(Received for publication, March 27, 1995; and in revised form, September 26, 1995)
From the
We previously reported on the purification of a serum
calcium-decreasing factor, referred to as caldecrin, from porcine
pancreas, that is thought to be a serine protease (Tomomura, A.,
Fukushige, T., Noda, T., Noikura, T., and Saheki, T.(1992) FEBS
Lett. 301, 277-281). In the present study, we purified
caldecrin from rat pancreas and determined its primary structure by
cDNA cloning. The predicted caldecrin protein is presumed to be
synthesized as a preproenzyme of 268 amino acids with a signal peptide
of 16 amino acids and an activation peptide of 13 amino acids, and is,
with the exception of a central region, almost identical to the
reported rat pancreatic elastase IV sequence. The caldecrin gene is
selectively expressed in the pancreas, as judged by Northern blot
analysis. After expression in BMT-10 cells, immunoreactive caldecrin
was found in the culture supernatant, and it inhibited the parathyroid
hormone-stimulated Ca release from cultured fetal long
bones. Catalytic site mutants were synthesized in a baculovirus system,
and recombinant mutants also decreased the serum calcium level of mice.
These data implicate caldecrin, a protease closely related to elastase
IV, in the regulation of blood calcium levels.
It is well known that blood calcium is regulated by several
calcitropic hormones, e.g. calcitonin, parathyroid hormone
(PTH), ()and 1,25-dihydroxy vitamin D
,
synthesized by the thyroid, parathyroid gland, and kidney,
respectively. However, the involvement of other organs in the
regulation of blood calcium level is not clearly understood. It has
been reported that some gastric factors reduce blood calcium levels and
that vagally mediated hypocalcemia is induced by hypothalamic
stimulation(1, 2, 3) . Another etiologic
organ is the pancreas. Acute pancreatitis causes hypocalcemia, which
suggests that pancreas tissue contains hypocalcemic
factors(4, 5) . Among pancreatic hormones, glucagon
and amylin are reported to have hypocalcemic effects (6, 7, 8) . Earlier we purified a serum
calcium-decreasing factor (caldecrin) from porcine
pancreas(9) . Caldecrin is a chymotrypsin-like serine protease
that lowers blood calcium levels in mice and inhibits in vitro PTH-stimulated bone resorption in long bone cultures.
Most serine proteases are synthesized as preproenzymes and secreted as proenzymes (zymogens) that require enzymatic cleavage of an amino-terminal peptide for conversion to active proteases. Recently, a proform of caldecrin (procaldecrin) was successfully purified from porcine pancreas(10) . Procaldecrin does not possess serum calcium-decreasing activity (SCDA), but acquires SCDA, as well as protease activity, upon trypsin treatment. Incubation of the activated caldecrin with the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF) destroys its protease activity, but has no effect on its SCDA(9, 10) .
The mammalian exocrine pancreas contains a predominant superfamily of serine proteases and isoforms of these enzymes. Two chymotrypsinogens, cationic chymotrypsinogen A and anionic chymotrypsinogen B, have been isolated from bovine and rat pancreas(11, 12, 13) . Anionic chymotrypsinogen C was isolated from porcine pancreas(14) . The nomenclature of the related elastase family is more complicated. Two elastase isozymes, elastases I and II, were found in pigs, humans, and rats(15, 16, 17, 18) . Rat and porcine elastase I cDNAs (19, 20) and rat, porcine, and human elastase II cDNAs (19, 21, 22) were successfully isolated. Anionic protease E was purified from the human, porcine, and bovine pancreas(23, 24, 25) , and it hydrolyzed synthetic elastase-specific substrates but failed to cleave elastin. Human protease E (26) and its isozyme (27) were found to be identical to elastase IIIB and IIIA(28) , respectively. Kang et al. reported the sequence of elastase IV cDNA amplified by the polymerase chain reaction (PCR) from rat pancreas RNA(29) .
In this paper, we report on the purification of a rat caldecrin that displays SCDA and the isolation of a rat caldecrin cDNA clone. On the basis of the predicted protein structure, caldecrin can be classified as an elastase-type protease. Recombinant rat caldecrin inhibited PTH-stimulated calcium release from cultured bones. Mutant caldecrins, which lacked protease activity, also decreased the serum calcium level of mice.
gt11 recombinant lysogens were prepared from positive
clones (rat pancreas caldecrin, pRPC) by the method of Huynh et al.(32) . Briefly, BNN103 cells were infected with the
gt11 clone. All clones that grew at 30 °C but lysed the cells
at 42 °C were cultured at 30 °C overnight, at 42 °C for 2
min, and at 37 °C for 2 h. After centrifugation, the precipitates
were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) and
Western blotting.
The cDNA library was rescreened with an EcoRI/PstI fragment of pRPC18 used as a probe. Positive clones were subcloned into pUC19, and both strands of inserted DNA were sequenced by use of Sequenase (version 2) (U. S. Biochemical Corp.) and an automatic DNA sequencer (Applied Biosystems).
For amino acid sequence analysis of proteolytic
fragments, purified caldecrin from rat and pig were digested at 60
°C for 1 h with metalloendopeptidase from Grifola frondosa (2.3 units/µg) (Seikagaku Kogyo) at a substrate to enzyme
ratio of 100:1 (w/w) in 50 mM ammonium acetate buffer (pH 10)
containing 5 mM PMSF. The digests were immediately loaded onto
a C18 reverse-phase HPLC column (Vyduc 0.46 25 cm) and eluted
with a linear gradient of 0-80% acetonitrile in 0.1%
trifluoroacetic acid. The amino acid sequences of the peptides were
determined with an automated gas-phase protein sequencer (Applied
Biosystems).
Figure 1: SDS-PAGE and Western blotting analysis of purified rat caldecrin. The eluate from the Superdex 75 column was subjected to SDS-PAGE and analyzed by Western blotting using anti-porcine caldecrin antibody. The molecular sizes of markers are indicated on the left.
Figure 2: Bioassay for dose-response curves of rat caldecrin before and after PMSF treatment. Purified rat caldecrin treated (closed circle) or not (open circle) with PMSF and porcine caldecrin (open square) were separately administered to mice, and the serum calcium concentration was determined 4 h after injection. PMSF pretreatment was as described under ``Experimental Procedures.'' Data are expressed as means ± S.D. values obtained from five mice.
Figure 3: Northern blot analysis of several rat tissues. Total RNA was isolated from different rat tissues by acid guanidinium thiocyanate-phenol-chloroform extraction, and equal amounts of RNA were subjected to Northern blot analysis with random primer-labeled rat caldecrin cDNA as the probe. The migration positions of 28 and 18 S ribosomal RNA are indicated.
Figure 4: Nucleotide and deduced amino acid sequences of rat caldecrin cDNA. The primary nucleotide sequence (upper row) and deduced amino acid sequence indicated by the single-letter code (second row) of rat caldecrin are shown. An open reading frame extends from the translation initiation site (ATG) to the termination site (TGA). This is followed by a 3`-untranslated region that ends with a polyadenylation signal (bold underlined) and the poly(A) tail. The vertical arrowheads indicate proteolytic processing sites. The deduced amino acid sequence is numbered sequentially from the amino terminus of the predicted active enzyme. The amino acid residues of the charge-relay system are circled(37) , and the residues determining the substrate binding specificity are boxed(15) . The corresponding amino acid sequences of fragments obtained by proteolytic cleavage of rat caldecrin are underlined with a dashed line. The amino acid sequences of fragments analyzed after proteolytic cleavage of porcine caldecrin are shown below the rat caldecrin sequences (third row). The deduced amino acid sequence of elastase IV (NBRF-PDB accession number JQ1473) is given in the bottom row. Asterisks indicate residues conserved between rat caldecrin and rat elastase IV.
Next, purified rat caldecrin was digested in the presence of PMSF with a metalloendopeptidase that cleaves specifically on the amino-terminal side of lysine residues. After separation of the resulting fragments by C18 reverse-phase HPLC, amino acid sequences of several major peptides were determined (Fig. 5A). All amino acid sequences of the six peptides examined were identical to the corresponding parts of the amino acid sequences deduced from the rat caldecrin cDNA (Fig. 4). Then, to compare at the molecular level the caldecrins purified from rat and porcine pancreas, we also subjected porcine caldecrin to the same metalloendopeptidase cleavage protocol. Again, all sequences of the nine fragments obtained showed high homology with the deduced amino acid sequence of rat caldecrin ( Fig. 4and Fig. 5B). These results indicate that the caldecrins purified from rat and porcine pancreas are both members of the same elastase IV subfamily.
Figure 5: Separation and sequencing of proteolytic fragments of rat and porcine caldecrin. Caldecrins purified from rat (A) and porcine (B) pancreas were digested with metalloendopeptidase, applied onto a C18 reverse-phase HPLC column, and eluted with a linear gradient of acetonitrile (dashed line). The effluent was monitored at 229 nm. Amino acid sequences were performed on the numbered peaks, and residues are indicated by the single-letter code. Unidentified amino acid residues are denoted by X. A, rat caldecrin fragments: 1, KDFTYRVGLG (corresponding to sequence of residues 49-58); 2, KDDTWRHTXGGSLITTSHVLT (residues 22-42); 3, KKPVVFTRVSAYNDWINE (residues 218-235); 4, KPVVFTRVSAYNDWINE (residues 219-235); 5, VVGGEDAVPNSWPWQVSLQYL (residues 1-21); 6, KWNRLFLWNDIAI (residues 83-95). B, porcine caldecrin fragments: 1, KWN (residues 83-85); 2, YLSGDT (residues 20-25); 3, KHTXG GTLITSTHVLTAAHXISNSRT (residues 27-52); 4, KHTXGGTLITSTHVLTAAHXISNSRTY (residues 27-53); 5, KWNSLLIRNDIA (residues 83-94); 6, RVSAYIDWIDQ (residues 225-235); 7, KWNSLLIRNDIAL (residues 83-95); 8, KNNLEVEDEEGSLVVGVDSIFV (residues 59-80) and KKPLVFARVSAYIDWIDQQ (residues 218-236); 9, VVGGENAVPHSWPWQISLQYLSGDT (residues 1-25).
Figure 6: Expression of rat caldecrin in BMT-10 cells and secretion into the medium. BMT-10 cells transfected with either pCAGGS vector alone (V), rat caldecrin cDNA in the antisense orientation (R), or rat caldecrin cDNA in the sense orientation (N) were cultured for 1 or 2 days. The cell lysates obtained from 2-day cultures, and the media from 1- and 2-day-old cultures, were analyzed by Western blotting using anti-porcine caldecrin antibody. Molecular sizes of markers are indicated on the left.
Recombinant rat caldecrin was also
examined for hypocalcemic activity. Medium conditioned by the cells
transfected with the caldecrin cDNA was used in the in vitro assay utilizing fetal long bone cultures, which cultures have been
shown to be a valid model system to study porcine caldecrin
activity(9) . As shown in Fig. 7, medium containing
recombinant rat caldecrin inhibited dose-dependently the PTH-stimulated
release of Ca from the bone. The conditioned medium caused
maximum inhibition at 20 µl/ml of assay medium and maintained this
level of inhibition at higher doses. The conditioned medium of cultured
cells transfected with antisense cDNA did not affect the PTH-stimulated
release of calcium from the bone.
Figure 7:
Inhibition of the PTH-stimulated Ca release from cultured long bones by recombinant rat
caldecrin. BMT-10 cells were transfected with rat caldecrin cDNA in the
sense (open circle) or antisense (open square)
orientation and cultured for 3 days. The conditioned media were
collected and added to the medium (0.5 ml) of long bone cultures, which
were then cultured for another 3 days in the presence of 10 nM PTH before measurement of
Ca release. The open
triangle indicates basal calcium release from
bone.
Figure 8: SCDA of recombinant wild-type and mutant caldecrin is not required for the protease activity. Samples of recombinant wild-type (proform), trypsin-activated wild-type, and mutant caldecrins were assayed for chymotrypsin activities, and others were injected into mice at a dose of 100 µg/kg body weight. Blood was taken 4 h after the injection, and the serum calcium concentration was then measured.
In this paper, we reported the primary structure of rat
caldecrin, a proteolytic enzyme from rat pancreas, and showed that it
is highly related or identical to elastase IV, a previously analyzed
protease of the chymotrypsin/elastase superfamily. This conclusion is
based on the following findings. (i) The rat caldecrin cDNA encodes a
serine protease zymogen consisting of a signal peptide, an activation
peptide, and a mature enzyme. Eight cysteine residues present at
positions 30, 46, 126, 157, 173, 183, 193, and 214 of the mature
protein are conserved in all elastase family members, which suggests
the formation of four disulfide bonds as observed in porcine elastase
I(15) . In addition, three cysteine residues are present at the
positions -14, -13, and 112, indicating that a cysteine
residue at position -14 or -13 may form a disulfide bond
with Cys-112. (ii) The amino-terminal four residues of the mature form
of pancreatic elastase, Val-Val-X-Gly, are highly conserved
and these residues follow an arginine residue. Table 1, part A,
corroborates that rat caldecrin is synthesized as a zymogen with a
signal peptide of 16 residues, and an activation peptide of 13 amino
acids. Moreover, these data suggest that a cysteine (at position
-13) of the activation peptide forms a disulfide link with an
internal cysteine, i.e. 112, and that the activation peptide
is associated with the enzyme after proenzyme activation, a situation
analogous to that seen with both elastase II and chymotrypsin
processing, as given in Table 1, part
B(11, 12, 13, 19, 22, 29, 37) .
(iii) The nucleotide sequences of rat caldecrin and rat elastase IV
genes are 99.3% identical, but the deduced amino acid sequences display
only 90.3% identity (Fig. 4). These results strongly suggest
that rat caldecrin could be an isoform of elastase IV generated by some
unusual splicing mechanism. Alternatively, and in our view more likely,
however, the differences between our protein sequence and the elastase
IV sequence published by Kang et al.(29) result from
a frameshift reading error in the corresponding region of the cDNA by
the latter group (see Fig. 4). Unfortunately, the elastase IV
protein has so far not been investigated. Further studies will be
required to resolve this problem. In any event, a stretch of the
deduced amino acid sequence of rat caldecrin cDNA, which is quite
different from that of elastase IV, was identical to the peptide
sequence of a fragment obtained from the purified rat caldecrin and had
a high homology with the corresponding peptide sequence of purified
porcine caldecrin, indicating that the caldecrin gene is really
translated. Moreover, the deduced amino acid sequence of the human
caldecrin cDNA that we recently cloned displays, within the region of
sequence divergence between caldecrin and elastase IV, a very high
identity with the rat caldecrin analyzed in a study to be published
elsewhere. ()
The amino acid residues characteristic of the serine protease catalytic triad (38) are retained at positions His-45, Asp-92, and Ser-187 of the deduced amino acid sequence. The other key amino acid residues, Gly-209 and Val-221, thought to contribute to the substrate specificity of such enzymes, may enlarge the substrate binding pocket to accommodate more bulky amino acid side chains(19) . The presence of an activation peptide similar to that found in chymotrypsin and elastase II suggests a chymotrypsin-like substrate preference and indicates that caldecrin may be an evolutionary link between chymotrypsin and elastase I. Anionic elastase possessing chymotrypsin activity has not been found in rat or porcine pancreas. The partial amino acid sequence of anionic chymotrypsin C has been reported, but its cDNA sequence has not. The partial amino acid sequence around an essential histidine of porcine chymotrypsin C (39) is not identical to that of porcine caldecrin.
So far, many proteases with elastolytic activity have been isolated from various tissues. Thus, careful consideration should be given to this nomenclature, in particular to elastase numbering. Rat and porcine caldecrins might be classified as elastase IV family members based on the current nomenclature rules, although the SCDA of these proteins apparently is not connected with protease activity. Yoneda et al.(40) have reported partial purification of a factor in a porcine pancreatic extract (PX) that decreases the blood calcium level, and they also showed that PX prevents progression of hypercalcemia and cachexia in mice inoculated with a carcinoma cell line(41) . Amino-terminal sequencing of PX indicates that it belongs to the elastase IIIB family, and recombinant elastase IIIB displays hypocalcemic activity that is dependent on its protease activity(42) . The reasons for this discrepancy between their results and ours are presently unclear. In any case, all presently available data show that same hypocalcemic factor(s) exists in the pancreas, and their structural analysis uncovers a novel regulatory function of these proteases. However, rat and porcine caldecrins treated with PMSF and even two kinds of the catalytic site mutants of rat caldecrin still displayed SCDA, suggesting that caldecrin may possess functional residues for SCDA that differ from those involved in the protease activity. The proform of caldecrin does not possess SCDA but acquires this property as well as protease activity after activation by trypsin treatment. These results suggest that the residues responsible for SCDA require a trypsin-induced conformational change to be come exposed at the outer surface of the molecule. The SCDA of caldecrin correlates with a decrease in serum hydroxyproline levels and with the inhibition of PTH-stimulated bone resorption(9) , suggesting that caldecrin suppresses osteoclastic activity through an as yet unknown mechanism. All these data indicate that caldecrin, a protease closely related or identical to elastase IV, is a multifunctional protein that may be implicated in the regulation of blood calcium levels.