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J. Biol. Chem., Vol. 278, Issue 36, 34467-34474, September 5, 2003
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From the Pioneer Hi-Bred International, A DuPont Company, Johnston, Iowa 50131
Received for publication, January 22, 2003 , and in revised form, June 20, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Calpains are a family of Ca2+-dependent cytosolic cysteine proteinases (12, 13) that consist of ubiquitous and tissue-specific isoforms in animals. Two mammalian calpains, m- and µ-calpain, are ubiquitously expressed and have been extensively characterized. Each of these forms a heterodimer comprising an 80-kDa catalytic subunit and a 30-kDa regulatory subunit. The µ- and m-isoforms differ in their in vitro requirements for Ca2+, the half-maximal activity in vitro for µ-calpain being 50 µM Ca2+ while m-calpain requires 300 µM Ca2+. The catalytic subunit of m-calpain is comprised of four domains; domain I which is involved in autolytic activation; domain II containing the catalytic triad residues (Cys-105, His-262, Asn-286 in m-calpain); domain III with similarity to a C2-domain and domain IV that has five EF-hand motifs (13, 14). The small, or regulatory subunit of calpain contains domain V, which is glycine- and proline-rich and domain VI that has several EF-hand motifs and is structurally very similar to domain IV of the catalytic subunit (15, 16).
More than a dozen calpain genes have been cloned and identified in animals. In addition to conventional calpain with a four domain structure, atypical calpains have been described where individual domains are replaced or deleted (17–19). Some of these calpains appear to function as monomers and do not associate with a regulatory calpain subunit. These different properties indicate that individual isoforms of calpain are regulated differently and may have unique functions. The physiological role of calpain is determined by the function of the protein targeted for proteolysis. Various types of proteins such as transcription factors, calmodulin-binding proteins, components of receptor-mediated signal transduction and cytoskeletal proteins have been identified as calpain substrates in vitro or in vivo (20–29). Calpains are reported to be involved in a wide range of cellular processes including cell proliferation, apoptosis, differentiation, and signal transduction. In addition, calpains have been implicated in endocytosis, exocytosis, and intracellular membrane fusion (30).
Although an understanding of calpain at the molecular, biochemical, and cellular levels has advanced greatly in animals since calpain was first described in 1964 (31), the functional significance of the DEK1 calpain in plants was not identified until 2001 (9). In maize, the phenotype of dek1 mutants suggests a role for Dek1 in global developmental regulation. In addition to the lack of aleurone cells, knockout of the Dek1 gene results in improper embryo axis formation and a missing shoot apical meristem (9). Also, as shown by mutant sector analysis, leaf differentiation, and in particular epidermis cell formation, requires Dek1 gene function (11). The predicted structure of DEK1 calpain suggests that it may function as a cysteine proteinase, and that its activity is regulated by extra cellular events mediated through the predicted membrane portion of the protein. In this study we investigate the structure of the calpain-like domain of DEK1 by using three-dimensional modeling and its biological activity by expressing recombinant DEK1 protein in bacteria. We also investigate the pattern of Dek1 transcript distribution in maize grains using in situ hybridization.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Cloning of cDNA Domains Encoding DEK1 Domain Proteins in Escherichia coli Expression Vectors—cDNA fragments corresponding to DEK1 calpain domains (9) were amplified by PCR. The flanking primers used for domain II&III and domain II were: 5'-GTCGACTTCACTGATCAAGAGTTCCCTC-3' with SalI site (forward) and 5'-GCGGCCGCTTAAACAGCCTCTAGTCTGATTGATG-3' with NotI site (reverse) and 5'-GTCGACTTCACTGATCAAGAGTTCCCTC-3' with SalI site (forward) and 5'-CGGCCGCTTAATAAACACGACAAACATATATTG-3' with NotI site (reverse), respectively. The cDNAs were ligated into pCR2.1 TOPO (Invitrogen) and then into pGEX-4T vector (Amersham Biosciences), which produced a glutathione S-transferase (GST) fusion at the N terminus. The recombinant plasmids were transformed into TOP 10 E. coli (Invitrogen). After insert sequences were verified by sequencing, the constructs were transformed into BL21 (DE3) E. coli (Invitrogen) for protein expression.
The Expression of DEK1 Domain II&III using
Fermentation—The Dek1 domain II&III pGEX 4T-3
construct was transformed into BL21 Codon + RP cells (Stratagene). A 5-liter
fermentor containing 5 liters of Terrific Broth (Yeast Extract 24 g/liter,
pancreatic digest of casein 12 g/liter, dipotassium, phosphate 9.4 g/liter,
monopotassium phosphate 2.2 g/liter, glycerol 4 ml/liter), 100 µg/ml
carbenicillin, and 5 ml of antifoam (Mazu DF 204 Defoamer) was inoculated with
50 ml of overnight culture grown at 37 °C. The fermentation culture was
grown at 37 °C until an OD600 of 1.0 was reached. The
temperature was then lowered to 30 °C. The dissolved oxygen was maintained
at greater than 30% throughout the run by controlling both stirring and
airflow. The pH was maintained at 7.2 with NH4OH. The culture was
induced with 0.1 mM IPTG
(isopropyl-1-thio-
-galactopyranoside) at an OD600 of 2.5.
Three hours after induction, the cells were harvested by centrifugation at
10,000 x g for 20 min. A pellet from 1 liter of fermentation
culture was resuspended in 20 ml of lysis buffer (50 mM Tris, pH
8.0, 200 mM NaCl, 5 mM dithiothreitol, Complete
Proteinase Inhibitor (Roche Applied Science), lysozyme 1 mg/ml, bensonase 1
unit/ml) and lysed by sonication. Cell debris was removed by centrifugation at
10,000 x g for 5 min. The supernatant was incubated with
glutathione Sepharose 4B beads prewashed with phosphate-buffered saline buffer
containing 140 mM NaCl, 2.7 mM KCl, 10 mM
Na2HPO4, and 1.8 mM
KH2PO4 (pH 7.3) with gentle agitation at 4 °C for
0.5–1 h. Proteins bound to the beads were eluted with a buffer of 50
mM Tris-HCl (pH 8.0) and 10 mM reduced glutathione. The
protein concentration was estimated by using a dye-binding protein assay kit
(Bio-Rad) with bovine serum albumin as a standard.
The Expression of DEK1 Domain II—E. coli BL21 cell cultures
were grown in LB medium (Difco Laboratories) with 100 µg/ml carbenicillin.
The transformed cells harboring Dek1 domain II were grown at 37
°Cto an absorbance of 
1.0 at 600 nm. 5 ml of the cells were
transferred into a 1-liter flask with 200 ml of LB medium containing 0.1
mM IPTG and incubated overnight at 18 °C. The cells were then
harvested for the GST-DEK1 domain II purification as mentioned above.
Site-directed Mutagenesis of DEK1—Site-directed mutagenesis of DEK1 domain protein was generated by a PCR-based overlapping method described by Ho et al. (33). The flanking primers were the same as those used for domain II&III. The internal primers used for generation of mutant were 5'-TCGGTTGGGAGACTCTTGGTTCCTAAGTG-3' and 5'-CACTTAGGAACCAAGAGTCTCCCAACCGA-3' (altered codon is underlined). The mutation was verified by sequencing.
DEK1 Activity Assay—The method used to determine the
proteolytic activity of DEK1 domains is based on an electrophoretic casein
degradation assay used in previous studies of animal m-clapain
(34). In our assays, a typical
reaction contained 2 µg of DEK1 domain protein, 3 µg of purified
-casein (Sigma-Aldrich C6905, more than 90% purity) in 20 µl of
reaction buffer (50 mM imidazole-HCl, pH 7.5, 10 mM
-mercaptoethanol). Reaction mixtures were incubated at 30 °C for a
variable time interval. The reaction was stopped by adding NuPAGE LDS sample
buffer (Invitrogen). After incubation at 70 °C for 10 min, the mixture was
separated by a NuPAGE 10% bis-tris gel and then stained with Colloidal Blue
kit (Invitrogen). Change in the intensity of the -casein band was used
to quantify DEK1 proteolytic activity. Three independent assays were carried
out to calculate standard deviation (S.D.) in these assays. Intensity was
measured by using the ChemiDoc system (Bio-Rad).
CD Studies—CD spectra were measured using a Jasco J-715
model spectropolarimeter. Far-UV spectra were recorded from 190 to 260 nm in a
0.1 mm pathlength quartz cuvette. Protein was dialyzed into 35 mM
potassium phosphate buffer (pH 7.6) and diluted to a concentration of 0.3
mg/ml. Data are reported as mean residue ellipticity. In Situ
Hybridization Analysis—Digoxigenin-11-UTP-labeled RNA probes were
used to localize Dek1 mRNA in cells according to the protocol
described by Jackson (35). RNA
probes were made using Digoxigenin-11-UTP labeled NTP mixture with SP6 and T7
RNA polymerases (Roche Applied Science). The 721-bp fragment of
3'-region Dek1 cDNA was subcloned into pSPORT I vector
(Invitrogen). The clone was linearized by XbaI (sense) and
PstI (antisense) and transcribed into RNA in vitro.
Unincorporated ribonucleotides were removed using Qiagen RNeasy purification
kit and probes were subjected to carbonate hydrolysis in order to reduce probe
length to 150 nucleotides. Microscopy evaluation was carried out in dark
field using a Nikon Eclipse E800 microscope.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Amino acid sequence alignment of DEK1 calpain and mcalpain demonstrates a
conserved domain organization for domain II and domain III
(Fig. 2). Indeed, this
architecture appears to be remarkably well conserved even in the modeled
three-dimensional structure of the protein
(Fig. 3A). In addition
to the overall fold, the conservation and arrangement of residues important
for catalysis and substrate binding in domain II are particularly significant.
As in m-calpain, the catalytic Cys-71 (Cys-105 in m-calpain) is located in the
-helix of domain IIa, on the opposite side of the interface with domain
IIb which harbors the two other residues of the catalytic triad, His-229 and
Asn-249 (corresponding to His-262 and Asn-286 in m-calpain,
Fig. 2). Importantly, the
distance of 10.3 Å between the catalytic Cys S
and the His
N
1 in the model compares favorably with the 10.5 Å
measurement for the same atoms in the calcium-free conformation of m-calpain
(14,
36) and validates the modeled
structure. Additional residues conserved within the context of the active-site
cleft comprising the subdomains of the catalytic domain include Gln-65,
Trp-72, Gly-160–161, Pro-250, and Trp-251
(Fig. 3B). Despite
this strong conservation in domain II, however, there are two significant
differences to be noted in domain III between m-calpain and DEK1. First, while
the basic loop comprising residues His-415-His-427 of m-calpain contains 4
Args and 1 Lys, the corresponding loop in DEK1 (residues 371–383 in
Fig. 2) contains just one Arg
residue. Even more importantly, in contrast to the very acidic loop of
m-calpain (residues 392–402) consisting of 10 negatively charged
residues, there is just one conserved Asp-347 in the DEK1 loop (residues
344–354 in Fig. 2;
asterisk in Fig.
3A).
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Expression and Purification of Recombinant DEK1 Wild-type and Mutated Domain Proteins—The partial cDNAs encoding DEK1 domain II&III and domain II alone were cloned into pGEX-4T-3 vector to produce the corresponding GST fusion proteins in E. coli. Under conditions normally used to express fusion proteins in E. coli, the majority of the expressed DEK1 domain II&III protein appears in inclusion bodies. Production of the wild-type and mutant (see below) versions of DEK1 domain II&III was therefore carried out by fermentation, yielding a significant amount of soluble domain II&III protein. The expressed DEK1 domain II&III protein was purified to near homogeneity with glutathione-Sepharose 4B beads (Fig. 4A). In contrast to DEK1 domain II&III protein, DEK1 domain II appears both in soluble and insoluble forms under standard expression conditions (Fig. 4A). The identity of the purified protein was confirmed by immunoblotting using an antibody raised against GST and by measuring GST activity (data not shown).
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The far-UV CD spectra demonstrate that DEK1 domain II&III, domain II and a mutant, C71S, of the DEK1 domain II&III protein all have a similar secondary structure (see below for details on the mutant protein) (Fig. 4B). These results indicated that no gross structural rearrangements had occurred in the mutated protein.
Characterization of DEK1 Domain II&III Proteinase
Activity—The proteolytic activity of DEK1 domain II&III was
tested in an assay in which purified -casein
(Fig. 5A, lane
2) was incubated with the DEK1 domain II&III protein under various
conditions (Fig. 5A,
lane 3). In the presence of 5 mM
Ca2+, casein degradation leveled off after approximately
9 h of incubation (Fig.
5A, lanes 4–9). This result suggests that
DEK1 domain II&III possesses an activity that is similar to that of
m-calpain. Surprisingly, however, DEK1 domain II&III also displays a
strong proteolytic activity in the absence of Ca2+. The
same proportion of -casein was degraded after 9 h as in the presence of
Ca2+ (Fig.
5A, lanes 10–15). Notably, in the presence
of Ca2+, DEK1 proteolysis of casein produced two bands
after 5 h of incubation (Fig.
5A, lane 6). In the absence of
Ca2+, the second breakdown product (lower molecular
weight) appeared as a weak band only after 12 h of incubation
(Fig. 5A, lane
15). From these observations we conclude that Ca2+
acts as an activator of DEK1 in this in vitro assay. Incubating
casein with different concentrations of purified DEK1 domain II&III
demonstrates that the casein degradation is concentration dependent
(Fig. 5, C and
D).
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Role of Cys-71 in DEK1 Domain II&III Proteolytic
Activity— The sequence alignment of DEK1 and m-calpain predicts
that Cys-71 is an active site residue of the catalytic triad. To verify
whether or not DEK1 is a true cysteine proteinase, we created a mutant in
which the Cys-71 was replaced with Ser as was previously done for animal
calpains (37,
38). The mutant protein was
expressed and purified in the same manner as wild-type DEK1 domain II&III
protein. This mutation did not cause secondary structural rearrangements as
evidenced by the CD spectrum (Fig.
4B). Activity measurements show that the mutant protein
is inactive compared with the wild-type protein
(Fig. 6A, lanes
4 and 5). This loss of activity clearly identifies the mutated
cysteine as part of the active triad of the DEK1 domain II protein, and is in
agreement with previous reports showing that the same mutation in m-calpain
abolishes this activity. To evaluate the effect of DEK1 domain III on the
proteinase activity of DEK1 domain II&III, DEK1 domain II alone was
incubated with -casein. This experiment showed that in vitro
DEK1 domain II alone was inactive (Fig.
6B, lane 6), demonstrating that domain
III is necessary for DEK1 proteolytic activity.
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Expression of Dek1 in Maize Grains—As shown previously, the Dek1 transcript is present at a low level in most plant tissues (9). To determine if Dek1 mRNA shows an aleurone preferred pattern of expression in endosperm, we carried out in situ hybridization experiments using sections from various grain developmental stages. These experiments show that the Dek1 transcript is detectable in all grain cell types, including the maternal pericarp, aleurone cells, and the starchy endosperm (Fig. 7, A and B). This result corresponds well with the previously reported LYNX MPSS data (9), suggesting that the activity of DEK1 in the aleurone layer is post-transcriptionally regulated.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Regulation in vivo of conventional animal calpains such as
m-calpain is complex, and operates at several levels, including signal
transduction, Ca2+ activation, subcellular relocation
from cytosol to, or near, the plasma membrane, interaction with phospholipids,
autocatalytic cleavage, and interaction with a regulatory small subunit.
Extensive studies of the three-dimensional structure of m-calpain and other
members of this cysteine proteinase family have revealed that the inactive
ground state of the enzyme is one in which the amino acids of the catalytic
triad are sterically separated. Calcium binding induces a conformational
change that assembles the catalytic triad of residues into its active
conformation (14,
36). In m-calpain, activation
involves a series of events including a relief of the conformational restraint
imposed by the interaction between domain I of the large subunit and the
penta-EF-hand domain VI of the regulatory subunit. Concomitantly,
Ca2+-induced conformational changes in domain IV are
transduced via domain III to the proteinase domain, domain II
(39,
40). These structural features
include an acidic loop consisting of 10 negatively charged residues in domain
III that makes direct contact with domain II
(13,
40,
41). The negative
electrostatic potential in the loop is somewhat counterbalanced by interdomain
salt bridges between some of the negatively charged residues and the basic
residues in domain II, including the Lys residues at positions 226, 230, 234,
354, 355, and 357 (Fig. 2).
Furthermore, it has been hypothesized that domain III itself can bind calcium
at this negative cluster, thereby further lowering the strongly negative
potential. The role of calcium binding in this model is to drive the
conformational change that simultaneously overcomes the considerable steric
hindrance from the Pro-287–Trp-288 loop in the active site interface and
permits movement of domains IIa and domains IIb toward each other to
"fuse" into the catalytically active domain. In this state, where
the competent catalytic triad is reassembled, the Cys S -His N 1
is reduced to 3.7 Å. This model explains why recombinant
conventional animal m-calpain is inactive in the absence of calcium, as well
as the underlying mechanism for the calcium-dependent activation of the
enzyme.
The lack of measurable activity of DEK1 domain II presented here is in accordance with the result reported for m-calpain domain II, showing less than 1% of full-length m-calpain activity (34). The similar characteristics of domain II from m-calpain and DEK1 appear reasonable considering the high similarity in their predicted structures (Fig. 3). In contrast, recombinant DEK1 domain II&III displayed significant activity in the absence of calcium, a characteristic that differs dramatically from that of m-calpain. In our interpretation of the DEK1 calpain 3D model (Fig. 3), the lack of an absolute calcium requirement for DEK1 calpain activity may be attributed to a ground state for the enzyme in which the catalytic triad is assembled close to its optimal configuration for activity. As mentioned above, several features of DEK1 calpain contribute to our conjecture. First, it has been suggested that a cluster of 4 negatively charged residues in m-calpain, Asp-96, Glu-172, Glu-320, and Glu-321 provide a strong repulsive force that prevents domains IIa and IIb from coming together and that charge compensation through calcium binding relieves this repulsive force and facilitates the fusion of the catalytic domain (41). In DEK1 calpain, only Asp-62 (corresponding to Asp-96 in m-calpain) is conserved, suggesting that this repulsive force is not operating in DEK1 calpain, thus making charge-compensation by calcium binding less needed. Secondly, DEK1 domain III is missing 9 of the 10 negatively charged residues in the loop corresponding to the acidic loop of m-calpain. Furthermore, key Lys residues at positions 226, 230, 234, 354, 355, and 357 of m-calpain are also absent in DEK1 domain III, making it unlikely that this domain can bind calcium and thereby regulate calpain activity (Fig. 2). We suggest that the increase in DEK1 calpain activity when calcium is added is probably caused by a slight repositioning of domain IIa and IIb, leading to an optimalization of the catalytic triad configuration. The mechanism, as well as the biological role of Ca2+ activation in DEK1 calpain functioning remains to be determined.
In addition to domain II&III discussed above, the conventional Ca2+-dependent calpain holoenzymes consist of a domain I and an EF-hand domain IV, as well as a regulatory small subunit. Animal calpains, however, are a diverse group of proteins with domain II as the main conserved feature. In addition, most calpains, including DEK1 have domain III. There are, however, exceptions to this rule, as calpain 10 lacks a penta-EF-hand domain, while neither a C2-like domain nor a penta-EF-hand domain is present in SOL or in SOLH (13). So far, a calcium requirement for calpain 10 or SOL has not been reported (17, 18), Interestingly, in the animal calpain super-family, the acidic loop exhibits high sequence diversity in terms of the number of negatively charged residues (see discussion above, Ref. 13). This variability may provide a plausible explanation for the differences in calcium sensitivities among members of the calpain family, also raising the possibility that some animal calpains may act in a Ca2+-independent manner.
In contrast to the diversified structure of animal calpains, plants appear to possess only one member of the calpain super family, namely DEK1, showing a high degree of conservation among plant species (9). Similar to animal calpains, the catalytic domain II of plant DEK1 homologues is the most highly conserved domain. For example, domain II, which is 302 amino acids long, is 100% identical between maize and sugarcane. A comparison of domain II sequences between maize and loblolly pine, a gymnosperm, reveals 79% similarity (9). The high conservation between all these sequences suggests an important function for the DEK1 protein in all plants. Most notably, DEK1 calpain represents the C-terminal domain of a 240-kDa protein that is predicted to be anchored in the plasma membrane by 21 transmembrane segments interrupted by a putative extra cytosolic loop domain (9). A membrane-anchoring domain is unusal for animal calpains, which mostly are cytosolic enzymes that are translocated to the plasma membrane upon activation (42, 43). The only known example of an animal calpain with a predicted membrane anchor is the Drosophila calpain CG3692 that has a transmembrane domain structure similar to DEK1 calpain. The calpain domains are linked to the membrane part of the DEK1 protein by a 600 amino acid segment with few recognizable features.
The Dek1 gene plays an important role in plant development, being essential for the proper development of the aleurone layer in maize grains, embryo shoot apical meristem function as well as leaf epidermis formation (14, 36). A second gene known to function in signal transduction in the same tissues is the TNFR-like receptor like kinase CR4 (4, 5). Recently, we cloned a third gene implicated in the same developmental pathways, Superal1 (supernumerary aleurone layer 1), encoding a plant homologue of CHMP1, a member of the E-vacuolar protein sorting family (44). This finding may suggest that regulation of aleurone cell fate involves CR4 receptor internalization through endosome trafficking and targeted proteolysis in vacuoles. Interestingly, animal calpains have been implicated in vesicle trafficking, including the formation of coated vesicles and vesicle fusion to endosomes (30). One possibility, therefore, is that DEK1 functions in the endosome trafficking pathway by modifying membrane proteins participating in the formation and targeted transport of membrane vesicles. The in situ hybridization results presented here suggest that Dek1 is transcribed in all cell types, despite the fact that in the endosperm, Dek1 function is essential only in the aleurone layer (9). We are currently exploring the possibility that the DEK1 calpain is activated only in epidermal cell layers mediated through interaction(s) between the predicted extracellular loop region of DEK1 and extracellular factors.
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FOOTNOTES |
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To whom correspondence should be addressed. Tel.: 515-253-2417; Fax:
515-254-2619; E-mail:
odd-arne.olsen{at}pioneer.com.
1 The abbreviations used are: dek1, defective kernel 1 gene; GST,
glutathione S-transferase; DAP, days after pollination; IPTG,
isopropyl-1-thio--D-galactopyranoside.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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