The Cloning and Expression of Mouse Na + /H + Exchanger 10

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2 The Cloning and Expression of Mouse Na + /H + Exchanger 10 A thesis submitted to the Miami University Honors Program in partial fulfillment of the requirements for University Honors with Distinction by Jessica Leigh McAfee May 2007 Oxford, Ohio

3 ABSTRACT THE CLONING AND EXPRESSION OF MOUSE NA + /H + EXCHANGER 10 By Jessica Leigh McAfee The Na+/H+ Exchanger (NHE) is a trans-membrane antiporter that regulates the intracellular ph of the cell. The sodium ion gradient established by the Na + /K + ATPase allows for the simultaneous exchange of a H + out of the cell while allowing Na + to fall back into the cell along its gradient, in a stoichiometric ratio of one to one through a NHE. Nine isoforms of the NHE have previously been characterized in eukaryotic cells, with NHE1 and NHE5 isoforms localized to sperm. A novel NHE isoform has recently been discovered and localized to mice sperm through a signal peptide screen. While the sequenced homology of the protein categorizes it as an NHE, it has yet to be characterized as a functional antiporter. Intracellular ph levels have been theorized to affect the motility of vertebrate sperm cells. The NHE utilizes the Na + /K + ATPase to pump out hydrogen ions produced by the metabolic pathways of the cell. Inhibition of the sperm specific 4 Na + /K + ATPase by ouabain results in a secondary inhibition of the sperm NHE and thus reduced motility, and sperm motility has been shown to be directly reduced when NHE is inhibited by EIPA. While the role of NHE5 in maintaining sperm motility is not yet known, the inhibition of NHE1 isoform alone results in functional mouse male fertility and motile sperm, suggesting a high level of ion exchange by NHE10 in sperm. To characterize the mouse NHE10 (mnhe10), its gene must first be cloned and inserted in a mammalian expression vector, and expressed in a cell line lacking any NHE. Once the expression of the mnhe10 has been determined by Northern and Western blots, its proton-extruding function can be characterized through the method of proton suicide via increased intracellular hydrogen levels. Future studies will include further characterization of the mnhe10 and its specific function in the mouse sperm cell, with a possibility of targeting the mnhe10 protein in future male infertility or contraceptive treatments. ii

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5 The Cloning and Expression of Mouse Na + /H + Exchanger 10 by Jessica McAfee Approved by: _/s/ Paul F James, Advisor Dr. Paul James _/s/ David Pennock, Reader Dr. David Pennock _/s/ Paul Harding, Reader Dr. Paul Harding Accepted by: _/s/ Carolyn Haynes, Director, University Honors Program iv

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7 ACKNOWLEDGEMENTS In grateful acknowledgement for the assistance and guidance given by Dr. Paul F. James, Arunima Sengupta, Santanu Chakraborty, Jayanthi Sanjee, and Deepti Kumar; as well as the support of Kevin J. Van Dyke. vi

8 TABLE OF CONTENTS Abstract p. ii Acknowledgements p. vi List of Figures p. viii Introduction p. 1 Materials and Methods p. 7 Summary and Discussion p. 14 References p. 15 vii

9 LIST OF FIGURES Concentration gradients of the Na,K-ATPase and NHE ion transport. p. 2 Localization of sperm specific mouse NHE 10. p. 4 Model of the coupled roles of the Na + /K + ATPase and the Na + /H + Exchanger in regulation of sperm motility. p. 5 Fragments of the mnhe10 cdna in topo vectors and their restriction enzyme sites. p. 7 Diagnostic digests with the EcoRI restriction enzyme. p. 8 Xba1 restriction enzyme digestion and extraction of vector and insert fragments. p. 9 Ligation diagram of mnhe10 gene in topo. p. 9 Results of Ligation p. 10 Expression vector p. 11 Proton suicide characterization. p. 12 viii 1

10 INTRODUCTION The Na + /H + Exchanger (NHE) is a critically important integral membrane protein necessary to maintain H + homeostasis. The NHE acts to regulate intracellular ph (ph i ), and changes to this ph i can affect much of the basic functioning of eukaryotic cells and biochemical systems. Optimal protein structure and function is dependent upon a specific ph i, affecting enzyme activity, structure, communication pathways, and many cellular mechanisms. The ph i also regulates gene expression and affects electrical and chemical gradients [5]. The NHE family of proteins regulate ph i through proton extrusion from the cell, as well as controlling ion flux, cell volume, and initiating changes in the growth stages of the cell [9]. Because charged molecules cannot passively move through the nonpolar hydrophobic cell membrane, ion transport must be facilitated by proteins embedded into the plasma membrane. The hydrophilic cytosolic and extracellular domains of the NHE allow for the binding of protons and Na +, respectively, and the ions are subsequently transported through the hydrophobic transmembrane domain [9]. As an antiporter, NHE transports one H + out of the cell against its gradient for every Na + that it allows to fall back into the cell along its gradient. While the NHE does not metabolize ATP itself, the ion exchange is dependent on high extracellular Na + concentrations, achieved by the active pumping of Na + outside of the cell by Na,K-ATPase, as shown in Figure 1. This transmembrane pump consumes energy as it shuttles three sodium ions from inside to

11 2 outside the cell against the gradient, and two potassium ions from outside to inside the cell against that gradient. Coupled together, the Na,K-ATPase and the NHE work in tandem to achieve the extrusion of protons from the cell to maintain a balanced ph i [4,5]. Figure 1. Concentration gradients of the Na,K-ATPase and NHE ion transport. The ion transport proteins are shown with their respective ion exchanges. The Na,K-ATPase pumps Na + out of the cell and the NHE couples the high concentration gradient established by the accumulated extracellular Na + to fall back into the cell to drive the extrusion of H + from the cell. Transport involving ion movement against its respective gradient is depicted with dotted lines, while ion transport along its gradient is depicted with a solid line. The NHE was first described in 1976 in the plasma membrane vesicles of the kidney and in 1982 the Na + /H + exchange system had been elucidated. When this NHE (isoform 1, or NHE1) was first cloned in 1989 from its cdna sequence, the protein could be localized and its function characterized [2,3,5]. The NHE1 has been found to be ubiquitously expressed in all mammalian cells, and is called the housekeeper isoform [8]. Nine isoforms have as yet been conclusively identified (NHE1-NHE9). These isoforms have between 25%-70% homology of amino acids and similar predicted

12 3 membrane topologies. The NHE2-NHE5 isoforms are localized to specific tissues, while the NHE6-NHE9 isoforms are also ubiquitously expressed [9]. A novel sperm specific NHE isoform has been recently identified in mice and its cdna clone sequenced [10]. Hydropathy plots indicate 14 possible transmembrane segments, and the amino terminus of the sperm-specific mouse NHE isoform (mnhe10) shows a region of 10 transmembrane segments conserved in all NHEs, which is the region of binding and transport of Na + and H +. The carboxyl tail of mnhe10 also appears to contain a domain in common with many NHE isoforms that interacts with protein kinases, phosphatases, cytoskeleton associated components, and possibly cyclic nucleotides for regulation of exchanger activity. Between these two domains lies a sequence similar to voltage sensing portions of voltage-gated ion channels that are unique to the mnhe10 out of the NHE protein family, suggesting a possible voltage regulation of the sodium-proton exchange [7]. The mnhe10 has been shown to be expressed in the mouse testis and sperm, and immunofluorescence microscopy localizes the mnhe10 to the midpiece of the sperm flagellum, as illustrated in Figure 2. Two other isoforms of NHE are also expressed in mammalian sperm, NHE1 and NHE5. Disruption of the mnhe10 gene causes infertile male animals with considerably reduced sperm motility, although testis morphology and sperm numbers were unaffected. NHE1-null mice have normal sperm motility, suggesting a specific role of mnhe10 in maintaining sperm motility [10]. Little is know about the role of NHE5 in sperm, which is primarily localized in neural tissues. To date, no mouse with a targeted deletion of NHE5 has been reported, and therefore little characterization of the NHE5 in sperm has been possible [1,4].

4 a. b. Figure 2. Localization of sperm specific mouse NHE 10. a) In experiments by Wang et al, testes and sperm proteins were separated and probed with anti-mnhe10 antibodies.

13 4 a. b. Figure 2. Localization of sperm specific mouse NHE 10. a) In experiments by Wang et al, testes and sperm proteins were separated and probed with anti-mnhe10 antibodies. b) Spermatozoa were probed with anti-sperm NHE, and the bound antibody was detected with fluorescent-labeled secondary antibodies (left image). The mnhe10 is shown to be localized to the midpiece of the flagella, as the labeling on the sperm heads was nonspecific (right image) [10]. For many years sperm motility has been thought to be regulated by ph i, and the coupled NHE and the Na,K-ATPase ion pumps suggest a model for the extrusion of protons from the spermatozoa, illustrated by Figure 3 [11]. As the mitochondria in the midpiece of the sperm flagellum generate protons during metabolism and energy production necessary for the movement of the spermatozoa, protons leak from the mitochondrial membranes into the cytoplasm. The Na,K-ATPase creates the sodium ion gradient that the NHE uses to pump these accumulating protons from the cell. If the protons are not removed from the cell, the flagellar movement of the sperm is inhibited by the deactivation of dynein molecules [11]. This model is supported as the inhibition of the sperm-specific 4 isoform of the Na,K-ATPase by oaubain in rat results in the diminished sperm motility likely via the secondary inactivation of the NHE and

14 decreased ph i. Likewise, sperm motility is also directly reduced with the addition of EIPA, a NHE inhibitor [8]. 5 Figure 3. Model of the coupled roles of the Na + /K + ATPase and the Na + /H + Exchanger in regulation of sperm motility. This proposed model hypothesizes that the Na + /H + exchanger in sperm cells utilizes the gradient established by the Na + /K + ATPase to eject out of the cell the protons leaked by the process of oxidative phosphorylation in the mitochondria. The suppression of the Na + /H + exchanger may result in disrupted activity of the flagellar microtubules and decreases in the sperm motility due to decreased ph levels. To characterize the novel mnhe10 and to begin to understand its specific role in maintaining sperm motility, I have undertaken the process of cloning and expression of the protein into a NHE-null cell line. With no other expressed NHE isoforms expressed, the activity and function of this specific antiporter can be discerned. One such approach to the characterization that will be used in mnhe10 experiments is the proton-suicide

15 6 method, based on techniques first described by Pouyssegur et al. in 1984 [6]. Characterization of this NHE isoform will help to elucidate its role in sperm motility and suggests future clinical applications for the development of male contraceptives or the treatment of infertility [10].

16 7 METHODS AND MATERIALS To begin the process of cloning and characterization of the function of mnhe10, we started with cdna that had previously been cloned by RT-PCR into pcr2.1-topo vectors. As cloning of the large mnhe10 cdna, almost 4 kilobases long, by RT-PCR is difficult, the cdna were cloned in two fragments into two topo vectors, shown in Figure 4. The topo vectors contain Kanamycin and Ampicillin-resistant markers so bacteria transfected with these vectors could be cultured and selected. As the restriction enzyme sites are known in both the cdna fragments of the mnhe10 and in the topo vectors, diagnostic digests were performed with EcoRI enzyme to ensure the presence of the correct mnhe10 fragments in the isolated vectors (Figure 5). As the band sizes of each fragment matched the previously calculated digested fragment size, known from the cdna and vector lengths, both topo vectors were determined to contain the correct fragment and could then be extracted and ligated together. Figure 4. Fragments of the mnhe10 cdna in topo vectors and their restriction enzyme sites. The mnhe10 cdna was cloned, by RT-PCR, in two pieces in the pcr2.1-topo vector, and were transfected into bacteria. Restriction enzyme sites are depicted in only in the mnhe10 fragments.

To extract the two fragments from their respective topo vector, another digest is performed. Xba1 digests the fragment 1 vector into two pieces, of which the 1.

3 kilobase band in the electrophoresis gel (Figure 6). These pieces were cut and purified from the agarose gel.

17 To extract the two fragments from their respective topo vector, another digest is performed. Xba1 digests the fragment 1 vector into two pieces, of which the 1.9 kilobase 8 segment contains the entire fragment 1 of mnhe10 cdna. The second vector is Fragment 2 is cut in one location, as seen by the presence of only one 5.3 kilobase band in the electrophoresis gel (Figure 6). These pieces were cut and purified from the agarose gel. Ligation of the two fragments occurred at the Xba1 complementary ends of each created by the Xba1 digest (Figure 7). The enzyme T4 DNA ligase joined fragment 1 into fragment 2 to achieve the complete cdna of mnhe10 in the topo vector. The topo vectors were then transfected into bacteria and were again screened for ampicillin resistance. The plasmid vectors of the colonies containing the vector were then isolated and diagnostically digested with EcoRV to ensure the whole mnhe10 cdna was correctly ligated and inserted into the vector (Figure 8a). As of yet, the digested plasmids of the screened colonies have only identified the incorrect backwards ligation of the cdna segments (Figure 8b,c) More screening is necessary to find the correct ligation of the fragments so the cloning and expression experiments of mnhe10 can continue. a. b. c Figure 5. Diagnostic digests with the EcoRI restriction enzyme. a) Digestion of each fragment with EcoRI restriction enzyme. The fragment 1 digest yielded the expected

a) The fragment 1, 1.9 kb segment was extracted from the gel as the insert. b) The fragment 2, 5.3 kb band was extracted as the vector.

18 three segments of 3.9, 1.1, and 0.75 kb. b) The digest of fragment 2 yielded the expected two segments of 3.9 and 1.6 kb. c) The 1 kb DNA ladder used as a reference, 1.0=1 kb. 9 a. b. c. Figure 6. Xba1 restriction enzyme digestion and extraction of vector and insert fragments. a) The fragment 1, 1.9 kb segment was extracted from the gel as the insert. b) The fragment 2, 5.3 kb band was extracted as the vector. c) The 1 kb DNA ladder used as a reference, 1.0=1 kb. a. b. Figure 7. Ligation diagram of mnhe10 gene in topo. a) Digestion of each fragment at respective Xba1 restriction enzyme site and b) ligation of fragments together with T4 DNA ligase.

19 10 a. b. c. Figure 8. Results of Ligation. a) If the fragments were ligated together correctly at the Xba1 site in the topo vector, the expected band size in a diagnostic digest with EcoRV (RV) would be 5152 and 2140 base pairs. b) A backwards ligation of the fragments would result in the band sizes of 7002 and 290 base pairs. c) A sample gel with band size showing probable backwards ligation (EcoRV digestion middle lane). d) The 1 kb DNA ladder used as a reference, 1.0=1 kb. Once the correct ligation of the mnhe10 fragments are found, the mnhe10 cdna will then be excised from the topo vector by restriction enzymes and will then be ligated into the expression vector pcdna3.1. This mammalian expression vector contains both neomycin and ampicillin selection markers as well as many restriction enzyme sites for the addition of the desired genetic material (Figure 9). Once the mnhe10 is cloned into the pcdna3.1, the expression vector will then be transfected into the mammalian PS120 cell line. The PS120 cells will then be cultured with G418, a neomycin analogue, to select for the colonies containing the expression vector and mnhe10. Northern and southern blotting techniques will then be used to screen for the mnhe10 mrna and cdna, respectively, as well as using the developed antibodies in western blotting to show the presence of the expressed protein.

20 11 Figure 9. Expression vector. The ligated mnhe10 will then be cloned into the expression vector pcdna3.1 and transfected into the mammalian cell line PS120. The cells containing the neomycin-resistant vector will be selected for using G418, a neomycin analog. As the PS120 cell line does not express any NHE, the function and activity of mnhe10 alone can be studied by its effects upon the cells. One method of characterization is the proton suicide experiment. In this technique, PS120 cells with the addition of the cloned pcdna3.1 vector are compared to cells containing an empty vector. Therefore, cells expressing mnhe10 and no other NHE are compared to cells that do not express any NHE isoform at all. In the first step of the experiment, NH 4 Cl is added to the extracellular environment of both cell types. In the aqueous solution the compound dissociates into the NH + 4 and Cl - ions. The NH4 + further dissociates into a proton and NH 3, and as a small, nonpolar molecule, NH 3 is able to diffuse across the cell membrane. Once inside a cell, the NH 3 can interact with an intracellular proton to again form NH + 4 (Figure 10a). As this increases the ph i, the regulatory mechanisms in the cell act to maintain homeostasis by moving protons from cellular compartments into the cytoplasm to maintain the physiological ph i value of 7.4. When NH + 4 concentrations equilibrate across the membrane, the NH 4 Cl-containing extracellular solution will then be

21 replaced with fresh buffer with physiological levels of Na +. In each of the cells types, the 12 removal of the NH 4 Cl (and therefore NH 3 from the extracellular fluid) will drive the NH 3 to diffuse back outside the cell, yet the charged protons will remain trapped in the cell. PS120 cells without an NHE will die from the increased intracellular acidity, while cells expressing mnhe10, if NHE10 is a functional NHE, should be able to extrude H + and will survive (Figure 10b). In addition to only comparing mnhe10 expressing cells against NHE-null cells, PS120 cells expressing each NHE1 and NHE5 isoforms will also be used in this experiment as positive controls. a. b. Figure 10. Proton suicide characterization. Figure 9. Proton suicide characterization. a) The addition of NH 4 Cl to the cell and its dissociation in the extracellular fluid allows NH 3 to diffuse inside the cell to interact with intracellular

22 13 protons. b) Removal of the NH 4 Cl and replacement with fresh buffer to the extracellular fluid drives the NH 3 out of the cell, leaving behind protons. Cells with NHE will be able to pump out the H + while PS120 cells will die from increased acidity.

23 14 SUMMARY AND DISCUSSION In conclusion, the cloning and expression of the mouse NHE10 has been critical in determining its role in sperm motility. Further work will be done to continue my efforts to produce a correctly ligated mnhe10 fragment in the topo vector, and the activity and function of the antiporter can then be determined. If the proton suicide experiment shows the PS120 cells expressing the mnhe10 to live after the treatment and removal of NH 4 Cl in contrast to the cell death of NHE-null PS120 cells, the proton extrusion activity of the mnhe10 will be confirmed. The proton exchange mechanism of this antiporter can then be compared to other NHE isoforms to determine which are necessary in sperm motility. While previous disruptions of the NHE1 isoform show no effect on sperm motility, further characterization of NHE5 is required to determine its specific role in sperm motility. In addition, the sperm specific 4 isoform of the Na,K- ATPase and its relationship to the mnhe10 will be further explored to provide a more complete understanding of the regulation of sperm motility due to intracellular ph. With this future research, clinical applications of the ph i regulatory mechanisms in sperm may potentially be developed in treatment of male infertility or for the development of male contraceptives. The mnhe10 may be a possible target protein in future treatments, as well as the Na,K-ATPase 4 in its hypothesized regulation of the function of mnhe10.

24 15 REFERENCES 1. Attaphitaya S, Park K, Melvin JE. Molecular Cloning and Functional Expression of a Rat Na+/H+ Exchanger (NHE5) Highly Expressed in Brain. The Journal of Biological Chemistry 1999; 274(7)12; Bianchini L, Pouyssegur J. Molecular Structure and Regulation of Vertebrate Na+/H+ Exchangers. J. exp. Biol 1994; 196: Collins JF, Honda T, Knobel S, Bulus NM, Conary J, DuBois R, Ghishan FK. Molecular Cloning, Sequencing, Tissue Distribution, and Functional Expression of a Na+/H+ Exchanger (NHE-2). Proceedings of the National Academy of Sciences of the United States of America 1993; 90(9): Hlivko JT, Chakraborty S, Hlivko TJ, Sengupta A, James PF. The Human Na,K-ATPase Alpha4 Isoform Is a Ouabain-Sensitive Alpha Isoform that Is Expressed in Sperm. Molecular Reproduction and Development 2006; 73: Malo ME, Fliegel L. Physiological role and regulation of the Na+/H+ exchanger. Can. J. Physiol. Pharmacol 2006; 84: Pouyssegur J, Sardet C, Franchi A, L Allemain G, Paris S. A specific mutation abolishing Na+/H+ antiport activity in hamster fibroblasts precludes growth at neutral and acidic ph. Proceedings of the National Academy of Sciences of the United States of America 1984; 81: Quill TA, Wang D, Garbers DL. Insights into sperm cell motility signaling through snhe and the CatSpers. Molecular and Cellular Endocrinology 2006; 250: Sengupta A, James PF. Unpublished Observations. 9. Slepkov ER, Rainey JK, Sykes BD, Fliegel L. Structural and functional analysis of the Na+/H+ exchanger. Biochem J. 2007; 401; Wang D, King SM, Quill TA, Doolittle LK, Garbers DL. A new sperm-specific Na+/H+ Exchanger required for sperm motility and fertility. Nature Cell Biology 2003; 5(12): Woo AL, James PF, Lingrel JB. Roles of the Na,K-ATPase 4 Isoform and the Na+/H+ Exchanger in Sperm Motility. Molecular Reproduction and Development 2002; 62:

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