Cell Calcium 7: 41-47, 1986

THE USE OF MONOCLONAL ANTIBODIES FOR THE SPECIES

AND TISSUES DISTRIBUTION OF PHOSPHOLAMBAN

Takashi Suzuki, Perry Lui and Jerry H. Wang

Cell Regulation Group, Department of Medical Biochemistry The University of Calgary, Calgary, Alberta, CANADA

(reprint requests to JHW)

ABSTRACT

Monoclonal antibodies have been raised against canine phospholamban. Two antibodies have been used in the study of phospholamban distribution in tissues, and in different animal species. The antibodies recognized the three different forms of phospholamban: non-phosphorylated, phospho- rylated, and dissociated forms. A survey of nine rabbit tissues revealed that phospholamban is a cardiac muscle specific protein. Phospholamban from different mammalian hearts were found to be identical with respect to molecular weight and antigenicity.

INTRODUCTION

The stimulation of (Ca2+, Mg2+)-ATPase and calcium uptake into the cardiac sarcoplasmic reticulum (SR) appears to be regulated through the phosphorylation of phospholamban (1,2). Phospholamban has been found in various cardiac tissues. The molecular weights reported for these proteins have varied from 20,000 to 27,000 (1). This variation may be due to the different intrinsic properties of the proteins or the different SDS-PAGE systems used in the studies. In addition, it is not known whether phospholamban is specific to cardiac tissue or not (3).

We have prepared two monoclonal antibodies against canine phospho- lamban, and used these antibodies to demonstrate that phospholamban from various mammalian hearts have virtually identical molecular weight and antigenicity, and that phospholamban exists only in the heart.

MATERIALS AND METHODS

Preparation of SR and partially purified phospholamban: Membrane fraction enriched in SR (crude SR) was obtained by a slight modification of the method described by Jones et al (3). Homogenization of tissues and subsequent SR preparation was carried out with a buffer containing 10 mM HEPES, pH 7.5, 0.3 M sucrose, 0.1 mM PMSF and 1 mM DTT (buffer A)

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instead of 10 mM NaHCO 3 solution.

Crude SR (5 mg/ml) was solubilized by the addition of 50 mg solid SDS/ mg protein followed by dialysis against 5 mM HEPES buffer, pH 8.0, contain- ing 0.1% SDS and 1 mM 2-mercaptoethanol. The solubilized sample (10 ml) was applied to a Sepharose CL-6B(5x125) column. Fractions containing phospholamban were pooled, dialyzed against 5 mM Tris-HCl, pH 7.5, contain- ing 0.05% bovine serum albumin and concentrated by Pro-Dicon. This parti- ally purified phospholamban was used for the ELISA screening of antibody production and the preparation of pure phospholamban for immunization of mice. The details of the production of monoclonal antibodies against phospholamban will be described elsewhere.

Preparation of crude membranes from tissues: Fresh tissues were immediately homogenized in 5 volumes of buffer A, followed by centrifuga- tion at 100,000 x g, and the resulting pellets (crude membrane fractions) were suspended in buffer A. An aliquot of the suspension (100 pg of protein) was mixed with SDS-sample buffer for SDS-PAGE.

Phosphorylation by catalytic subunit of CAMP-dependent protein kinase: Phosphorylation was carried out at 30°C for 6 min. in a 0.1 ml reaction mixture containing 25-50 ug of protein, 3 ug of the catalytic subunit, 50 mM histidine-HCl, pH 7.0, 5 mM MgC12, and 0.2 mM [Y-~*P]ATP (specific activity, 100-200 dpm/pmol). The reaction was terminated by the addition of 100 ~1 of SDS-sample buffer.

SDS-polyacrylamide gel electrophoresis, immunotransblotting and autoradiography: SDS-gel electrophoresis (SDS-PAGE) was performed with a 5-20% polyacrylamide gel according to Laemmli (4). After SDS-PAGE, the proteins on the gel were electrophoretically transferred to Zeta- probe membrane at 3'C for 2 hr in a buffer consisting of 25 mM Tris-HCl, pH 8.3, 192 mM glycine, and 0.03% SDS. The membrane was then soaked in 100 ml of Tris-saline buffer (20 mM Tris-HCl, pH 7.5, 500 mM NaCl) containing 10% fetal calf serum (FCS) and incubated at room temperature overnight. After removal of excess FCS by washing with Tris-saline, the membrane was incubated with anti-phospholamban antibody followed by incubation with either horseradish conjugated anti-mouse IgG or 1251- labelled anti-mouse IgG. Immuno-reactive proteins were visualized either by horseradish peroxidase activity using 4-chloro-1-napthol as the subst- rate or autoradiography.

RESULTS AND DISCUSSION

Several monoclonal antibodies have been produced by using a SDS-gel electrophoresis purified canine cardiac phospholamban for the immunization of mice and a relatively impure phospholamban preparation (< 5%, Fig. lA, lane C) in the ELISA screening of antibody-producing hybridomas. To provide positive identification of phospholamban antibodies, we used

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SR I PP-PLB I 8R I Pp-PLB I SR , PP-PLB losphorywon - - + + - - + + - - + + , - - + + - - + + - - + +

bO,,,~ _ + - + , - + - + , - + - + , - + - + , - + - t, - + - +

Fig. 1. Specificity of monoclonal antibody against phospholamban. Canine cardiac sarcoplasmic reticulum (SR 50 iig> and partially purified phospho- lamban (PP-PLB; 25 ug) were subjected to SDS-PAGE (A), transferred to Zeta-probe membrane, and incubated with anit-phospholamban antibody. Immuno-reactive proteins and phosphorylated proteins were visualized by incubation with horseradish peroxidase conjugated anti-mouse IgG (B) and autoradiography (C), respectively. (+> indicates samples treated by either phosphorylation using catalytic subunit of CAMP-dependent protein kinase (PK) or boiling in 1% SDS prior to electrophoresis. (-1 indicates samples not subjected to these treatments. Abbreviations used:

PHLH, intact phospholamban; PHLL, dissociated phospholamban; and [32P]PHL, phosphorylated PHLH and PHLL.

Western immunoblotting technique to examine selected culture media, which showed positive ELISA reaction. Phospholamban exhibits slightly lower electrophoretic mobility upon phosphorylation ([3z~l~~~~>, and converts to a much lower molecular weight species upon boiling in 1% SDS(PHLL) (6). By taking advantage of these characteristic SDS-PAGE mobility shifts of phospholamban, immunoblotting provided unambiguous identification of phospholamban antibodies (Fig. 1). Cells producing phospholamban antibody were subcloned by limiting dilution, monoclones were then cultured and injected into mice to provide ascites fluids in pristane-primed mice. For the present work, ascites fluid with high titers were used.

To characterize the specificity of the anti-phospholamban antibodies, crude canine cardiac SR and partially purified phospholamban with or with- out phosphorylation were subjected to SDS-PAGE (Fig. ZA), immunotransblot- ting (Fig. lB), and autoradiography, (Fig. lC>. Immunoreactive protein bands exhibited the exact characteristic SDS-PAGE mobility shifts of phospholamban upon phosphorylation, and boiling in SDS solution. In addition, when phosphorylated phospholamban was used, the radioactive band corresponded exactly to the immuno-reactive band. Among multitudes of protein bands observed on SDS gel patterns of crude membrane

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fractions, the antibody recognized only one protein, phospholamban, showing high immuno-reactivity towards phospholamban. Both antibodies reacted with the three different forms of phospholamban.

Phospholamban was originally found in canine cardiac SR(5), but similar proteins have also been reported in cardiac tissues from various animals (1). These proteins show similar characteristic SDS-PAGE mobility shifts as canine cardiac phospholamban, however, their reported molecular weights vary from 20,000 to 27,000 (1). By using immunotransblotting technique, a comparison of phospholambans in several mammalian hearts was carried out. The immunoautoradiography revealed that phospholambans from different animals are virtually identical, at least with respect to molecular weight and antigenicity. (Fig. 2)

31K-P-F PHL,*

PHL y

A B C I D

boiling - + - + - + 1 - +

Fig. 2. Demonstration of phospholamban in different mammalian cardiac tissues by immunotransblotting. Crude canine SR (50 ug, lane A) and proteins (100 ug) from 100,000 x g particulate fractions of rabbit (lane B), bovine (lane C),and rat hearts (lane D) were subjected to SDS-PAGE, transferred to Zeta-probe membrane, and incubated with anti-phospholamban antibody followed by 1251-anti-mouse IgG. Symbols are the same as in Fig. 1. 31K-P indicates an immuno-cross-reactive protein to anti-phospho- lamban antibody.

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Previous studies have indicated that phospholamban is not present in skeletal muscle (6,7). However, the possible existence of phospho- lamban in other tissues has not been systematically investigated. We have checked for the presence of phospholamban by immunoblotting of crude membrane fractions from various rabbit tissues. Of the eight tissues surveyed, heart was found to be the only tissue containing phospholamban (Fig. 3). Although the results indicated that phospholamban was a cardiac-

ABCDEFGHI

Fig. 3. Autoradiogram of immunotransblot showing tissue distribution of phospholamban in rabbit. Crude canine SR (50 ug, lane A) and proteins (100 ug) of particulate fractions from rabbit heart (lane B), leg white muscle (lane C>, brain (lane D), lung (lane E), liver (lane F), spleen (lane G), kidney (lane H), and platelets (lane I> were subjected to SDS- PAGE, immunoblotted using 1251-labelled second antibody and then auto- radiographed.

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specific protein, it was possible that the other tissues contained isoproteins of phospholamban which did not cross-react with the anti- body. A platelet protein has been postulated to be phospholamban-like based on its similar molecular weight and phosphorylatability (8). We did not detect any immuno-reactive protein in platelets. It is, therefore, not clear whether this protein is a phospholamban or not. The platelet protein also did not show SDS-PAGE mobility change upon boiling in 1% SDS solution (8). It is interesting to note that a 31-kDa protein in very crude membrane preparations of cardiac tissues was found to cross- react with both phospholamban antibodies, albeit weakly, but it did not show a characteristic mobility shift upon boiling as in phospholamban (Fig. 2). This protein was not detected in pure SR (data not shown).

In the present communication, we demonstrate that phospholamban is a cardiac specific protein and that phospholambans in various cardiac tissues are indistinguishable, in terms of antigenicity and SDS-PAGE mobilities. The availability of specific phospholamban monoclonal antibodies would facilitate the study of various aspects of this regulatory protein.

ACKNOWLEDGEMENTS

This work is supported by an operating grant from the Medical Research Council of Canada, and an establishment grant from the Alberta Heritage Foundation for Medical Research (AHFMR). T.S. is a postdoctoral fellow of the AHFMR, P.L. is recipient of AHFMR studentship, and J.H.W. is an AHFMR Medical Scientist. We are grateful to Dr. R.K. Sharma for the generous supply of catalytic subunit of CAMP-dependent protein kinase.

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2.

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4.

REFERENCES

Tada, M. and Katz, A.M. (1982) Phosphorylation of the sarcoplasmic reticulum and sarcolemma. Annu. Rev. Physiol. 44. 401-423.

Ambudkar, I.S. and $hamoo, A.E. (1984) Role of Phospholamban in Regulating Cardiac Sarcoplasmic Reticulum Calcium Pump. Membrane Biochem, 5_, 119-130.

Jones, L.R., Besch, H.R., Fleming, J.W., McConnaughey, M.M. and Watanabe, A.M. (1979) Separation of Vesicles of Cardiac Sarcolemma from Vesicles of Cardiac Sarcoplasmic Reticulum. J. Biol. Chem. 254, 530-539.

Laemmli, U.K. (1970) Cleavageof Structural Proteins during the Assembly of the Heat of Bacteriophabe T4. Nature, (London) 227, 680-685.

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Tada, M. Krichberger, M. and Katz, A.M. (1973) Phosphorylation of a 22,000 Dalton Component of the Cardiac Sarcoplasmic Reticulum by Adenosine 3':5'-monophosphate-dependent Protein Kinase. J. Biol. Chem. 250, 2640-2647.

Inui, M., Kodama, M. and Tada, M. (1985) Purification and Characteri- zation of Phospholamban from Canine Cardiac Sarcoplasmic Reticulum. J. Biol. Chem. 260, 3708-3715.

Kirchberger, M.A., Tada, M. (1976) Effects of Adenosine 3':5'-mono- phosphate-dependent Protein Kinase on Sarcoplasmic Reticulum Isolated from Cardiac and Slow and Fast Contracting Skeletal Muscles. J. Biol. Chem. 251, 725-729.

Kgser-Glanzmann, R., Jakabova, M., George, J.N. and Lsscher, E.F. (1977) Stimulation of Calcium Uptake in Platelet Membrane Vesicles by Adenosine 3',5'-cyclic Monophosphate and Protein Kinase. Biochim. Biophys. Acta. 466, 429-440.

Received 28.8.85 Revised version received and accepted 27.11.85

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