(Received for publication, August 25, 1995; and in revised form, January 29, 1996)
From the
Cyclic inositol phosphohydrolase is a phosphodiesterase that cleaves the cyclic bond of cyclic inositol monophosphate. In 1990, Ross et al. (Ross, T. S., Tait, J. F., and Majerus, P. W.(1990) Science 248, 605-607) purified this enzyme from human placenta and reported that cyclic inositol phosphohydrolase is identical to annexin III. Independent confirmation of this finding has not been provided. The relative distribution of annexin III and cyclic inositol phosphohydrolase activity in rat kidney and spleen indicated that annexin III can be dissociated from cyclic inositol phosphohydrolase activity. Rat spleen contains large quantities of annexin III, but has very little cyclic inositol phosphohydrolase activity. In contrast, rat kidney, one of the richest sources of cyclic inositol phosphohydrolase activity, possesses very little (immunohistochemistry) or no (Western blot) annexin III. Similar to cytosol of human placenta, cytosol of guinea pig kidney contains both annexin III and cyclic inositol phosphohydrolase. On SDS-gel electrophoresis, guinea pig kidney annexin III has a slightly different mobility than the human placental annexin III. Human placental annexin III co-migrates with cyclic inositol phosphohydrolase on ion exchange chromatography, while guinea pig kidney annexin III is clearly dissociated from cyclic inositol phosphohydrolase on ion exchange chromatography. Both guinea pig kidney annexin III and human placental annexin III pellet with the addition of calcium and centrifugation, while cyclic inositol phosphohydrolase activity in both of these tissues remains in the supernatant. Our studies clearly show that cyclic inositol phosphohydrolase and annexin III are two different proteins.
Phospholipase C hydrolysis of phosphoinositide results in the
generation of both cyclic and noncyclic inositol
phosphates(1) . Cyclic inositol mono- and polyphosphate esters
have been observed in intact tissue, and their accumulation in response
to agonist activation has been described in mouse pancreas(2) ,
kidney(3, 4) ,
platelets(5, 6, 7) , parotid
gland(8) , and SV40 transformed cells(9) . Dawson and
co-workers (10) identified a phosphodiesterase that
specifically hydrolyzes the cyclic bond of cyclic inositol
monophosphate (cIP) ()to yield inositol 1-phosphate. It is
currently believed that cyclic inositol polyphosphate is first
converted to cyclic inositol monophosphate by stepwise
dephosphorylation, before the cyclic bond is cleaved by cyclic inositol
phosphohydrolase (cIPH). Kidney has the highest activity of
cIPH(10, 11) . More recently, Ross and Majerus (12) have purified and characterized cIPH from human placenta
and later identified the placental cIPH as identical with lipocortin
III(13) . They have further reported increased cIPH activity
following transfection of lipocortin III (14) and reported
identification of another phosphodiesterase that converts cIP to
inositol 2-phosphate(15) .
Lipocortin III has also been
referred to as placental anticoagulant protein III(16) ,
calcimedin 35-(17) , and annexin
III(18, 19) . In accordance with recent agreement
among 41 researchers working in this field(19) , this protein
will be referred to as annexin III. Annexin III is one of the 12
members of the annexin family of proteins that have been identified so
far. Proteins belonging to this family have the following two
characteristics: (a) Conserved 70-amino acid domain repeated
either four or eight times in the overall structure and (b)
ability to bind to phospholipids in a calcium-dependent manner.
Annexins have been implicated in various cellular functions such as
exocytosis(20) , formation(21, 22) or
modulation of ion channels(23) , and membrane attachment of
cytoskeletal elements(24, 25, 26) .
During the course of investigating cyclic inositol phosphate metabolism, we reinvestigated the putative identity of cIPH as annexin III(13) . Evidence provided in this paper, when taken in conjunction with data already reported in the literature, clearly support our conclusion that annexin III and cIPH are two different proteins.
Inositol dehydrogenase, bathophenanthroline, inositol, cyclic
inositol monophosphate, and NAD were obtained from
Sigma; phenazine was from Aldrich; ferric chloride was from
Mallinckrodt; rabbit polyclonal antibody to rat spleen annexin III was
a gift from Dr. John Dedman, University of Cincinnati, Cincinnati, OH;
and rabbit polyclonal antibody raised against human neutrophil annexin
III was a gift from Dr. Joel Ernst, University of California at San
Francisco. All other reagents and chemicals used were of analytical
grade. Kidney and spleen were obtained from Sprague-Dawley male rats
(100-150 g), guinea pig tissues were obtained from male albino
(350-450 g), and human placenta following normal delivery.
Figure 1: Distribution of annexin III and cIPH activity in rat spleen and kidney. A, Western blot-tissue homogenate protein (25 µg) from spleen, renal medulla, and renal cortex was separated by SDS-PAGE and transferred to Immobilon. Proteins were probed with annexin III antibody raised against rat spleen. B, tissue homogenate protein (20 µg) was assayed for cIPH activity using the nonradioactive assay (see ``Materials and Methods''). Results are calculated as nanomoles of inositol released per min per mg of protein, based on a 5-min incubation and expressed as mean ± S.D. (n = 3). Similar results were obtained in three other experiments.
Figure 2:
Immunostaining of rat spleen and kidney
cortex and medulla with annexin III antibody. A, rat spleen
( 100) stained for annexin III using a polyclonal antibody and
Biogenix detection system; arrows point toward staining of red
pulp. B, higher magnification of A (
200); arrow points toward staining of red pulp. C, rat
kidney cortex (
200) stained for annexin III using a polyclonal
antibody and biogenix detection system; arrow demonstrates the
only staining in Bowman's capsule of the glomerulus. D,
rat kidney medulla (
400) stained for annexin III; arrow demonstrates a small amount of staining in basal area of
collecting ducts.
Antibody raised against human neutrophil annexin III recognizes a specific band on the Western blot of both guinea pig spleen and kidney as well as in human placenta and neutrophil (Fig. 3A). Presence of annexin III on the Western blot is consistent with the earlier report indicating that 1% of the cytosolic protein in neutrophil is annexin III(32) . Interestingly, while significant cIPH activity is present in both guinea pig kidney and human placenta, no cIPH activity is detected in human neutrophil (Fig. 3B). This observation further dissociates cIPH activity from annexin III.
Figure 3: Distribution of annexin III and cIPH activity in guinea pig kidney, guinea pig spleen, human placenta, and neutrophil. A, annexin III Western blot, cytosol of guinea pig spleen (GPS), guinea pig kidney (GPK), human placenta (HP), and the homogenate of human neutrophil (HN) were separated by SDS-PAGE and transferred to Immobilon-P. Proteins probed with annexin III antibody were raised against human neutrophil annexin III. B, cIPH activity, guinea pig spleen cytosol (40 µg), guinea pig kidney cytosol (50 µg), human placenta cytosol (200 µg), and human neutrophil homogenate (200 µg) were assayed for cIPH activity by nonradioactive assay (see ``Materials and Methods''). Results are calculated as nmol of inositol released per min per mg of protein, based on a 15-min incubation.
Annexin III from both guinea pig kidney and spleen, on SDS-gel electrophoresis consistently (five different experiments) migrate slightly slower than the annexin III obtained from human neutrophil and placenta (Fig. 3A). Interestingly, a slower migrating form of annexin III has also been observed in human monocytes(33) . Guinea pig annexin III appears to be immunologically distinct from rat annexin III, as rat spleen annexin III antibody fails to recognize annexin III in either guinea pig spleen or kidney (data not shown).
Figure 4: Fractionization of human placental cytosol on ion exchange chromatography. Human placental cytosol (10 mg of protein) was injected onto a perceptive ion exchange chromatography (POROS HQ/H) attached to a Biosprint HPLC. Sample (3 ml) in Tris/MES pH 7.0 buffer was loaded onto the column, followed by washing with 5 column volumes of buffer and elution with 30 column volumes of a linear gradient to 0.2 M sodium chloride. Flow rate was 4 ml/min, and 0.5-min fractions were collected. Fractions 5 to 16 (0.5-min fractions) were assayed for both annexin III and cIPH activity. A, annexin III Western blot. L and S refer to the original sample prior to injection and the standard annexin III, respectively. B, cIPH activity of the ion exchange fractions by radioactive method. 100 µl of the fraction were incubated with 55 µM labeled cIP for 60 min. Results are shown as disintegrations/min of inositol released in the supernatant.
Figure 5: Fractionization of guinea pig kidney cytosol by ion exchange chromatography. Guinea pig kidney cytosol (10 mg of protein) was injected onto a perceptive ion exchange chromatography (POROS HQ/H) attached to a Biosprint HPLC, conditions identical with that in Fig. 4. Fractions 5 to 16 (0.5-min fractions) were assayed for both annexin III and cIPH activity. A, annexin III Western blot. L and S refer to the original sample prior to injection and the standard annexin III, respectively. B, cIPH activity of the ion exchange fractions by radioactive method. 100 µl of the fraction were incubated with 55 µM labeled cIP for 60 min. Results are shown as disintegrations/min of inositol released in the supernatant.
Figure 6:
Distribution of annexin III and cIPH
activity following calcium precipitation. Cytosol from guinea pig
kidney and human placenta were treated with calcium and centrifuged as
described under ``Materials and Methods.'' A,
annexin III Western blot, protein (10 µg) from both guinea pig
kidney and placenta for each of the three fractions, S (untreated cytosol), CaP, and CaS are pellet and
supernatant obtained following calcium treatment and 100,000 g spin, probed with antibody to human neutrophil annexin III. B, cIPH assay performed on the same fractions, S, CaP, and CaS by nonradioactive cIPH assay (see
``Materials and Methods''). Protein (50 µg) used for
guinea pig kidney and (100 µg) for human placental samples. Results
are calculated as nanomoles of inositol released per min per mg of
protein, based on a 30-min incubation. Similar results were obtained in
two other experiments.
Ross and Majerus (12) in 1986 purified cIPH from
human placenta and reported in 1990 that cIPH is identical with annexin
III(13) . In a later publication, they reported having
expressed cyclic inositol phosphohydrolase activity by transfecting
annexin III cDNA(14) . Consistent with the earlier
observations(10, 11) , renal homogenate possessed
significant cIPH activity (Fig. 1B), but to our
surprise failed to bind rat annexin III antibody on a Western blot (Fig. 1A). The same antibody clearly recognized annexin
III from rat spleen (Fig. 1A). Rat spleen, which is
rich in annexin III and gives a strong positive signal both on Western
blot and immunohistochemical staining with annexin III antibody, has
20-fold lower cIPH activity compared to the kidney. The above results
were fully consistent with what was reported in the literature. In an
earlier study (17) (where annexin III has been referred to as
calcimedin 35-), a significant amount of annexin III could be
detected on a Western blot of rat spleen, but none could be detected on
the Western blot of rat kidney. The above discrepancy in the relative
distribution of cIPH and annexin III, as well as the fact that none of
the groups working with annexin III have independently confirmed the
cIPH activity of annexin III, raised the possibility that cIPH may have
been misidentified as annexin III.
A closer look at the original (12, 13) purification scheme suggests that the first ion exchange chromatography step may have had poor resolution capacity. In the study of Ross and Majerus(12) , 816 mg of human placental cytosol protein was loaded on a Bio-Gel TSK DEAE 5 PW HPLC anion exchange column. Even though it is a preparative column, the total load was severalfold higher than the maximum column capacity for that column. Direct loading of such a large amount of the crude preparation, without preliminary purification, on an HPLC will likely lead to poor resolution. When we loaded 10 mg of crude cytosolic placental protein (the maximum capacity for this column) onto a perceptive ion exchange column, to determine whether it is able to resolve annexin III and cIPH, human placental annexin III and cIPH appeared in the same fractions (Fig. 4). Interestingly, under identical conditions, in guinea pig kidney, annexin III cannot be detected in Western blot in fractions demonstrating cIPH activity (Fig. 5).
In their original report, Ross et al.(13) identified the cIPH activity as annexin III based on a single Western blot following final purification, with polyclonal antibody generated against a 12-amino acid amino-terminal peptide of annexin III. One of the well established properties of annexins is that they precipitate with calcium. When guinea pig kidney cytosol and human placental cytosol were treated with calcium, cIPH activity remained in the supernatant, while annexin III is seen in the pellet (Fig. 6). But the pellet containing annexin III had no cIPH activity. We suggest that misidentification of cIPH as annexin III resulted because human placenta contains both cIPH and annexin III in cytosol, which comigrated during purification (not unusual), and the purification procedure did not employ any specific step to deplete annexin III.
We have no logical explanation for the small cIPH activity (0.02 µmol/min/mg of protein) reported with purified annexin III (13) or the small amount of activity detected following transfection of Swiss 3T3 cells with the cDNA of annexin III(14) . As the assay conditions used in the transfections studies (14) were reported to be similar to their original paper(12) , we can make the following calculations from their work. The total amount of substrate, based on a final substrate concentration of 75 µM and the incubation volume of 25 µl, was 1.87 nmol of cIP. In the only time course reported(14) , the annexin III transfected cells were reported to generate 30 nmol of inositol monophosphate per mg of protein, over the entire incubation period of 120 min. If we assume they used 0.3 mg of protein (based on Fig. 1of (15) ), this amounts to 9 nmol of cIP being hydrolyzed during the 120-min incubation, which is 5-fold higher than the total cIP present at the beginning of the incubation. These calculations make it highly unlikely that they were measuring true cIPH activity.
Neutrophil provides an interesting test case. If annexin III indeed possessed cIPH activity, one would expect a very high level of cIPH activity in neutrophils, as 1% of the neutrophil cytosolic protein is annexin III(32) . In a single observation, Ross and Majerus (34) reported that neutrophils have a cIPH activity of 12,000 pmol/min/mg of protein, which would be consistent with the above hypothesis. However, the authors do not describe the exact conditions used for this single point measurement. If we assume that incubation conditions were similar to those described earlier (10-min incubation in the presence of 0.3 mg of protein), it would result in consumption of 36,000 pmol of substrate during the course of this incubation, 19-fold higher substrate than what was actually provided at the beginning of the assay.
In our hands, three
different experiments with neutrophils (Fig. 2) as well as time
course studies (data not shown) detected no cIPH activity, which is
consistent with our finding that annexin III and cIPH are two different
proteins. Further support for such a conclusion is obtained from the
work of Riddle et al.()where overexpression of the
gene coding annexin III in Escherichia coli is not accompanied
by enhanced cIPH activity. Annexin III is much better characterized
protein (36) than cIPH, and this ``presumed
identity'' between cIPH and annexin III has dampened progress in
understanding the role of cIPH and cyclic inositol phosphate
metabolism. The importance of our finding is that it paves the way for
purification, characterization, cloning, and determining the real
sequence of cIPH. Such studies should provide impetus to clarify the
role of cIPH in kidney and placenta as well as determine its
significance in cell growth and proliferation.