Tissue-specific Expression of the Na,K-ATPase β3 Subunit

Abstract

The Na,K-ATPase belongs to a family of P-type ion-translocating ATPases sharing homologous catalytic subunits (α) that traverse the membrane several times and contain the binding sites for ATP and cations. In this family, only Na,K- and H,K-ATPases have been shown to have a second subunit, a single-span glycoprotein called β. Recently a new isoform (β3) has been identified in mammals. Here we describe structural features and tissue distribution of the β3 protein, utilizing an antiserum specific for its N terminus. β3 was the only β detected in Na,K-ATPase purified from C6 glioma. Treatment with N-glycosidase F confirmed that β3 is a glycoprotein containing N-linked carbohydrate chains. Molecular masses of the glycosylated protein and core protein were estimated to be 42 and 35 kDa, respectively, which are different from those of the β1 and β2 subunits. Detection of β subunits has historically been difficult in certain tissues. Sensitivity was improved by deglycosylating, and expression was evaluated by obtaining estimates of β3/α ratio. The proportion of β3 protein in the rat was highest in lung and testis. It was also present in liver and skeletal muscle, whereas kidney, heart, and brain contained it only as a minor component of the Na,K-ATPase. In P7 rat, we found skeletal muscle and lung Na,K-ATPase to be the most enriched in β3 subunit, whereas expression in liver was very low, illustrating developmentally regulated changes in expression. The substantial expression in lung and adult liver very likely explains long-standing puzzles about an apparent paucity of β subunit in membranes or in discrete cellular or subcellular structures. The Na,K-ATPase belongs to a family of P-type ion-translocating ATPases sharing homologous catalytic subunits (α) that traverse the membrane several times and contain the binding sites for ATP and cations. In this family, only Na,K- and H,K-ATPases have been shown to have a second subunit, a single-span glycoprotein called β. Recently a new isoform (β3) has been identified in mammals. Here we describe structural features and tissue distribution of the β3 protein, utilizing an antiserum specific for its N terminus. β3 was the only β detected in Na,K-ATPase purified from C6 glioma. Treatment with N-glycosidase F confirmed that β3 is a glycoprotein containing N-linked carbohydrate chains. Molecular masses of the glycosylated protein and core protein were estimated to be 42 and 35 kDa, respectively, which are different from those of the β1 and β2 subunits. Detection of β subunits has historically been difficult in certain tissues. Sensitivity was improved by deglycosylating, and expression was evaluated by obtaining estimates of β3/α ratio. The proportion of β3 protein in the rat was highest in lung and testis. It was also present in liver and skeletal muscle, whereas kidney, heart, and brain contained it only as a minor component of the Na,K-ATPase. In P7 rat, we found skeletal muscle and lung Na,K-ATPase to be the most enriched in β3 subunit, whereas expression in liver was very low, illustrating developmentally regulated changes in expression. The substantial expression in lung and adult liver very likely explains long-standing puzzles about an apparent paucity of β subunit in membranes or in discrete cellular or subcellular structures. The Na,K-ATPase 1The abbreviation used is: Na,K-ATPase, sodium- and potassium-exchanging adenosine triphosphatase.catalyzes the active uptake of K+ and efflux of Na+ ions, thus controlling ionic gradients through the enzymatic hydrolysis of ATP. It is a heterodimer of two different kinds of subunit, a large α subunit with multiple membrane spans and a smaller glycoprotein subunit, β, with just one span and the bulk of its mass in the extracellular space. Both subunits of Na,K-ATPase are encoded by multigene families. In the rat, three isoforms of α subunit and two isoforms of β subunit have been identified and substantially characterized (1Levenson R. Rev. Physiol. Biochem. Pharmacol. 1994; 123: 1-39Crossref PubMed Google Scholar, 2Chow D.C. Forte J.G. J. Exp. Biol. 1995; 198: 1-17Crossref PubMed Google Scholar). Although the major ATPase characteristics are assigned to the α subunit, the β subunit is required for folding and transport of α,β-heterodimers to the plasma membrane (2Chow D.C. Forte J.G. J. Exp. Biol. 1995; 198: 1-17Crossref PubMed Google Scholar). Nonetheless, there have been a number of reports in which little or no β1 or β2 subunit or mRNA was detected despite significant levels of α or its mRNA (3Brown T.A. Horowitz B. Miller R.P. McDonough A.A. Farley R.A. Biochim. Biophys. Acta. 1987; 912: 244-253Crossref PubMed Scopus (38) Google Scholar, 4Emanuel J.R. Garetz S. Stone L. Levenson R. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 9030-9034Crossref PubMed Scopus (87) Google Scholar, 5Shyjan A.W. Cena V. Klein D.C. Levenson R. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1178-1182Crossref PubMed Scopus (101) Google Scholar, 6Hubert J.J. Schenk D.B. Skelly H. Leffert H.L. Biochemistry. 1986; 25: 4156-4163Crossref PubMed Scopus (30) Google Scholar), or alternatively, in which α and β mRNA or protein did not appear to colocalize in tissue sections (7Simon F.R. Leffert H.L. Ellisman M. Iwahashi M. Deerinck T. Fortune J. Morales D. Dahl R. Sutherland E. Am. J. Physiol. 1995; 269: C69-C84Crossref PubMed Google Scholar, 8Simon F.R. Fortune J. Alexander A. Iwahashi M. Dahl R. Sutherland E. J. Biol. Chem. 1996; 271: 24967-24975Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 9Crump R.G. Askew G.R. Wert S.E. Lingrel J.B. Joiner C.H. Am. J. Physiol. 1995; 269: L299-L308PubMed Google Scholar). Recently several cDNA fragments having homology with Na,K-ATPase β subunits were identified in the GenBank™ expressed sequence tag library. The full-length human clone revealed 59% sequence identity with the β3 subunit of Xenopus laevis and 38 and 48% identity with human β1 and β2 subunits, respectively, and was named β3 (10Malik N. Canfield V.A. Beckers M.C. Gros P. Levenson R. J. Biol. Chem. 1996; 271: 22754-22758Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Full-length clones were independently isolated from cDNA libraries from C6 rat glioma cells (GenBank™ D84450) and mouse retina (11Besirli C.G. Gong T.L. Lomax M.I. Biochim. Biophys. Acta. 1997; 1350: 21-26Crossref PubMed Scopus (20) Google Scholar). Northern blot analysis and representation in the expressed sequence tag data bank has revealed β3 message in a wide variety of tissues. Expression of the β3 subunit at the protein level and its association with the Na,K-ATPase has not been demonstrated yet however. Here we accomplish these aims with an antibody, specific for the rat β3 subunit, that will be a useful addition to an existing panel of isoform-specific antibodies to the Na,K-ATPase. Antipeptide-directed antiserum RNTβ3 (rat N terminus of β3) was raised at Quality Control Biochemicals, Inc. (Hopkinton, MA), against the 13-mer peptide TKTEKKSFHQSLAC-(NH2), corresponding to the N-terminal sequence of the rat β3 isoform (GenBank™ D84450) plus a terminal cysteine residue. The peptide was conjugated to keyhole limpet hemocyanin as a carrier, and two rabbits were immunized. The immune response was tested with an enzyme-linked immunosorbent assay with bovine serum albumin-coupled peptide on solid phase. The titer of crude sera against the peptide was >200,000 for the better rabbit. Other antibodies were generous gifts of other investigators: antiserum against the KETYY peptide representing the C terminus of all α subunits of Na,K-ATPase from R. Bayer and J. Kyte (University of California, San Diego); antipeptide antiserum 757 against sheep β1 from W. J. Ball, Jr. (University of Cincinnati); anti-fusion protein antiserum FPβ1 against rat β1 from A. McDonough (University of Southern California); antiserum SpETβ2 against human β2 from P. Martin-Vasallo (University de la Laguna, Tenerife, Spain); and monoclonal antibody SM-GP50 for β2 from J. Gurd (University of Toronto) and P. Beesley (Royal Holloway and Bedford New College, Egham, United Kingdom). The C6 glioma cell line was obtained from the American Type Culture Collection. Cells were maintained in Dulbecco's modified Eagle's medium, supplemented with 10% fetal bovine serum and penicillin/streptomycin. Membrane preparations from rat tissues were made as follows. Brain, lung, liver, kidney, and testis were minced and homogenized with a motor-driven Teflon homogenizer in 10 volumes of buffer containing 250 mm sucrose, 30 mm imidazole, pH 7.2, 1 mm EDTA, and 1 mm phenylmethylsulfonyl fluoride. Unhomogenized material and nuclei were removed by low-speed centrifugation at 7,000 rpm for 15 min, and membranes were sedimented at 33,000 rpm for 30 min. For cardiac and skeletal muscle preparations, the tissues were minced and homogenized in 40 volumes of buffer containing 250 mm sucrose, 20 mm Tris-HCl, pH 7.2, 1 mm EDTA, and 1 mm phenylmethylsulfonyl fluoride, followed by further disruption with an Omni homogenizer for 30 s at the maximal setting. The homogenates were centrifuged at 14,000 rpm for 30 min. Sarcolemma-enriched fractions were obtained by centrifugation of the supernatant at 33,000 rpm for 30 min. Protein concentration was determined according to Lowry. Partial purification of the Na,K-ATPase from rat brain microsomes and C6 crude membranes by extraction with SDS was performed as described elsewhere (12Sweadner K.J. Methods Enzymol. 1988; 156: 65-71Crossref PubMed Scopus (25) Google Scholar). Gel electrophoresis and Western blots were performed as described earlier (13Felsenfeld D.P. Sweadner K.J. J. Biol. Chem. 1988; 263: 10932-10942Abstract Full Text PDF PubMed Google Scholar), except that detection was by chemiluminescence using luminol-based reagents (Pierce). Blots were scanned with a Molecular Dynamics laser densitometer. Deglycosylation was performed as follows. Membranes were resuspended at 1–2 mg/ml in 0.25 m sodium phosphate buffer, pH 7.4, denatured with 0.1% SDS and 1% β-mercaptoethanol for 30 min at 37 °C, followed by addition of 1–2 units of N-glycosidase F (Boehringer Mannheim) and up to 0.5% Triton X-100. The reaction was performed overnight at room temperature or at 37 °C. To detect β3 protein, we generated an antiserum based on the N-terminal sequence deduced from the rat cDNA sequence. To test its reactivity with β3, rat C6 glioma cells were used, since the clone had been isolated from a C6 cDNA library. The antiserum stained C6 cell plasma membranes by immunofluorescence (data not shown). To determine whether β3 was associated with the Na,K-ATPase, the enzyme was partially purified from C6 crude membranes using SDS treatment followed by equilibrium centrifugation on a sucrose density gradient. During such treatment most other membrane proteins are solubilized, while Na,K-ATPase remains associated with plasma membrane. As illustrated in Fig. 1 A, the RNTβ3 antibody stained a fuzzy band with a molecular mass of about 42–45 kDa in preparations that had a Na,K-ATPase specific activity of 100–150 μmol of Pi/mg/h (equivalent to 30–50-fold purification). Since both β1 and β2 subunits have been shown to be glycoproteins containing several N-linked oligosaccharide chains (14Treuheit M.J. Costello C.E. Kirley T.L. J. Biol. Chem. 1993; 268: 13914-13919Abstract Full Text PDF PubMed Google Scholar, 15Fahrig T. Schmitz B. Weber D. Kucherer-Ehret A. Faissner A. Schachner M. Eur. J. Neurosci. 1990; 2: 153-161Crossref PubMed Scopus (43) Google Scholar),N-glycosidase F treatment was performed to determine whether β3 was also glycosylated. As seen in Fig. 1 A, this resulted in a decrease in apparent molecular mass to 35 kDa. Fig.1 B demonstrates that β3 is the only known β isoform of the Na,K-ATPase purified from C6 glioma cells, contrasting with the β1 and β2 isoforms found in Na,K-ATPase purified from rat brain. Different dilutions of sample were added to facilitate comparisons. The content of α (α1 only for C6; all three α subunits for rat brain) was determined by staining the blots with the KETYY antibody against the C-terminal peptide of all α subunits. Equal amounts of the sodium pump α subunits were loaded for rat brain and C6 samples, but the C6 membranes had no β1 or β2 and were more than four times more enriched in β3 than rat brain enzyme. Removal of oligosaccharide chains often facilitates glycoprotein detection on blots. As seen in Fig. 2, fully glycosylated β3 could be detected in rat brain microsomes only when 25–50 μg of protein were loaded. In deglycosylated samples, the binding was almost linear within a 5–50 μg range, however. Consequently, in further experiments we routinely used glycosidase treatment. Molecular masses of the mature and core β3 protein calculated from their mobilities on SDS gels were different from those of β1 and β2 subunits. As seen in Fig. 3 A, the mature β3 protein migrated faster than the mature β2 subunit. This was not surprising since rat β3 has only two potentialN-linked glycosylation sites in the extracellular domain, whereas rat β2 has seven. As shown in Fig. 3 B (in an experiment where no compensation was made for the smaller amount of β3 than β1 or β2), the core β3 protein migrated significantly slower than the core β2 protein and slightly slower than the core β1 subunit. Average values for glycosylated proteins were 46–50 kDa for β1 and β2 and 42–45 kDa for β3. Average values for core proteins were 34–37 kDa for β1, 32–34 kDa for β2, and 35–38 kDa for β3. The apparent size of core β3 is larger than predicted from the cDNA clone (30 kDa); the biochemical basis for the discrepancy remains to be determined. Fig. 3 B also illustrates an incidental peculiarity of glycoprotein staining. β1 and β2 were each stained with two different antibodies, and certain antibodies were substantially less sensitive to the glycosylated protein than to the core. This is likely to reflect differences in the proximity of their epitopes to glycosylation sites. The species specificity of the RNTβ3 antibody was examined with blots of 50 μg of deglycosylated brain microsomes from several species. β3 protein was detected readily in rat and mouse brain samples and more weakly in guinea pig samples (data not shown). All other samples, including human, chicken, rabbit, dog, cat, sheep, calf, monkey (Macaca), frog (Rana), and goldfish brain membranes, were negative. There is only 69% identity within the first 13 amino acids among mammalian β3 subunits, and even less for other named β2, this isoform of β from the is with β3 by analysis of (2Chow D.C. Forte J.G. J. Exp. Biol. 1995; 198: 1-17Crossref PubMed Google that the the amino acids and that of for in guinea pig β3 is weakly but of in human β3 RNTβ3 RNTβ3 has also been to be on human β3 expressed in N. panel of adult rat tissues was examined for the of the Na,K-ATPase β3 subunit on blots. In Fig. A, of samples were to levels of the α subunit, as detected with β3 staining was in lung, and liver when as as μg of microsomes were was also obtained with whereas less was detected in rat brain a little β3 was seen in and skeletal muscle Blots were and Fig. B the of the β3 protein within the Na,K-ATPase estimated by the β3 and α subunit staining. found lung and testis to have the highest proportion of β3 isoform expression Na,K-ATPase among the adult rat tissues β3 content was in skeletal muscle, liver, and and in and brain Na,K-ATPase it only a minor It be that the data are expressed to the β3/α in It is known that lung also β1 protein Am. J. Physiol. PubMed Google Scholar), and the proportion of the two isoforms has not yet been determined. The tissues were at Fig. C illustrates that as in the the expression of β3 protein in lung was but it was almost in the the for β3 was in membranes from skeletal Since there was almost no in the amount of α subunit lung and skeletal muscle samples as by this that skeletal muscle has the highest proportion of β3 in the P7 rat of the tissues of Fig. B and illustrates that brain and from rat, skeletal muscle, were more enriched in the β3 protein Na,K-ATPase than those tissues from adult expression of the β3 subunit of the Na,K-ATPase is to in several tissues. In several it has been to active of Na,K-ATPase or α subunits with different β subunits, even The is that of β subunits has on the enzymatic of the most on the for K+ or S. Schachner M. S. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar, P. J. Physiol. 1994; PubMed Scopus Google Scholar, Farley R.A. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar, Farley R.A. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, Biochemistry. 1995; PubMed Scopus Google Scholar, Biochemistry. 1995; PubMed Scopus Google Scholar). This that or developmentally differences in have by of β2 has also been in in the S. H. Sweadner K.J. S. R. M. Schachner M. J. Biol. 1990; PubMed Scopus Google Scholar). Since the β isoforms in the number and of their carbohydrate (14Treuheit M.J. Costello C.E. Kirley T.L. J. Biol. Chem. 1993; 268: 13914-13919Abstract Full Text PDF PubMed Google Scholar, 15Fahrig T. Schmitz B. Weber D. Kucherer-Ehret A. Faissner A. Schachner M. Eur. J. Neurosci. 1990; 2: 153-161Crossref PubMed Scopus (43) Google Scholar, Biochim. Biophys. Acta. 1993; PubMed Scopus Google Scholar, Proc. Natl. Acad. Sci. U. S. A. 1990; 87: PubMed Scopus Google Scholar), such differences in differences in be in The that β3 is expressed at the highest level to α in liver and lung long-standing In the liver, β1 subunit has been detected only density purification Am. J. Physiol. PubMed Google in it was not seen J.J. Schenk D.B. Skelly H. Leffert H.L. Biochemistry. 1986; 25: 4156-4163Crossref PubMed Scopus (30) Google Scholar, A.W. Levenson R. Biochemistry. PubMed Scopus Google Scholar). mRNA for β1 has been or (3Brown T.A. Horowitz B. Miller R.P. McDonough A.A. Farley R.A. Biochim. Biophys. Acta. 1987; 912: 244-253Crossref PubMed Scopus (38) Google Scholar, 4Emanuel J.R. Garetz S. Stone L. Levenson R. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 9030-9034Crossref PubMed Scopus (87) Google Scholar, P. W. J.R. Levenson R. J. Biol. Chem. Full Text PDF PubMed Google Scholar, D. J. Biol. 1993; PubMed Scopus Google Scholar), it was in liver from and was by and by J.R. Garetz S. Stone L. Levenson R. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 9030-9034Crossref PubMed Scopus (87) Google Scholar, V. J. J. T. 1995; PubMed Google Scholar, Leffert H.L. J. Biol. Chem. Full Text PDF PubMed Google Scholar). of the could in be to but an analysis of the distribution of Na,K-ATPase in liver a more In there were the subcellular distribution of reaction for Na,K-ATPase activity and in H.L. Schenk D.B. Skelly H. M. R. Ellisman M. PubMed Scopus Google Scholar). The on whether the pump was on or subcellular membranes, which is to the of and transport the of a while antibody staining a The was by that membrane Na,K-ATPase activity and that activity in the E. Leffert H.L. H. L. F.R. Proc. Natl. Acad. Sci. U. S. A. 1988; PubMed Scopus Google Scholar). by the that Na,K-ATPase subunits were found in both but the β1 subunit was found in the (7Simon F.R. Leffert H.L. Ellisman M. Iwahashi M. Deerinck T. Fortune J. Morales D. Dahl R. Sutherland E. Am. J. Physiol. 1995; 269: C69-C84Crossref PubMed Google Scholar). a less characterized antibody stained the The distribution of reactivity for β1 with the that the of β1 the enzyme this that the β1 seen was to the and of the F.R. Fortune J. Alexander A. Iwahashi M. Dahl R. Sutherland E. J. Biol. Chem. 1996; 271: 24967-24975Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). of the membrane Na,K-ATPase activity in the in β1 was but not in that from β1 was there appear to be differences in Na,K-ATPase on whether or not β1 is expressed there F.R. Fortune J. Alexander A. Iwahashi M. Dahl R. Sutherland E. J. Biol. Chem. 1996; 271: 24967-24975Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). that the β subunit in adult is β3 and that it is for the or no mRNA for β3 has been detected on Northern blots of adult mouse or rat liver (10Malik N. Canfield V.A. Beckers M.C. Gros P. Levenson R. J. Biol. Chem. 1996; 271: 22754-22758Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, C.G. Gong T.L. Lomax M.I. Biochim. Biophys. Acta. 1997; 1350: 21-26Crossref PubMed Scopus (20) Google Scholar), but this is likely to be to the level of expression of Na,K-ATPase subunit, that be by the of β3 in rat liver is a of differences in from and liver Am. J. Physiol. PubMed Google Scholar). Although both of these enzyme preparations contained and β1, the liver had significantly for and The of β3, as as β1, in the liver enzyme for the β3 for K+ than β1 with subunits expressed in Xenopus J. Biol. Chem. Full Text PDF PubMed Google Scholar, P. J. Physiol. 1994; PubMed Scopus Google Scholar). The on Na,K-ATPase expression in lung also has Na,K-ATPase activity the of its in In lung, and β1 mRNA levels at and to a level maintained in the adult R.G. Askew G.R. Wert S.E. Lingrel J.B. Joiner C.H. Am. J. Physiol. 1995; 269: L299-L308PubMed Google Scholar, J. Lingrel J.B. J. Biol. Chem. 1988; 263: Full Text PDF PubMed Google H. Am. J. Physiol. 1993; PubMed Google Scholar). Both in and cells have levels of Na,K-ATPase J. Physiol. 1986; PubMed Scopus Google Scholar, E. J.B. J. 87: PubMed Scopus Google Scholar). The most were obtained by mRNA levels with in R.G. Askew G.R. Wert S.E. Lingrel J.B. Joiner C.H. Am. J. Physiol. 1995; 269: L299-L308PubMed Google Scholar, E. J.B. J. 87: PubMed Scopus Google Scholar). During the mRNA in but not in while β1 mRNA in both at β1 a in while that the of the be associated with β3, since β1 to be in specific cells of the adult mRNA for β3 has been detected in adult rat and mouse lung (10Malik N. Canfield V.A. Beckers M.C. Gros P. Levenson R. J. Biol. Chem. 1996; 271: 22754-22758Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, C.G. Gong T.L. Lomax M.I. Biochim. Biophys. Acta. 1997; 1350: 21-26Crossref PubMed Scopus (20) Google Scholar). In the of β3 associated with Na,K-ATPase in tissues a more of this isoform and to differences for the of several tissues. It has not been that β3 also with a by the of β subunits in expression The of β subunits on α and on the or of the that β has a