- Email: [email protected]
[29]Spec proteins: Calcium-binding proteins in the embryonic ectoderm cells of sea urchins
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[29] S p e c P r o t e i n s : C a l c i u m - B i n d i n g P r o t e i n s in t h e E m b r y o n i c E c t o d e r m Cells o f S e a U r c h i n s
By WILLIAM H. KLEIN, SUSAN H. HARDIN, and BRUCE P. BRANDHORST
Introduction During its embryonic and larval development, the sea urchin Strongylocentrotus purpuratus synthesizes and accumulates a set of calciumbinding proteins detectable exclusively in dorsal (aboral) ectoderm cells.1 These proteins, termed Spec proteins, are related to the more extensively characterized calcium-binding proteins such as calmodulin, myosin light chains, troponin C, and parvalbumins.l The in oivo function of the Spec proteins is unknown but it has been hypothesized that they play a role in changing the shape of the ectodermal cells.l Supporting this hypothesis is the observation that two actin genes, cylIIa and cylllb, are restrictively expressed in the same cells and at the same time in development as are the Spec genes. 2 This suggests a structural and/or functional relationship between the Spec proteins and the cylII actin genes. There are two periods in embryonic and larval development when dorsal ectoderm cells are morphologically reshaped: After gastrulation when the cuboidal ectoderm cells stretch and flatten to become the squamous epithelial epidermis of the pluteus larva; and at metamorphosis, when microfilaments present in the dorsal epithelium contract, resulting in the collapse of the larval structure and release of the rudimentary juvenile sea urchin. 3 It is possible the Spec proteins are involved in either or both of these morphogenetic processes. Whatever their precise function, given the preservation of their calcium-binding domains it seems likely that the Spec proteins participate in some cellular process mediated by calcium ions. The Spec proteins are likely to be of interest in investigations of the evolution and role of the troponin C superfamily of calcium-binding proteins.
C. P. 2 R. 3 R.
D. Carpenter, A. M. Bruskin, P. E. Hardin, M. J. Keast, J. A n s t r o m , A. L. Tyner, B. Brandhorst, and W. H. Klein, Cell (Cambridge, Mass.) 36, 663 (1984). J. Shott, J. J. Lee, R. J. Britten, and E. H. Davidson, Dev. Biol. 101, 295 (1984). A. C a m e r o n and R. T. Hinegardner, J. Morphol. 157, 21 (1978).
METHODS IN ENZYMOLOGY,VOL. 139
Copyright © 1987by AcademicPress, Inc. All rights of reproduction in any form reserved.
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Spec Genes and mRNAs The Spec gene family is composed of two subfamilies, Spec 1 and Spec 2. Spec 1 is a single gene that encodes a 1.5-kb mRNA, the predominant mRNA of the family (0.6% of the mRNA of the gastrula stage embryo) and the Spec 1 protein is the most prevalent of the Spec proteins) ,4 In contrast to Spec 1, there are about 10 Spec 2 genes, which encode a set of mRNAs of about 2.2 kb that are much less prevalent than Spec 1 mRNA. Accumulation of the Spec 1 and Spec 2 mRNAs and proteins begins in late cleavage and is highly restricted to dorsal ectoderm cells) Several Spec genes have now been cloned and all show strong similarities in their exon/intron structure to genes encoding calcium-binding proteins in other organisms. These include chicken calmodulin, for which the resemblance is particularly striking, and myosin light chains 1 and 3 in chickens and mice. While the nucleotide sequence conservation is weak, the placement of the introns is conserved. Nevertheless, the exon/intron placement does not correspond to any known structural or functional domains in the proteins. 4 Preparation and Handling of Sea Urchin Embryos S. p u r p u r a t u s sea urchins are obtained from Pacific Biomarine (Venice, CA) or Marinus (Westchester, CA). Gravid adults are generally available from December through May. They may be held for several months at 8-12 ° in natural or artificial sea water (Instant Ocean, from Aquarium Systems, Eastlake, OH) in tanks utilizing filtration through calcareous gravel and activated charcoal, extensive gas exchange via airstones, and oxidation of ammonia by nitrifying bacteria. While tap water is acceptible for making sea water in some locations, we normally pass the water through activated carbon and mixed bed deionizing columns. Shedding of gametes is induced by intracoelomic injection of a total of 0.5-2.0 ml 0.5 M KC1 through several sites on the peristomial membrane on the oral side. Semen (cream colored) is collected " d r y " in a 0.1-ml serological pipet or capillary tube using a Clay-Adams pipetting aid and can be stored for several days at 0-4 ° . Eggs (orange) are collected by inverting the female over a beaker filled with cold (4-8 °) artificial sea water (ASW). A recipe for AWS,6 made up using glass-redistilled water
4 s. H. Hardin, C. D. Carpenter, P. E. Hardin, A. M. Bruskin, and W. H. Klein,J. Mol. Biol. 186, 243 (1985). s A. M. Bruskin,P.-A. Bddard,A. L. Tyner,R. M. Showman,B. P. Brandhorst,and W. H. Klein, Dev. Biol. 91, 317 (1982). 6R. T. Hinegardner, in "Methods in DevelopmentalBiology" (F. H. Wilt and N. K. Wessels, eds.), p. 139. Crowell-Collier,New York, 1967.
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and reagent grade chemicals, is (in g/liter): NaCI, 28.31, KC1, 0.77, MgCI2 • 6H20, 5.41, MgSO4-7H20, 7.13, CaCI2, I. 18, to which solution is added 0.2 g/liter NaHCO3; the pH should be adjusted to 8.2. Artificial sea water lacking calcium and magnesium (CMFSW) consists of (in g/liter): NaC1, 27, Na2SO4, 1.0, KC1, 0.8, and NaHCO3, 0.18. Embryos can also be cultured in natural sea water or Instant Ocean filtered through 0.22-/.~m nitrocellulose filters. AWS can be pasteurized but not autoclaved. After the eggs settle, excess ASW is removed by aspiration, and the eggs are resuspended in ASW and filtered through several layers of cheesecloth into a large volume of cold ASW (200-fold excess). After settling, eggs are resuspended in 100 vol cold ASW and fertilized by addition of 1/100 vol of semen prediluted 1/100 in ASW. After 2-3 min egg samples are examined by bright-field microscopy for elevation of the fertilization envelope; if fertilization is less than 95% another aliquot of sperm suspension is added. The fertilized eggs are allowed to settle twice through a large volume of cold ASW and then resuspended in ASW at 15° (containing 80 U/ml penicillin and 50/zg/ml streptomycin sulfate to inhibit bacterial growth) at a concentration of 1-5 × 103 eggs/ml (determined by counting a sample of the egg suspension drawn into a 10-/zl capillary tube; there are about 2 x l06 dejellied 70-/xm eggs in 1 ml after centrifugation at a few hundred gravities). Embryos are cultured at 14-16 °, stirred at 60 rpm with an acrylic paddle attached to a clock motor; for cultures of several liters compressed air is bubbled through the culture. First cleavage occurs about 90 min after suspension in 16° water, hatching from the fertilization envelope occurs at about 18 hr, gastrulation begins at about 36 hr and is completed by 48 hr, and plutei capable of feeding appear after 3-4 days. For optimum cultures, embryos' are collected by filtration through 44-/~m nylon mesh (Nitex, Tetco, NY) after hatching and resuspended at 100-1000/ml. Newly synthesized proteins can be labeled by incubation of 20,000 embryos/ml in a fiat-bottom vessel with 50-100/xCi/ml [35S]methionine (1300 Ci/mmol, Amersham) for 30-50 min, providing occasional agitation. Fractionation of embryos into tissue layers can result in changes in patterns of labeling of proteins, including the failure to label the Spec proteins; in part this is the result of changes in amino acid uptake by aboral ectoderm cells (P.-A. Bedard and B. P. Brandhorst, unpublished observations). Preparation of Ectoderm and Endoderm Fractions As first exploited by McClay and Marchase, 7 ectoderm cells can be selectively dissociated from embryos by dispersion of the enmeshing hya7 D. McClay and R. B. Marchase, Dev. Biol. 71, 289 (1979).
[29]
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line layer in media lacking divalent cations8; a residual basal laminar bag contains endoderm cells, as well as a few mesoderm cells. Cultures of pluteus stage embryos are harvested by filtration through 44-/xm nylon mesh. Embryos are resuspended in 40 vol cold ASW, collected by light centrifugation (medium speed in a clinical or hand centrifuge: 100-300 g, 1 min), and resuspended in 1 M glycine, 2 mM EDTA for 3 min on ice (optimal time is stage and batch dependent), collected by light centrifugation, and resuspended in 20 vol CMFSW. The ectoderm cells are dissociated from the basal lamina by repeated gentle aspiration through a Pasteur pipet or by passage through a 10-ml syringe lacking a needle. Dissociation is monitored by light microscopy, and continued until most ectoderm cells are released. The resuspension is then filtered through 28- or 35-/xm nylon mesh to collect a crude endoderm/mesoderm fraction. The ectoderm cells in the filtrate are collected by centrifugation at 5000 rpm for 5 min in a Beckman JA-20 rotor (3000 g maximum). The endoderm/mesoderm fraction can be further purified by resuspension and agitation in CMFSW followed by refiltration. Brief trituration of the bags in CMF at this step leads to the release of many of the mesoderm cells while the endoderm (archenteron) remains intact. 7 As an alternative to filtration (particularly at stages prior to spicule elongation when the bags tend to penetrate the filter), differential sedimentation can be employed. Endoderm/mesoderm is collected at 300 rpm in a Sorvall GLC rotor (116 g maximum) for 15 sec; several repeated centrifugations of both the pellet and the supernatant are required to obtain fractions of high purity. Alternatively, cells can be separated by isopycnic centrifugation on Percoll gradients. 9 This is particularly useful when epithelial cells (presumptive endoderm and ectoderm) are dissociated from basal laminar bags of mesenchyme blastulas, resulting in a fraction containing mesoderm cells. Screening for Nucleotide Sequences Encoding Calcium-Binding Proteins Several eDNA libraries are available that have been constructed from RNA of S. purpuratus and Lytechinus pictus embryos. These include egg, blastula, gastrula, and plutcus stage libraries made from total or polysomal polyadenylated RNA. The recombinants are packaged in either phage or plasmid-harboring Escherichia coli. Spec sequences, as well as other cell or stage-specific sequences, can be readily isolated from such libraries by routine differential screening procedures. These libraries are available upon request. 8 R. E. Kane, Exp. Cell Res. 81, 301 (1973). 9 M. A. Harkey and A. H. Whiteley, Wilhelm Roux's Arch. Dev. Biol. 198, 111 (1980).
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A probe has been constructed to isolate any nucleotide sequence encoding a calcium-binding protein belonging to the troponin C superfamily. A comparison of published nucleotide sequences of several calcium-binding protein genes shows that a 43-bp region covering the third calciumbinding domain is the most conserved region of the molecule. The 43-base consensus sequence T A A G (5'-ACTGACGGAGCCGTTGCCATCCTTGTCGAAGACACGCAAAGCT-Y)
G
C
has approximately 85% match to the corresponding region of most of the reported calcium-binding protein genes and is complementary to the transcribed sequence (S. H. Hardin and W. H. Klein, unpublished observations). Under appropriate conditions, this probe hybridizes specifically to various genes and mRNAs in the troponin C superfamily and should be of use in isolating and characterizing genes for calcium-binding proteins. A probe consisting of 0.1 /zg DNA labeled to high specific activity with [32p]ATP and T4 polynucleotide kinase 1° is hybridized to a DNA or RNA sample blotted onto a nitrocellulose filter. The filter is prewashed in a sealed plastic bag for 1 hr at 68° in 50 ml of 10x Denhardt's (0.2% bovine serum albumin, 0.2% Ficoll, 0.2% polyvinylpyrrolidone-40), 0.1% SDS, 0.2 M sodium phosphate, pH 7.0, and 5x SET (0.75 M sodium chloride, 0.15 M Tris-HC1, pH 8, 10 mM EDTA). This solution is removed and the filter is then prehybridized at 37° in 5 to 10 ml of 1 x Denhardt's, 20 mM sodium phosphate (pH 7.0), 5x SET, 10% Dextran sulfate, and sheared calf thymus DNA (100 ~g/ml). After 1 hr the probe is added and allowed to hybridize for 10 to 12 hr. The hybridization buffer is removed and the filter is washed for 1 hr at 45 ° with 50 ml of 5× SET, 0.1% sodium pyrophosphate, 25 mM sodium phosphate (pH 7.0), and 0.1% SDS. The filter is then washed for 1 hr at 45 ° with 50 ml of 1x SET, 1 x Denhardt's, 0.1% sodium pyrophosphate, and 25 mM sodium phosphate and exposed to X-ray film. Two-Dimensional Eleetrophoretic Analysis of Spec Proteins The position of the Spec proteins compared to other newly synthesized proteins is shown in Fig. 1. Spots 1 and 2 are products of the Spec 1 gene, while the various spots identified by arrows are ectoderm specific proteins which are mostly or entirely the products of Spec 2 genes. Spot 1 has the same pI as the major sea urchin embryonic form of actin, which 10 T. Maniatis, E. F. Fritsch, and J. Sambrook, "Molecular Cloning: A Laboratory Manual," p. 122. Cold Spring Harbor Lab., Cold Spring Harbor, New York, 1982.
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-5O Mr
(X 10-3)
25
7
6
5 pH
FIG. 1. Two-dimensional electrophoretic properties of Spec proteins. Proteins of plutei labeled with [35S]methionine were analyzed as described. Arrows identify small, acidic proteins synthesized only in ectoderm; most, possibly all, of these are encoded by Spec genes. Positive identification of some spots is impossible since the cell-free translation products of Spec 2 mRNAs do not all comigrate with proteins synthesized in vivo. Spots 1 and 2 are encoded by the Spec I gene. The position of the major actin isoform is indicated by the letter A.
comigrates with m o u s e fl-actin H (pI of about 5.7 in 9.5 M urea). Comparison of relative mobilities with m a r k e r s indicate a range of apparent molecular weights o f 14,000-17,000 for the Spec proteins, as shown in Fig. 1. T h e relative intensity of spots 1 and 2 is variable and appears to depend on sample preparation; this variation is also seen a m o n g cell-free translation products, giving the impression that one is derived from the other by postranslational modification. C o m p a r i s o n o f Spec proteins to calmodulin requires the use of a m o r e acidic isofocusing range. Sea urchin calmodulin comigrates with bovine calmodulin and shows the same differences in mobility in the presence it D. S. Durica and W. R. Crain, Dev. Biol. 92, 428 (1982).
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and absence of calcium) 2 Some of the Spec proteins also show C a 2+ mobility shifts, but these are much less striking than for calmodulin. Analytical two-dimensional electrophoresis is carried out as described by O'Farrell, 13 using the following conditions. Samples are prepared by suspension of 10,000 embryos in 200-/A lysis buffer 13 containing 0.02% sodium dodecyl sulfate (Bio-Rad or IBI) followed by vigorous agitation and a round of freeze-thawing. After centrifugation for 2 min in an Eppendorf microcentrifuge to collect insoluble residue, samples (5-25/zl) are loaded onto the first-dimension gels (11 cm in length and 2.8 mm in diameter), usually containing 1.6% pH 5-7 ampholytes (Ampholines, LKB) and 0.4% pH 3.5-10 Ampholines. Isoelectric focusing is carried out according to O'FarrelP 3 (400 V for 12 hr, 800 V for 1 hr), though the modification of Duncan and Hershey 14 have been found to improve resolution and consistency. For analysis of calmodulin, pH 3.5-5 Ampholines are substituted for the pH 5-7 Ampholines) 2 The isofocusing gels are extruded into 5 ml SDS sample buffer and allowed to equilibrate for 1-2 hr, using three changes of solution. For analysis of mobility shifts resulting from binding of calcium, the equilibration buffers contain in addition 5 mM CaCI2 or 5 mM EGTA. n Equilibrated gels are loaded onto polyacrylamide slab gels; these normally contain exponential gradients of equal volumes of l0 and 16% acrylamide13 (IBI or Bio-Rad; the ratio of acrylamide to bisacrylamide is 36.5 : 1); 15% acrylamide is also suitable, and electrophoresis is carried out at 15 mA/gel constant current for slabs (0.8 mm thick) until the bromphenol blue tracking dye reaches the bottom of the gel. Dried stained gels are exposed to Kodak AR1 film for 1.8 × 101° disintegrations for autoradiography. Purification of Spec Proteins for Immunization Since there is no assay for the activity of Spec proteins, their purification must be monitored by two-dimensional electrophoretic analysis. Separations by one-dimensional electrophoresis in SDS does not permit resolution from other proteins of similar mobility, such as calmodulin. The separation of embryos into ectoderm and endoderm fractions does not result in sufficient enrichment of Spec proteins to be worth the effort. The Spec polypeptides (1 and 2) are predominantly precipitated from aqueous extracts by addition of ethanol to 50% (along with most of the mass of soluble proteins), while the Spec 2 polypeptides are predominantly precipitated from 80% ethanol. 12 E. E. Floyd, F. Gong, B. P. Brandhorst, and W. H. Klein, Dev. Biol. 113, 501 (1986). 13 p. H. O'Farrell, J. Biol. Chem. 250, 4007 (1975). 14 R. Duncan and J. W. B. Hershey, Anal. Biochem. 138, 144 (1984).
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For preparation of Spec proteins 1 and 2 in quantities sufficient to raise antisera, a set of two-dimensional electrophoretic separations was prepared. The conditions for preparation electrophoresis are the same as for analytical electrophoresis, except for the modifications in sample preparation and loading described below; these conditions have been found to improve resolution on heavily loaded gels. An excessive load of protein on the isofocusing gel results in a distortion of the pH gradient formed, complicating identification of spots. The samples include an aliquot of an extract of embryos labeled with [35S]methionine for autoradiographic analysis to simplify identification and positioning of spots to be eluted. Stained gels are dried and stored in a vacuum desiccator and can be reused for preparation of other proteins.~5 Embryos washed twice by light centrifugation through ASW are homogenized in 10 vol cold 0.01 M Tris-HC1, pH 7.4, in a Dounce glass homogenizer (Kontes) using a tight-fitting pestle. Aliquots are set aside for protein determination, ~6and 50/zg/ml pancreatic RNase A (Sigma) is added and incubated on ice for 30 min, followed by addition of 50 ~g/ml DNase I (Sigma) for 5 rain. The samples are then quick frozen over dry ice and lyophilized. The residue is immediately dissolved in lysis buffer containing 0.2% SDS to a concentration of 3 mg/ml. Approximately 160/xg protein is applied to each isofocusing gel; an embryo contains about 40 ng total protein. ~7 Two-dimensional electrophoresis is performed as described above. Gels are stained for 30-60 rain in 0.2% Coomassie blue R250 in 50% methanol, 10% acetic acid, and destained in 30% methanol, 7% acetic acid. Gels are dried onto Whatman 3MM paper and autoradiographed. For elution of proteins, a spot (about 0.4 cm 2) from 30-35 gels is rehydrated in 3 ml 1% SDS, and the filter paper removed with forceps. The gel fragments are crushed in a ground glass homogenizer and incubated overnight at 37°. The gel fragments are collected by centrffugation at 1000 g for 5 rain; the gel fragments are reextracted twice for several hours, and the supernatants combined. The extract is cooled on ice for 1 hr to precipitate SDS, which is removed by centrifugation (10,000 g, 20 rain), and the supernatant lyophilized. The residue is dissolved in 1 ml water containing 100 /zg preimmune serum as a carrier; bovine serum albumin has been used as a carrier, but some sea urchin proteins crossreact with antisera raised against it. The protein is precipitated by addition of cold 80% acetone and collected by centrifugation (10,000 g, 20 min). The pellet is dried under vacuum and dissolved in 0.3 ml phosphatei~ L. M. Hougan, M.Sc. Thesis, McGill University, Montreal (1984). 16 M. Bradford, Anal. Biochern. 72, 248 (1978). 17 B. J. Fry and P. R. Gross, Dev. Biol. 21, 125 (1970).
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buffered saline (10 mM Na2HPO4, 0.14 M NaCI, pH 7.6), prior to emulsification with Freund's complete adjuvant (Gibco) for primary immunization. The SDS remaining in the residue after lyophilization is lethal when injected into mice, but is removed by the acetone precipitation step. Recovery of several radioactive proteins eluted from gels has been about 90%. Expression of Spec 1 Protein in E. coli Studies of the Spec protein will be aided by the availability of larger amounts of pure protein than can be readily obtained from embryos. To this end we have constructed a plasmid, pMAM 20, that consists of 386 bp of the Spec 1 open reading frame (amino acids 14 through 142) inserted into the single PvuII site of a ColE1 plasmid expression vector. 18 The vector carries the ColE1 rop gene under the control of the thermally inducible hPL promoter. The rop gene product is 61 amino acids long and is a negative regulator of ColE1 DNA replication. High-level expression of rop is lethal to cells, but cells harboring a plasmid with an insert in the rop gene grow normally under the inducible conditions. The induced ropSpec fusion protein consists of the N-terminal 51 amino acids of the rop protein, 128 amino acids of the Spec 1 protein, and 50 C-terminal amino acids representing out-of-frame sequences generated by the ligation of the Spec fragment into the vector PvuII site. When the cells are induced, the fusion protein comprises 20-25% of the E. coli proteins. We have shown that antibodies against Spec 1 protein react with the rop-Spec fusion protein, and antibodies made against the fusion protein react with the Spec 1 protein, thus allowing for purification using immunoaffinity procedures. 18In addition, the fusion protein has also been shown to bind calciums ions.~8 Cells harboring the pMAM20 plasmid and antibodies against the fusion protein are available upon request as well as the original vector and a set of three vectors modified with EcoRI linkers to allow any EcoRI fragment to be readily inserted in any reading frame. A colony of K12 AH1Atrp cells harboring pMAM 20 is grown in 5-10 ml of 2x YT (0.16 g Bacto tryptone, 0.1 g yeast extract, 0.05 g NaCI/10 ml) and 100/zg ampicillin overnight at 30°. A 1/40 dilution of these cells is then added to 5-10 ml of Mg-enriched media [44 ml H20, 5 ml 10x M9 salts (50 ml of 10x M9 salt: 3 g Na2HPO4, 1.5 g KH2PO4, 0.25 g NaC1, 0.5 g NH4CI), 50/~1 1 M MgSO4" 7H20, 16.5 /zl 0.3 M CaC12' 0.5 ml 20% glucose, 250 gl 10% caseamino acids, 50/xl 10% yeast extract, 250/zl Ltryptophan (10 mg/ml), 0.5 ml ampicillin (10 mg/ml)], and grown overnight is M. Muesing, C. D. Carpenter, W. H. Klein, and B. Polisky, Gene 31, 155 (1984).
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at 30°. A 1/40 dilution of these cells is added to 100 ml of M9-enriched media (prewarmed to 30° and lacking ampicillin) and grown to an A550 of 0.3. The culture is shifted rapidly to a 41° water bath with fairly fast shaking to ensure adequate aeration, incubated for 5-6 hr at this temperature, cooled on ice for 5 min, and the cells are harvested by centrifugation. The cells can be stored at - 8 0 ° until the fusion protein is extracted with a buffer of choice.
[30] C l o n i n g a n d E v o l u t i o n o f C a l c i u m - D e p e n d e n t P r o t e a s e , cDNA Cloning of a Novel Family of Calcium-Binding Proteins
By
SHIGEO OHNO, YASUFUMI EMORI, HIDEMITSU SUGIHARA. SHINOBU IMAJOH, and KOICH SUZUKI
Calcium-dependent protease (calcium protease, EC 3.4.22.17) is an intracellular nonlysosomal protease which requires calcium for its proteolytic activity.l-3 Two types of calcium protease, calcium protease I and II, respectively, requiring micro- and millimolar orders of Ca 2÷, exist in mammalian tissues. However, in avian tissues, only a single species of calcium protease is found. Calcium protease is composed of two subunits, large (MW 80K) and small (MW 30K) subunits. The large subunit is a catalytic subunit, but the small subunit, which is common to both types of enzyme, is indispensable for activity, though the function is not fully understood. Both types of calcium protease are converted by "autodigestion" to more Ca2+-sensitive forms and these enzymes may play physiological roles. The amino acid sequences of avian and mammalian calcium protease subunits determined recently by cDNA cloning4-9 have t T. Murachi, "Calcium and Cell Fucntion," Vol. 4, p. 377. Academic Press, New York, 1983. 2 L. Waxman, this series, Vol. 80, p. 664. 3 K. Suzuki, S. Kawashima, and K. Imahori, "Calcium Regulation in Biological Systems," p. 213. Academic Press, New York, 1984. 4 S. Ohno, Y. Emori, S. Imajoh, H. Kawasaki, M. Kisaragi, and K. Suzuki, Nature (London) 312, 566 (1984). 5 T. Sakihama, H. Kakidani, K. Zenita, N. Yumoto, T. Kikuchi, T. Sasaki, R. Kannagi, S. Nakanishi, M. Ohmori, K. Takio, K. Titani, and T. Murachi, Proc. Natl. Acad. Sci. U.S.A. 82, 6075 (1985). 6 y. Emori, H. Kawasaki, S. Imajoh, S. Kawashima, and K. Suzuki, J. Biol. Chem. 261, 9472 (1986).
METHODS IN ENZYMOLOGY, VOL. 139
Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
CLONING OF Ca-BINDING PROTEINS,
cDNAs,
AND GENES
[29]
[29] S p e c P r o t e i n s : C a l c i u m - B i n d i n g P r o t e i n s in t h e E m b r y o n i c E c t o d e r m Cells o f S e a U r c h i n s
By WILLIAM H. KLEIN, SUSAN H. HARDIN, and BRUCE P. BRANDHORST
Introduction During its embryonic and larval development, the sea urchin Strongylocentrotus purpuratus synthesizes and accumulates a set of calciumbinding proteins detectable exclusively in dorsal (aboral) ectoderm cells.1 These proteins, termed Spec proteins, are related to the more extensively characterized calcium-binding proteins such as calmodulin, myosin light chains, troponin C, and parvalbumins.l The in oivo function of the Spec proteins is unknown but it has been hypothesized that they play a role in changing the shape of the ectodermal cells.l Supporting this hypothesis is the observation that two actin genes, cylIIa and cylllb, are restrictively expressed in the same cells and at the same time in development as are the Spec genes. 2 This suggests a structural and/or functional relationship between the Spec proteins and the cylII actin genes. There are two periods in embryonic and larval development when dorsal ectoderm cells are morphologically reshaped: After gastrulation when the cuboidal ectoderm cells stretch and flatten to become the squamous epithelial epidermis of the pluteus larva; and at metamorphosis, when microfilaments present in the dorsal epithelium contract, resulting in the collapse of the larval structure and release of the rudimentary juvenile sea urchin. 3 It is possible the Spec proteins are involved in either or both of these morphogenetic processes. Whatever their precise function, given the preservation of their calcium-binding domains it seems likely that the Spec proteins participate in some cellular process mediated by calcium ions. The Spec proteins are likely to be of interest in investigations of the evolution and role of the troponin C superfamily of calcium-binding proteins.
C. P. 2 R. 3 R.
D. Carpenter, A. M. Bruskin, P. E. Hardin, M. J. Keast, J. A n s t r o m , A. L. Tyner, B. Brandhorst, and W. H. Klein, Cell (Cambridge, Mass.) 36, 663 (1984). J. Shott, J. J. Lee, R. J. Britten, and E. H. Davidson, Dev. Biol. 101, 295 (1984). A. C a m e r o n and R. T. Hinegardner, J. Morphol. 157, 21 (1978).
METHODS IN ENZYMOLOGY,VOL. 139
Copyright © 1987by AcademicPress, Inc. All rights of reproduction in any form reserved.
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Spec Genes and mRNAs The Spec gene family is composed of two subfamilies, Spec 1 and Spec 2. Spec 1 is a single gene that encodes a 1.5-kb mRNA, the predominant mRNA of the family (0.6% of the mRNA of the gastrula stage embryo) and the Spec 1 protein is the most prevalent of the Spec proteins) ,4 In contrast to Spec 1, there are about 10 Spec 2 genes, which encode a set of mRNAs of about 2.2 kb that are much less prevalent than Spec 1 mRNA. Accumulation of the Spec 1 and Spec 2 mRNAs and proteins begins in late cleavage and is highly restricted to dorsal ectoderm cells) Several Spec genes have now been cloned and all show strong similarities in their exon/intron structure to genes encoding calcium-binding proteins in other organisms. These include chicken calmodulin, for which the resemblance is particularly striking, and myosin light chains 1 and 3 in chickens and mice. While the nucleotide sequence conservation is weak, the placement of the introns is conserved. Nevertheless, the exon/intron placement does not correspond to any known structural or functional domains in the proteins. 4 Preparation and Handling of Sea Urchin Embryos S. p u r p u r a t u s sea urchins are obtained from Pacific Biomarine (Venice, CA) or Marinus (Westchester, CA). Gravid adults are generally available from December through May. They may be held for several months at 8-12 ° in natural or artificial sea water (Instant Ocean, from Aquarium Systems, Eastlake, OH) in tanks utilizing filtration through calcareous gravel and activated charcoal, extensive gas exchange via airstones, and oxidation of ammonia by nitrifying bacteria. While tap water is acceptible for making sea water in some locations, we normally pass the water through activated carbon and mixed bed deionizing columns. Shedding of gametes is induced by intracoelomic injection of a total of 0.5-2.0 ml 0.5 M KC1 through several sites on the peristomial membrane on the oral side. Semen (cream colored) is collected " d r y " in a 0.1-ml serological pipet or capillary tube using a Clay-Adams pipetting aid and can be stored for several days at 0-4 ° . Eggs (orange) are collected by inverting the female over a beaker filled with cold (4-8 °) artificial sea water (ASW). A recipe for AWS,6 made up using glass-redistilled water
4 s. H. Hardin, C. D. Carpenter, P. E. Hardin, A. M. Bruskin, and W. H. Klein,J. Mol. Biol. 186, 243 (1985). s A. M. Bruskin,P.-A. Bddard,A. L. Tyner,R. M. Showman,B. P. Brandhorst,and W. H. Klein, Dev. Biol. 91, 317 (1982). 6R. T. Hinegardner, in "Methods in DevelopmentalBiology" (F. H. Wilt and N. K. Wessels, eds.), p. 139. Crowell-Collier,New York, 1967.
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AND GENES
[29]
and reagent grade chemicals, is (in g/liter): NaCI, 28.31, KC1, 0.77, MgCI2 • 6H20, 5.41, MgSO4-7H20, 7.13, CaCI2, I. 18, to which solution is added 0.2 g/liter NaHCO3; the pH should be adjusted to 8.2. Artificial sea water lacking calcium and magnesium (CMFSW) consists of (in g/liter): NaC1, 27, Na2SO4, 1.0, KC1, 0.8, and NaHCO3, 0.18. Embryos can also be cultured in natural sea water or Instant Ocean filtered through 0.22-/.~m nitrocellulose filters. AWS can be pasteurized but not autoclaved. After the eggs settle, excess ASW is removed by aspiration, and the eggs are resuspended in ASW and filtered through several layers of cheesecloth into a large volume of cold ASW (200-fold excess). After settling, eggs are resuspended in 100 vol cold ASW and fertilized by addition of 1/100 vol of semen prediluted 1/100 in ASW. After 2-3 min egg samples are examined by bright-field microscopy for elevation of the fertilization envelope; if fertilization is less than 95% another aliquot of sperm suspension is added. The fertilized eggs are allowed to settle twice through a large volume of cold ASW and then resuspended in ASW at 15° (containing 80 U/ml penicillin and 50/zg/ml streptomycin sulfate to inhibit bacterial growth) at a concentration of 1-5 × 103 eggs/ml (determined by counting a sample of the egg suspension drawn into a 10-/zl capillary tube; there are about 2 x l06 dejellied 70-/xm eggs in 1 ml after centrifugation at a few hundred gravities). Embryos are cultured at 14-16 °, stirred at 60 rpm with an acrylic paddle attached to a clock motor; for cultures of several liters compressed air is bubbled through the culture. First cleavage occurs about 90 min after suspension in 16° water, hatching from the fertilization envelope occurs at about 18 hr, gastrulation begins at about 36 hr and is completed by 48 hr, and plutei capable of feeding appear after 3-4 days. For optimum cultures, embryos' are collected by filtration through 44-/~m nylon mesh (Nitex, Tetco, NY) after hatching and resuspended at 100-1000/ml. Newly synthesized proteins can be labeled by incubation of 20,000 embryos/ml in a fiat-bottom vessel with 50-100/xCi/ml [35S]methionine (1300 Ci/mmol, Amersham) for 30-50 min, providing occasional agitation. Fractionation of embryos into tissue layers can result in changes in patterns of labeling of proteins, including the failure to label the Spec proteins; in part this is the result of changes in amino acid uptake by aboral ectoderm cells (P.-A. Bedard and B. P. Brandhorst, unpublished observations). Preparation of Ectoderm and Endoderm Fractions As first exploited by McClay and Marchase, 7 ectoderm cells can be selectively dissociated from embryos by dispersion of the enmeshing hya7 D. McClay and R. B. Marchase, Dev. Biol. 71, 289 (1979).
[29]
Spec PROTEINS
357
line layer in media lacking divalent cations8; a residual basal laminar bag contains endoderm cells, as well as a few mesoderm cells. Cultures of pluteus stage embryos are harvested by filtration through 44-/xm nylon mesh. Embryos are resuspended in 40 vol cold ASW, collected by light centrifugation (medium speed in a clinical or hand centrifuge: 100-300 g, 1 min), and resuspended in 1 M glycine, 2 mM EDTA for 3 min on ice (optimal time is stage and batch dependent), collected by light centrifugation, and resuspended in 20 vol CMFSW. The ectoderm cells are dissociated from the basal lamina by repeated gentle aspiration through a Pasteur pipet or by passage through a 10-ml syringe lacking a needle. Dissociation is monitored by light microscopy, and continued until most ectoderm cells are released. The resuspension is then filtered through 28- or 35-/xm nylon mesh to collect a crude endoderm/mesoderm fraction. The ectoderm cells in the filtrate are collected by centrifugation at 5000 rpm for 5 min in a Beckman JA-20 rotor (3000 g maximum). The endoderm/mesoderm fraction can be further purified by resuspension and agitation in CMFSW followed by refiltration. Brief trituration of the bags in CMF at this step leads to the release of many of the mesoderm cells while the endoderm (archenteron) remains intact. 7 As an alternative to filtration (particularly at stages prior to spicule elongation when the bags tend to penetrate the filter), differential sedimentation can be employed. Endoderm/mesoderm is collected at 300 rpm in a Sorvall GLC rotor (116 g maximum) for 15 sec; several repeated centrifugations of both the pellet and the supernatant are required to obtain fractions of high purity. Alternatively, cells can be separated by isopycnic centrifugation on Percoll gradients. 9 This is particularly useful when epithelial cells (presumptive endoderm and ectoderm) are dissociated from basal laminar bags of mesenchyme blastulas, resulting in a fraction containing mesoderm cells. Screening for Nucleotide Sequences Encoding Calcium-Binding Proteins Several eDNA libraries are available that have been constructed from RNA of S. purpuratus and Lytechinus pictus embryos. These include egg, blastula, gastrula, and plutcus stage libraries made from total or polysomal polyadenylated RNA. The recombinants are packaged in either phage or plasmid-harboring Escherichia coli. Spec sequences, as well as other cell or stage-specific sequences, can be readily isolated from such libraries by routine differential screening procedures. These libraries are available upon request. 8 R. E. Kane, Exp. Cell Res. 81, 301 (1973). 9 M. A. Harkey and A. H. Whiteley, Wilhelm Roux's Arch. Dev. Biol. 198, 111 (1980).
358
CLONING OF Ca-BINDING PROTEINS,
cDNAs,
AND GENES
[29]
A probe has been constructed to isolate any nucleotide sequence encoding a calcium-binding protein belonging to the troponin C superfamily. A comparison of published nucleotide sequences of several calcium-binding protein genes shows that a 43-bp region covering the third calciumbinding domain is the most conserved region of the molecule. The 43-base consensus sequence T A A G (5'-ACTGACGGAGCCGTTGCCATCCTTGTCGAAGACACGCAAAGCT-Y)
G
C
has approximately 85% match to the corresponding region of most of the reported calcium-binding protein genes and is complementary to the transcribed sequence (S. H. Hardin and W. H. Klein, unpublished observations). Under appropriate conditions, this probe hybridizes specifically to various genes and mRNAs in the troponin C superfamily and should be of use in isolating and characterizing genes for calcium-binding proteins. A probe consisting of 0.1 /zg DNA labeled to high specific activity with [32p]ATP and T4 polynucleotide kinase 1° is hybridized to a DNA or RNA sample blotted onto a nitrocellulose filter. The filter is prewashed in a sealed plastic bag for 1 hr at 68° in 50 ml of 10x Denhardt's (0.2% bovine serum albumin, 0.2% Ficoll, 0.2% polyvinylpyrrolidone-40), 0.1% SDS, 0.2 M sodium phosphate, pH 7.0, and 5x SET (0.75 M sodium chloride, 0.15 M Tris-HC1, pH 8, 10 mM EDTA). This solution is removed and the filter is then prehybridized at 37° in 5 to 10 ml of 1 x Denhardt's, 20 mM sodium phosphate (pH 7.0), 5x SET, 10% Dextran sulfate, and sheared calf thymus DNA (100 ~g/ml). After 1 hr the probe is added and allowed to hybridize for 10 to 12 hr. The hybridization buffer is removed and the filter is washed for 1 hr at 45 ° with 50 ml of 5× SET, 0.1% sodium pyrophosphate, 25 mM sodium phosphate (pH 7.0), and 0.1% SDS. The filter is then washed for 1 hr at 45 ° with 50 ml of 1x SET, 1 x Denhardt's, 0.1% sodium pyrophosphate, and 25 mM sodium phosphate and exposed to X-ray film. Two-Dimensional Eleetrophoretic Analysis of Spec Proteins The position of the Spec proteins compared to other newly synthesized proteins is shown in Fig. 1. Spots 1 and 2 are products of the Spec 1 gene, while the various spots identified by arrows are ectoderm specific proteins which are mostly or entirely the products of Spec 2 genes. Spot 1 has the same pI as the major sea urchin embryonic form of actin, which 10 T. Maniatis, E. F. Fritsch, and J. Sambrook, "Molecular Cloning: A Laboratory Manual," p. 122. Cold Spring Harbor Lab., Cold Spring Harbor, New York, 1982.
[29]
Spec PROTEINS
359
-5O Mr
(X 10-3)
25
7
6
5 pH
FIG. 1. Two-dimensional electrophoretic properties of Spec proteins. Proteins of plutei labeled with [35S]methionine were analyzed as described. Arrows identify small, acidic proteins synthesized only in ectoderm; most, possibly all, of these are encoded by Spec genes. Positive identification of some spots is impossible since the cell-free translation products of Spec 2 mRNAs do not all comigrate with proteins synthesized in vivo. Spots 1 and 2 are encoded by the Spec I gene. The position of the major actin isoform is indicated by the letter A.
comigrates with m o u s e fl-actin H (pI of about 5.7 in 9.5 M urea). Comparison of relative mobilities with m a r k e r s indicate a range of apparent molecular weights o f 14,000-17,000 for the Spec proteins, as shown in Fig. 1. T h e relative intensity of spots 1 and 2 is variable and appears to depend on sample preparation; this variation is also seen a m o n g cell-free translation products, giving the impression that one is derived from the other by postranslational modification. C o m p a r i s o n o f Spec proteins to calmodulin requires the use of a m o r e acidic isofocusing range. Sea urchin calmodulin comigrates with bovine calmodulin and shows the same differences in mobility in the presence it D. S. Durica and W. R. Crain, Dev. Biol. 92, 428 (1982).
360
CLONING OF Ca-BINDING PROTEINS,
cDNAs,
AND GENES
[29]
and absence of calcium) 2 Some of the Spec proteins also show C a 2+ mobility shifts, but these are much less striking than for calmodulin. Analytical two-dimensional electrophoresis is carried out as described by O'Farrell, 13 using the following conditions. Samples are prepared by suspension of 10,000 embryos in 200-/A lysis buffer 13 containing 0.02% sodium dodecyl sulfate (Bio-Rad or IBI) followed by vigorous agitation and a round of freeze-thawing. After centrifugation for 2 min in an Eppendorf microcentrifuge to collect insoluble residue, samples (5-25/zl) are loaded onto the first-dimension gels (11 cm in length and 2.8 mm in diameter), usually containing 1.6% pH 5-7 ampholytes (Ampholines, LKB) and 0.4% pH 3.5-10 Ampholines. Isoelectric focusing is carried out according to O'FarrelP 3 (400 V for 12 hr, 800 V for 1 hr), though the modification of Duncan and Hershey 14 have been found to improve resolution and consistency. For analysis of calmodulin, pH 3.5-5 Ampholines are substituted for the pH 5-7 Ampholines) 2 The isofocusing gels are extruded into 5 ml SDS sample buffer and allowed to equilibrate for 1-2 hr, using three changes of solution. For analysis of mobility shifts resulting from binding of calcium, the equilibration buffers contain in addition 5 mM CaCI2 or 5 mM EGTA. n Equilibrated gels are loaded onto polyacrylamide slab gels; these normally contain exponential gradients of equal volumes of l0 and 16% acrylamide13 (IBI or Bio-Rad; the ratio of acrylamide to bisacrylamide is 36.5 : 1); 15% acrylamide is also suitable, and electrophoresis is carried out at 15 mA/gel constant current for slabs (0.8 mm thick) until the bromphenol blue tracking dye reaches the bottom of the gel. Dried stained gels are exposed to Kodak AR1 film for 1.8 × 101° disintegrations for autoradiography. Purification of Spec Proteins for Immunization Since there is no assay for the activity of Spec proteins, their purification must be monitored by two-dimensional electrophoretic analysis. Separations by one-dimensional electrophoresis in SDS does not permit resolution from other proteins of similar mobility, such as calmodulin. The separation of embryos into ectoderm and endoderm fractions does not result in sufficient enrichment of Spec proteins to be worth the effort. The Spec polypeptides (1 and 2) are predominantly precipitated from aqueous extracts by addition of ethanol to 50% (along with most of the mass of soluble proteins), while the Spec 2 polypeptides are predominantly precipitated from 80% ethanol. 12 E. E. Floyd, F. Gong, B. P. Brandhorst, and W. H. Klein, Dev. Biol. 113, 501 (1986). 13 p. H. O'Farrell, J. Biol. Chem. 250, 4007 (1975). 14 R. Duncan and J. W. B. Hershey, Anal. Biochem. 138, 144 (1984).
[29]
Spec PROTEINS
361
For preparation of Spec proteins 1 and 2 in quantities sufficient to raise antisera, a set of two-dimensional electrophoretic separations was prepared. The conditions for preparation electrophoresis are the same as for analytical electrophoresis, except for the modifications in sample preparation and loading described below; these conditions have been found to improve resolution on heavily loaded gels. An excessive load of protein on the isofocusing gel results in a distortion of the pH gradient formed, complicating identification of spots. The samples include an aliquot of an extract of embryos labeled with [35S]methionine for autoradiographic analysis to simplify identification and positioning of spots to be eluted. Stained gels are dried and stored in a vacuum desiccator and can be reused for preparation of other proteins.~5 Embryos washed twice by light centrifugation through ASW are homogenized in 10 vol cold 0.01 M Tris-HC1, pH 7.4, in a Dounce glass homogenizer (Kontes) using a tight-fitting pestle. Aliquots are set aside for protein determination, ~6and 50/zg/ml pancreatic RNase A (Sigma) is added and incubated on ice for 30 min, followed by addition of 50 ~g/ml DNase I (Sigma) for 5 rain. The samples are then quick frozen over dry ice and lyophilized. The residue is immediately dissolved in lysis buffer containing 0.2% SDS to a concentration of 3 mg/ml. Approximately 160/xg protein is applied to each isofocusing gel; an embryo contains about 40 ng total protein. ~7 Two-dimensional electrophoresis is performed as described above. Gels are stained for 30-60 rain in 0.2% Coomassie blue R250 in 50% methanol, 10% acetic acid, and destained in 30% methanol, 7% acetic acid. Gels are dried onto Whatman 3MM paper and autoradiographed. For elution of proteins, a spot (about 0.4 cm 2) from 30-35 gels is rehydrated in 3 ml 1% SDS, and the filter paper removed with forceps. The gel fragments are crushed in a ground glass homogenizer and incubated overnight at 37°. The gel fragments are collected by centrffugation at 1000 g for 5 rain; the gel fragments are reextracted twice for several hours, and the supernatants combined. The extract is cooled on ice for 1 hr to precipitate SDS, which is removed by centrifugation (10,000 g, 20 rain), and the supernatant lyophilized. The residue is dissolved in 1 ml water containing 100 /zg preimmune serum as a carrier; bovine serum albumin has been used as a carrier, but some sea urchin proteins crossreact with antisera raised against it. The protein is precipitated by addition of cold 80% acetone and collected by centrifugation (10,000 g, 20 min). The pellet is dried under vacuum and dissolved in 0.3 ml phosphatei~ L. M. Hougan, M.Sc. Thesis, McGill University, Montreal (1984). 16 M. Bradford, Anal. Biochern. 72, 248 (1978). 17 B. J. Fry and P. R. Gross, Dev. Biol. 21, 125 (1970).
362
CLONING OF Ca-BINDING PROTEINS, c D N A s , AND GENES
[29]
buffered saline (10 mM Na2HPO4, 0.14 M NaCI, pH 7.6), prior to emulsification with Freund's complete adjuvant (Gibco) for primary immunization. The SDS remaining in the residue after lyophilization is lethal when injected into mice, but is removed by the acetone precipitation step. Recovery of several radioactive proteins eluted from gels has been about 90%. Expression of Spec 1 Protein in E. coli Studies of the Spec protein will be aided by the availability of larger amounts of pure protein than can be readily obtained from embryos. To this end we have constructed a plasmid, pMAM 20, that consists of 386 bp of the Spec 1 open reading frame (amino acids 14 through 142) inserted into the single PvuII site of a ColE1 plasmid expression vector. 18 The vector carries the ColE1 rop gene under the control of the thermally inducible hPL promoter. The rop gene product is 61 amino acids long and is a negative regulator of ColE1 DNA replication. High-level expression of rop is lethal to cells, but cells harboring a plasmid with an insert in the rop gene grow normally under the inducible conditions. The induced ropSpec fusion protein consists of the N-terminal 51 amino acids of the rop protein, 128 amino acids of the Spec 1 protein, and 50 C-terminal amino acids representing out-of-frame sequences generated by the ligation of the Spec fragment into the vector PvuII site. When the cells are induced, the fusion protein comprises 20-25% of the E. coli proteins. We have shown that antibodies against Spec 1 protein react with the rop-Spec fusion protein, and antibodies made against the fusion protein react with the Spec 1 protein, thus allowing for purification using immunoaffinity procedures. 18In addition, the fusion protein has also been shown to bind calciums ions.~8 Cells harboring the pMAM20 plasmid and antibodies against the fusion protein are available upon request as well as the original vector and a set of three vectors modified with EcoRI linkers to allow any EcoRI fragment to be readily inserted in any reading frame. A colony of K12 AH1Atrp cells harboring pMAM 20 is grown in 5-10 ml of 2x YT (0.16 g Bacto tryptone, 0.1 g yeast extract, 0.05 g NaCI/10 ml) and 100/zg ampicillin overnight at 30°. A 1/40 dilution of these cells is then added to 5-10 ml of Mg-enriched media [44 ml H20, 5 ml 10x M9 salts (50 ml of 10x M9 salt: 3 g Na2HPO4, 1.5 g KH2PO4, 0.25 g NaC1, 0.5 g NH4CI), 50/~1 1 M MgSO4" 7H20, 16.5 /zl 0.3 M CaC12' 0.5 ml 20% glucose, 250 gl 10% caseamino acids, 50/xl 10% yeast extract, 250/zl Ltryptophan (10 mg/ml), 0.5 ml ampicillin (10 mg/ml)], and grown overnight is M. Muesing, C. D. Carpenter, W. H. Klein, and B. Polisky, Gene 31, 155 (1984).
[30]
CLONING OF Ca-DEPENDENT PROTEASE
363
at 30°. A 1/40 dilution of these cells is added to 100 ml of M9-enriched media (prewarmed to 30° and lacking ampicillin) and grown to an A550 of 0.3. The culture is shifted rapidly to a 41° water bath with fairly fast shaking to ensure adequate aeration, incubated for 5-6 hr at this temperature, cooled on ice for 5 min, and the cells are harvested by centrifugation. The cells can be stored at - 8 0 ° until the fusion protein is extracted with a buffer of choice.
[30] C l o n i n g a n d E v o l u t i o n o f C a l c i u m - D e p e n d e n t P r o t e a s e , cDNA Cloning of a Novel Family of Calcium-Binding Proteins
By
SHIGEO OHNO, YASUFUMI EMORI, HIDEMITSU SUGIHARA. SHINOBU IMAJOH, and KOICH SUZUKI
Calcium-dependent protease (calcium protease, EC 3.4.22.17) is an intracellular nonlysosomal protease which requires calcium for its proteolytic activity.l-3 Two types of calcium protease, calcium protease I and II, respectively, requiring micro- and millimolar orders of Ca 2÷, exist in mammalian tissues. However, in avian tissues, only a single species of calcium protease is found. Calcium protease is composed of two subunits, large (MW 80K) and small (MW 30K) subunits. The large subunit is a catalytic subunit, but the small subunit, which is common to both types of enzyme, is indispensable for activity, though the function is not fully understood. Both types of calcium protease are converted by "autodigestion" to more Ca2+-sensitive forms and these enzymes may play physiological roles. The amino acid sequences of avian and mammalian calcium protease subunits determined recently by cDNA cloning4-9 have t T. Murachi, "Calcium and Cell Fucntion," Vol. 4, p. 377. Academic Press, New York, 1983. 2 L. Waxman, this series, Vol. 80, p. 664. 3 K. Suzuki, S. Kawashima, and K. Imahori, "Calcium Regulation in Biological Systems," p. 213. Academic Press, New York, 1984. 4 S. Ohno, Y. Emori, S. Imajoh, H. Kawasaki, M. Kisaragi, and K. Suzuki, Nature (London) 312, 566 (1984). 5 T. Sakihama, H. Kakidani, K. Zenita, N. Yumoto, T. Kikuchi, T. Sasaki, R. Kannagi, S. Nakanishi, M. Ohmori, K. Takio, K. Titani, and T. Murachi, Proc. Natl. Acad. Sci. U.S.A. 82, 6075 (1985). 6 y. Emori, H. Kawasaki, S. Imajoh, S. Kawashima, and K. Suzuki, J. Biol. Chem. 261, 9472 (1986).
METHODS IN ENZYMOLOGY, VOL. 139
Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.