition reveals a complex expression pattern. Subunit DmHbB is present in high amounts at all oxygen concentrations studied. Subunits DmHbE and DmHbG are present only at high oxygen concentrations and decrease under hypoxia. * Corresponding author. Tel.: þ49-251832-3851; fax: þ49-2518323876. E-mail address: [email protected] (B. Zeis). 0968-4328/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2003.10.015
Among the subunits induced by hypoxia, subunits DmHbC and DmHbF already increase at moderate hypoxia. Increased amounts of subunits DmHbA and DmHbD are observed only at severe hypoxia of 4.1 kPa and below (Fig. 1, bottom). Hb oxygen affinity is not changed within the wide range of 73.6– 7.2 kPa, half-maximal saturation being achieved at an oxygen partial pressure of about 1 kPa. At severe hypoxia, rising amounts of subunits DmHbA, DmHbD and DmHbF coincide with elevated oxygen affinity of the hemoglobin (P50 values of 0.5 kPa were measured in Hb from animals kept at a Po2 of 2.1 kPa; Zeis et al., 2003b). For analysis of the time-course of hemoglobin expression animals from normoxic culture were transferred to hypoxic medium (Po2 of 3.1 kPa). Within an 11-day period Hb concentration, its affinity and subunit composition were studied daily. Newly synthesized hemoglobin was detected within eighteen hours. A pronounced increase of hemoglobin concentration occurred around the third and fourth day and after 6 days, a constantly elevated level of hemoglobin concentration was reached. Increases in affinity can be aligned with these quantitative changes. Twodimensional electrophoresis reveals the domination of subunits DmHbB, DmHbE and DmHbG in Hb of normoxically raised animals. Upon the transfer to hypoxic medium, the newly expressed Hb subunits show a transient increase of subunit DmHbC and after 11 days the Hb pool is dominated by subunits DmHbD and DmHbF. The dynamics of Hb oxygen affinity elevation coincides with the increase of the latter subunits. Subunit DmHbA is not present at any time (Zeis et al., 2003a). Acclimation of D. magna to different temperatures (10, 20, 30 8C) also results in an increase of Hb concentration and
48
B. Zeis et al. / Micron 35 (2004) 47–49
Fig. 1. Functional and structural characteristics of D. magna hemoglobin as a function of oxygen tension in the culture medium. The subunit composition of D. magna hemoglobin (bottom) changes along with an increase in concentration at lower oxygen availability. The oxygen affinity is unchanged in a wide Po2 range and only at severe hypoxia P50 decreases. The elevated oxygen affinity is correlated with an induction of Hb subunits DmHbA, DmHbD and DmHbF.
affinity along with increased temperature. Since oxygen solubility is inversely correlated to temperature, changes of these parameters can be compared to the alterations as a function of oxygen concentration. The effect of temperature on Hb concentration and affinity is only partly explained by the change in oxygen availability. Subunit composition of the expressed hemoglobin species reveals an increase of subunit DmHbA at elevated temperature, its presence being correlated with the observed increase in oxygen affinity (Lamkemeyer et al., 2003).
The effects of oxygen and temperature on each individual subunit are summarized in Table 1 along with the apparent values of molecular mass and pI determined from 2D gels. Analysis of similarities and differences in subunit composition and resulting functional characteristics in these three sets of experiments can be used to correlate the presence of individual subunits with effects on oxygen affinity. From subunit composition changes as a function of oxygen, the three potential candidates responsible for increased oxygen affinity are DmHbA, DmHbD and DmHbF. It seems
Table 1 Effect of environmental changes on the contribution of individual subunits of D. magna hemoglobin to the Hb pool Subunit
Molecular weight (kDa)
pI
Oxygen conditions for induction
Hypoxic induction
Dynamics
Induction at high temperature
DmHbA DmHbB DmHbC DmHbD DmHbE DmHbF DmHbG
36.2 ^ 0.9
8.1 ^ 0.2 7.5 ^ 0.2 7.1 ^ 0.1 6.8 ^ 0.2 6.5 ^ 0.1 6.7 ^ 0.2 7.2 ^ 0.1
Severe hypoxia All oxygen conc Moderate hypoxia Severe hypoxia Normoxia, hyperoxia Moderate hypoxia Normoxia, hyperoxia
þþ þ þ þ þþ þ – þþ –
Slow
þ þþ þ þ
40.6 ^ 1.6 37.9 ^ 1.2
Transient Fast Fast
2 þ 2
B. Zeis et al. / Micron 35 (2004) 47–49
that in the case of elevated temperature, the change in affinity is brought about by the increase in the share of subunit DmHbA alone, since only minor changes in DmHbD and DmHbF contribution to the Hb pool are observed. However, this subunit (DmHbA) is not induced within the time-course studied, although a considerable increase in affinity takes place. Here, subunits DmHbD and DmHbF are increased most, so a higher oxygen affinity of these subunits compared to subunits DmHbB, DmHbE and DmHbG can also be postulated. Thus adaptation to different oxygen partial pressures leads to the expression of different D. magna hemoglobin subunits increasing the oxygen affinity. The oxygen threshold for the induction of each Hb subunit is assumed to be constant on the cellular level. However, the thresholds for the expression of the different subunits vary over a broad range of oxygen concentrations in the medium, some subunits already being present at high oxygen concentrations, some being expressed at moderate hypoxia and some being induced only at severe hypoxia. A discussion of these observed variations in subunit expression with oxygen supply must keep in mind the different positions of the two Hb synthesizing tissues (Goldmann et al., 1999). The centrally located fat cells are influenced by oxygen consuming neighbouring tissues (gut, ovaries) and their oxygen supply depends on the animals’ circulation. So these cells could experience oxygen deficiency at moderate medium hypoxia, they are the possible origin for subunits expressed already under these conditions. In contrast, the epithelia of the epipodites (which are leg appendices) face the medium directly, lacking oxygen only at low medium concentrations. These cells might be the origin of Hb subunits rising in concentration only at severe hypoxia. Thus, the acclimatisation to environmental changes is achieved by differential synthesis of hemoglobin of adequate functional properties (functional isoform multiplicity), the high heterogeneity of Daphnia hemoglobin being the result of a complex expression pattern of seven
49
subunit types in synthesizing cells sensing different oxygen concentrations. Adaptation at the macromolecular level provides Hb of adequate oxygen affinity, rather than requiring modulation of oxygen affinity by low-molecular weight effectors. This seems to be the regulatory strategy to optimize quantity and quality of the respiratory protein for altered oxygen transport requirements in D. magna due to its restricted homoiostasis capabilities.
References Fox, H.M., Gilchrist, B.M., Phear, E.A., 1951. Functions of haemoglobin in Daphnia. Proc. R. Soc. Lond. B138, 514–528. Goldmann, T., Becher, B., Wiedorn, K.H., Pirow, R., Deutschbein, M.E., Vollmer, E., Paul, R.J., 1999. Epipodite and fat cells as sites of hemoglobin synthesis in the branchiopod crustacean Daphnia magna. Histochem. Cell Biol. 112, 335–339. Kobayashi, M., Hoshi, T., 1982. Relationship between the haemoglobin concentration of Daphnia magna and the ambient oxygen concentration. Comp. Biochem. Physiol. 72A, 247–249. Kimura, S., Tokishita, S., Ohta, T., Kobayashi, M., 1999. Heterogeneity and differential expression under hypoxia of two-domain hemoglobin chains in the water flea Daphnia magna. J. Biol. Chem. 274, 10649–10653. Lamkemeyer, T., Zeis, B., Paul, R.J., 2003. Temperature acclimation to moderate influences temperature-related behaviour as well as oxygen transport physiology and biochemistry in the water flea Daphnia magna. Can. J. Zool. 81, 237–241. Pirow, R., Ba¨umer, C., Paul, R.J., 2001. Benefits of hemoglobin in the cladoceran crustacean Daphnia magna. J. Exp. Biol. 204, 3425–3441. Weber, R.E., Vinogradov, S.N., 2001. Nonvertebrate hemoglobins, functions and molecular adaptations. Physiol. Rev. 81, 569–628. Zeis, B., Becher, B., Lamkemeyer, T., Rolf, S., Pirow, R., Paul, R.J., 2003a. The process of hypoxic induction of Daphnia magna hemoglobin: subunit composition and functional properties. Comp. Biochem. Physiol. B 134, 243–252. Zeis, B., Becher, B., Goldmann, T., Clark, R., Vollmer, E., Bo¨lke, B., Bredebusch, I., Lamkemeyer, T., Pinkhaus, O., Pirow, R., Paul, R.J., 2003b. Differential haemoglobin gene expression in the crustacean Daphnia magna exposed to different oxygen partial pressures. Biol. Chem. 384, 1133–1145.