Rat brain synthesizes two ‘vitamin D-dependent’ calcium-binding proteins

Brain Research, 345 (1985) 251-256 Elsevier

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Rat Brain Synthesizes Two 'Vitamin D-Dependent' Calcium-Binding Proteins R. POCHET 1, M. PARMENTIER 1, D. E. M. LAWSON 2 and J. L. PASTEELS 1 ILaboratoire d'Histologie, FacultOde M~decine, Universit~ Libre de Bruxelles, 2 rue Evers, 1000 Bruxelles, (Belgium) and :Dunn Nutritional Laboratory, University of Cambridge and Medical Research Council, Milton Road, Cambridge CB4 1XJ ( U. K. ) (Accepted January 8th, 1985) Key words: brain mRNA - - rat cerebellum - - rat kidney - - in vitro translation - - western blotting - - gel electrophoresis - molecular weight determination - - vitamin D-dependent calcium binding protein

Two proteins from rat brain reacting against anti-chick intestinal vitamin D-dependent calcium-binding protein were characterized in terms of their mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and their molecular size. The proteins were present in the isolated cytoplasm and were produced following translation of brain mRNA in the rabbit reticulocyte lysate system. Their apparent molecular weight was 29,000 and 27,000 daltons whereas rat kidney contained only one protein cross-reacting with this antiserum and with a molecular weight of 27,000 daltons.

INTRODUCTION A vitamin D - d e p e n d e n t calcium-binding protein (D-CaBP) was first described by W a s s e r m a n and Taylor in chick intestinal mucosa20. Later, a protein with the same molecular weight (27,000 daltons) cross-reacting immunologically with chick intestinal D - C a B P , was found in kidney, brain, placenta and pancreas from birds and m a m m a l s 19. In addition another vitamin D - d e p e n d e n t calcium binding protein having a different m o l e c u l a r weight (11,000 daltons) and showing no cross-reactivity with the 27,000-dalton form was found in the intestinal mucosa of m a m mals 4. The two calcium-binding proteins seem to play an important role in calcium absorption from the gastrointestinal tract 14 and from kidney tubules since they are located in the kidney at the same site, distal convoluted tubules, at which Ca is reabsorbed6.:2.13 but their wide tissue distribution suggests that they might be involved in o t h e r aspects of calcium metabolism as well. Brain D - C a B P has been localized by immunohistochemistry in chick 11, rat 3 and h u m a n I and synthesized in vitro from the m R N A :6. The n a m e D - C a B P will be used in this p a p e r with reference to the well-

known vitamin D d e p e n d e n c y of that protein in kidney and intestine. It does not imply vitamin D dependency in brain, a m a t t e r still to be assessed. The present work, using both in vitro translation and Western blot analysis reports evidence for the synthesis of two distinct D - C a B P by the brain of the rat. MATERIALS AND METHODS Purification o f chick intestinal D - C a B P and antisera These were p r e p a r e d as described previously ~. Isolation o f p o l y ( A +) m R N A f r o m brain Fresh cerebelli were collected from 10-week-old anesthetized S p r a g u e - D a w l e y rats and immediately frozen in liquid nitrogen, homogenized in 5 vols. of 10 m M Na acetate ( p H 5.0), 3 M LiC1, 6 M urea, 0.5% sodium dodecyl sulfate (SDS), 200 zlg/ml heparim and stored overnight at 0 °C. The pellet from a 16,000 g centrifugation was washed by 4 M LiCI/8 M urea and incubated for 30 min at 37 °C with 1/*g/ml proteinase K. Total R N A was extracted by the phenol/chloroform method. The p o l y ( A ) rich R N A was purified by p o l y ( U ) -

Correspondence: R. Pochet, Laboratoire d'Histologie, Facult6 de M6decine, 2. rue Evers, 1000 Bruxelles. Belgium. 0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

252 Sepharose chromatography and separated through a linear sucrose gradient centrifugation (5-311% sucrose in 10 mM Tris HCI (pH 7.5), 10 mM NaCI and 1 mM EDTA) at 60,000 rpm for 5 h on a SW50Ti rotor. Twenty-five fractions of 200 B! were collected under ultraviolet monitoring, precipitated with ethanol, lyophilized and further used for in vitro translation. The same procedure was used for rat kidney.

Cell-free translation One hundred to 300 ng of mRNA and 25 ~tCi of [35S]methionine (Amersham) were added t o 10 ¢tl of rabbit reticulocyte lysate (N 90 Amersham) and incubated for 90 min at 30 °C. The reaction was stopped by adding 200/A of 1% deoxycholic acid (DOC), 1% Triton X-100 and 0.1% methionine. Measurement of trichloracetic acid (TCA) precipitable radioactivity in released protein was performed as described in ref. 9. The remainder of the translation mixture was adjusted to 50 mM Tris HCI (pH 8.5), 150 mM NaCI and double immunoprecipitation was carried out. The first incubation was performed overnight at 4 °C using rabbit anti-(chick intestinal CaBP) or preimmune serum at a 1/1000 final dilution. Sheep anti(rabbit IgG) was further added to a 1/1000 dilution and the mixture incubated in the same conditions. Immunoprecipitates were recovered using a Beckman microfuge B, and washed 3 times in distilled water, dissolved in 70 #1 of electrophoresis sample buffer (0.1 M Tris HCI (pH 8.0), 2 mM EDTA, 1% SDS, 2% mercaptoethanol, 20% sucrose, 0.1% phenol red) and heated for 2 min at 100 °C. Five-/A aliquots were counted in a liquid scintillation counter. 30-~1 aliquots were used for a SDS discontinuous potyacrylamide gel electrophoresis on a 12.5% slab gel. Electrophoresis gels were treated with Enhancer solution (NEN), dried and exposed for 5 weeks at -80 °C with XIAR5 Kodak films.

Electrophoretic transfer was performed at 313 V for 5 h. The blots were soaked for 1 h in a Coons Veronal buffered saline (CVBS) solution (pH 7.2) containing 3% gelatin, to saturate unoccupied protein binding sites. They were rinsed with CVBS containing 0.2% Triton X-100, preincubated with 5% normal goat serum (NGS) for 15 min and incubated for 48 h at 4 °C with rabbit anti-chick intestinal CaBP antiserum (1:1000) in CVBS. Blots were further rinsed twice in CVBS X-100, incubated with goat anti-rabbit gammaglobulin (1:100), rinsed twice and incubated with soluble peroxidase antiperoxidase complex (1:600) (PAP) for 10 min at room temperature. The blots were washed as described above, and incubated with a substrate solution containing 3,3'-diaminobenzidine tetrahydrochloride (DAB) (0.5 mg/ml), and 0.01% hydrogen peroxide in a citrate phosphate buffer (H 6.2). The brown bands corresponding to protein appeared within a few minutes, and the reaction was terminated after 10 min by washing with water. Calibration of the gel was performed by incorporating in the same gel, tritium-labeled proteins of known molecular weights (Amersham). The stained blot was soaked for 10 min in Enlighting solution (NEN), air dried, affixed to a sheet of filter paper and exposed to Kodak X1AR5 film for 1 week at -80 °C.

Degradation experiments The homogenates were incubated at 37 °C in 20 mM Tris HC1 pH 7.4 medium for various times in the presence or absence of a mixture of protease inhibitors. The protease inhibitor cocktail contained iodoacetamide (100 ~tM), phosphoramidon (2/xg/ml), Trasylol (100 U/ml), antipain (20 ~zg/ml), leupeptin (20/xg/ml) and EDTA (1.35 mg/ml). Phosphoramidon, leupeptin and antipain were provided by Peptide Institute, Japan. Trasylol was obtained from Bayer, Germany. RESULTS

Western blot analyses D-CaBP immunoreactive proteins were visualized by immunoperoxidase staining with the modified method of Towbin et a1.17. Electrophoresis was performed in a 12.5% polyacrylamide separating gel containing 0.1% SDS 5. The proteins were then electrophoretically transferred to nitrocellulose paper as described by Burnette2 in a BioRad Transblot Cell.

Cell-free synthesis Fresh rat cerebellum (7 g) extracted as described in the previous section gave 4.36 mg of total RNA from which 96 beg of Poly(A) containing RNA were recovered after poly(U) Sepharose chromatography. The mRNA profile after sucrose gradient centrifugation (Fig. 1) showed a classical pattern of undegraded

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Fig. 1. Sucrose gradient centrifugation profile of rat poly A-RNA (---) and total mRNA activity. Twenty-two fractions were collected and used for in vitro synthesis using [35S]methionine and reticulocyte lysate. Total protein synthesis was measured by counting hot TCA-precipitable materials.

m R N A with an additional 18-S p e a k due to a small degree of contamination with r R N A . The position of cerebellar r R N A in the sucrose gradient was found in the same run (data not shown). The fractions collected from the gradient were used for m e a s u r e m e n t of in vitro translation activity and the p a t t e r n of incorporation of radioactivity into hot T C A precipitable material is also shown in Fig. 1. The radioactivity profile was superimposable on the m R N A profile, with the expected exception of the 18-S r R N A peak. The m a x i m u m incorporation of [3sS]methionine into the T C A precipitate (6.25% of the a d d e d radioactivity) occurred in fraction 13. To further characterize the T C A - p r e c i p i t a b l e material, it was i m m u n o p r e c i pitated as described above and the proteins separated by polyacrylamide gel electrophoresis. The aut o r a d i o g r a p h y of the gels (Fig. 2) clearly r e v e a l e d the presence of two specific bands with a p p a r e n t molecular weights of 27,000 and 29,000 daltons (Table I). A third less intense band, corresponding to a molecular weight of 45,000 daltons, was non-specific material since it coprecipitated both with i m m u n e and nonimmune serum. The molecular weight values were the mean of 3 i n d e p e n d e n t estimations o b t a i n e d by corn-

parison with t4C-methylated standard proteins run on the same slab gel. A u t o r a d i o g r a p h s were run on a scanning d e n s i t o m e t e r (Shimadzu CS-930). Surface calculation of the D - C a B P peaks indicated that two proteins synthesized by the lysate were present in equal amounts. The same experiments were perf o r m e d with rat kidney m R N A but in this case only one band was specifically i m m u n o p r e c i p i t a t e d (not shown). Its a p p a r e n t molecular weight (27,000 daltons) was identical to the smaller D - C a B P form synthesized in vitro from rat brain m R N A s . Western blot analysis D - C a B P from rat brain or kidney h o m o g e n a t e was specifically recognized after S D S - P A G E and electroTABLEI Brain D-CaBPs molecular weights" The molecular weights were calculated after blotting experiments and using [14C]methylated proteins with known molecular weights (Amersham CFA 626). Part B of the table shows the values obtained from 3 different experiments. (A)

D-CaBP (daltons)

Proposed nomenclature

Rat kidney degraded form* Rat brain I II

27,099 _+.+_322 25.970 + 293 27,223 + 368 28,848 _+406

n= n= nn-

16 6 16 16

D-CaBP-27 D-CaBP-27 D-CaBP-29

(B)

Expr 1

Expr 2

Expr 3

Rat kidney Rat brain I II Rat kidney degraded form

27,245 _+208

27,461 _+98

27,039 _+202

26,802 _+50 28,433 _+ 176

27,316 + 138 27,204 _+ 151 29,022 + 184 28,637 _+210

25,794 + 60

26,186 _+ 358

* This form appeared only after 4 h incubation at 37 °C

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Degradation experiments" The stability of D - C a B P has been evaluated by incubation of cerebellar and kidney homogenates at 37 °C in Tris buffer (pH 7.4). At various times aliquots of the incubation solution were submitted to electrophoresis and Western blotting. D-CaBP was extremely stable both in kidney and brain homogenate. Only after 4 h incubation at 37 °C was a smaller molecular weight band detected in the kidney (Fig. 4 and Table I). The addition to the incubation medium of a mixture of protease inhibitors prevented this degradation. In brain the ratio between the two CaBP proteins remained constant even after 10 h incubation without protease inhibitors. DISCUSSION

Fig. 3. Western blotting. Transfer of total homogenate proteins from 12.5% SDS-PAGE electrophoresis to nitrocellulose sheet and immunostained using a-CaBP. 1, rat cerebellum 4-Mghomogenate proteins; 2, rat cerebellum 40-#g homogenate proteins; 3, rat kidney 3 ~g homogenate proteins; 4, rat kidney 30ug homogenate proteins.

phoretical transfer to nitrocellulose paper. After incubation with rabbit anti-chick CaBP, both brain and kidney D - C a B P were visualized by immunoperoxidase staining (Fig. 3). The results show the presence in brain of the two bands reacting with a n t i D - C a B P compared to only one in kidney. Their apparent molecular weights were calculated as before and were identical with the sizes estimated for the protein synthesized in vitro (27,000 and 29,000 daltons for brain and 27,000 daltons for kidney).

In this study we have identified and characterized in terms of molecular weight two rat cerebellar proteins. These two proteins are presumably different forms of the protein used to raise the antiserum: indeed the smaller protein seems to be very similar to that in rat kidney and chick intestine since they are all the same size t27.000 daltonsj and react against the same antiserum Degradation of the D - C a B P in intestine was observed from the first isolation of this protein. In the case of bovine intestinal CaBP the degradation is known to involve in one case the loss of the N-terminal amino-acid and in the other case the loss of two N-terminal amino acids:. In this study, we have excluded that degradation was responsible for the existence of two forms of CaBP. We have shown that even at 37 °C. the two forms of the protein are stable for several hours and do not degrade into lower molecular weight forms. Further, the two forms were identified with unrelated methods, the first using extracted brain m R N A s and their translation in rabbit reticulocyte lysate system, and the second by electrophoretic analysis of the cytosolic proteins from whole cerebellar homogenate. Although the larger protein could be a precursor form of the other no evidence for such a system has been seen for intestinal CaBP whose synthesis is known in some detail. Consequently our results suggest that rat cerebellum contains two m R N A coding for two D-CaBPs. The existence of these two

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Fig. 4. Degradation assay. Total homogenates from rat kidney (1 and 2) or cerebellum (3 and 4 ') were incubated at 37 °C in 20 mM Tris HCI pH 7.4 for various times in the presence (1 and 3) or absence (2 and 4) of protease inhibit6 .rs then electrophoresed, transferred to nitrocellulose paper and immunostained using a-CaBP.

m R N A s could be explained by two different mechanisms. On the one hand, the proteins can be expressed by two different genes, one being specifically expressed in brain and not in kidney. On the other hand, they might result from organ-specific processing of D - C a B P m R N A precursors. This latter mechanism has already been demonstrated for many proteins such as immunoglobulin 7, myosin light chain s, calcitonin m, preproopiomelanocortin 21. The first possibility might be correlated to the recent finding that a 16 kdalton specific transcript only present in rat brain is able to activate in vitro the polymerase II and through this mechanism activate genes usually silent in other organs ~5. We are still far from demonstrating what exact mechanism is responsible for the existence of two rat brain D-CaBP but our next step will be to clone rat brain D-CaBP in order to reach such a goal. We recently reported preliminary data on comparative studies in rats, chicken, lizards, frogs and a mormyrid fish, using both Western blotting and im-

munohistochemistry a's described in the present work. In every specie s except fish, two D - C a B P cross-reacting with the ct'aiel~n antigen were found in the brain, whereas only ~ me was present in intestine and/or kidney. Interesting' ly in ~ e fish, no CaBP was found in kidney and gut, u hile al least one molecular form was detected in the b iain, This suggest that the first functional role of D-C~tI3P is retafed to net!tonal activity rather than calcium a b s o r l ~ ~. kidney or intestine. ACKNOWLEDGEMENTS We thank C. Hubeau and M. Chirnoaga for helpful technical assistance anLd P. Miroir for typing the manuscript. This work ha:~ been supported by Foundation Reine Elisabettl, by F.R.S.M. Grant 3.4501.84 to J.L.P. and by the Minist6re de l'Emploi et du Travail. M.P. is an ~ s p i r a n t du Fonds National de la Recherche Scientifiq~ Je de Belgique.

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