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Isolation and characterization of mosquito ferritin and cloning of a cDNA that encodes one subunit.

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Archives of Insect Biochemistry and Physiology 29:293-307 (1995)
Isolation and Characterization of Mosquito
Ferritin and Cloning of a cDNA That Encodes
One Subunit
Boris C. Dunkov, Dianzheng Zhang, Kyril Choumarov, Joy J. Winzerling,
and John H. Law
Department of Biochemistry and the Center for Insect Science, Uriiversity of Arizona,
Tucson, Ar-izona
Ferritin, an iron storage protein, was isolated from larvae and pupae of Aedes
aegypti grown in an iron-rich medium. Mosquito ferritin i s a high molecular
weight protein composed of several different, relatively small, subunits. Subunits of molecular mass 24, 26, and 28 kDa are equally abundant, while that
of 30 kDa is present only in small amounts. The N-terminal sequence of the 24
and 26 kDa subunits are identical for the first 30 amino acids, while that of the
28 kDa subunit differs. Studies using antiserum raised against a subunit mixture showed that the ferritin subunits were present i n larvae, pupae, and adult
females, and were increased in animals exposed to excess iron.
The antiserum also was used to screen a cDNA library from unfed adult
female mosquitoes. Nine clones were obtained that differed only in a 27 bp
insertion in the 3' end. Rapid amplification of cDNA ends (RACE) was used to
obtain the complete protein coding sequence. A putative iron-responsive element (IRE) is present in the 5'-untranslated region. The deduced amino acid
sequence shows a typical leader sequence, consistent with the fact that most
insect ferritins are secreted, rather than cytoplasmic proteins. The sequence
encodes a mature polypeptide of 20,566 molecular weight, smaller than the
estimated size of any of the subunits. However, the sequence exactly matches
the N-terminal sequences of the 24 and 26 kDa subunits as determined by
Edman degradation. Of the known ferritin sequences, that of the mosquito is
most similar to that of somatic cells of a snail. o 1995 ~ i ~ e y - ~Inc.
is,
Key words: ferritin, cDNA sequence, Aedes aegypti, mosquito, cloning, expression
Acknowledgments: The authors are grateful to Dr. Henry Hagedorn, Department of Entomology
and the Center for Insect Science, University of Arizona, for the helpful discussions in the course
of this work and to Drs. John Andersen and Ren6 Feyereisen, Department of Entomology and
the Center for Insect Science, University of Arizona, for kindly providing the RACE kit. This
work was supported by grants from the John D. and Catherine T. MacArthur Foundation, 8900408,
and the U.S. Public Health Service, A132595 and GM29328.
Received October 26, 1994; accepted February 15, 1995.
Address reprint requests to John H. Law, Ph.D., Department of Biochemistry, University of Arizona, Tucson, AZ 85721.
The sequence reported in this paper has been deposited in the GenBank data base (accession
no. L37082).
0 1995 Wiley-Liss, Inc.
294
Dunkov et al.
INTRODUCTION
Iron is an essential element for all living organisms. However, ionic iron in
the presence of oxygen can form free radicals that are destructive to biological materials. Consequently, ferric ions in biological systems are chelated with
small molecules or sequestered by specific proteins.
The principal biological iron storage protein is ferritin. This protein consists of 24 small subunits (-20 kDa) arranged in a cage-like sphere in which
ferric iron is stored as a crystalline mineral (Crichton and Ward, 1992;Meldrum
et al., 1992). Although vertebrate ferritin is a cytoplasmic protein, a glycosylated form occurs at low levels in blood (Campbell et al., 1989; Theil, 1990a).
Iron metabolism has been extensively studied in vertebrates, and ferritins
have been isolated from a wide variety of organisms, including bacteria and
plants (Andrews et al., 1991, 1992). In contrast, iron metabolism in insects
has been a relatively neglected field. To date, ferritins from two insect species (Heubers et al., 1988; Nichol and Locke, 1989; Winzerling et al., 1995)
have been reported. Locke and Nichol (1992) noted several important differences between vertebrate and insect ferritins. Insect ferritins have more mass
(660 kDa) than vertebrate ferritins (440 kDa), exist primarily in the vacuolar
system rather than in cytoplasm, are abundant in insect blood (hemolymph),
and are usually glycosylated with mannose-rich chains.
Hemaphagous insects transmit several vertebrate diseases; thus the study
of iron metabolism in these organisms could have value in designing important control strategies. Further, insects that feed on vertebrate blood receive a
diet rich in iron, and therefore might have a distinctive iron metabolism. For
these reasons, we are investigating iron metabolism of the yellow fever mosquito, Aedes aegypti. We report here the isolation and properties of a ferritin
from this insect and we have successfully cloned a cDNA that encodes one
of the ferritin subunits.
MATERIALS AND METHODS
Insect Culture and Iron Loading
A. aegypti of the Rockefeller strain were raised at 27"C, 80% humidity with
a 12h:12h 1ight:dark photoperiod according to Shapiro and Hagedorn (1982).
Larvae were iron loaded by adding 0.05 M FeC13 to the culture water for a
final concentration of 0.005 M FeC& (Nichol and Locke, 1990). Late fourth
instar larvae or pupae were collected, rinsed, and immediately frozen at -80°C.
Isolation of A. aegypti Ferritin
A. aegypfi larvae or pupae were ground in 50 mM HEPES, pH 7.4, containing 5 mM phenylthiourea and 1 mM PMSF* (5 ml buffer/g insects) using a
*Abbreviations used: ELISA = enzyme-linked immunosorbent assay; FlTC = fluorescein isothiocyanate; IRE = iron-responsive element; IRP = iron-regulatory protein; NFDM = non-fat dry
milk; PBS = 0.1 M sodium hosphate, 0.15 M NaCI, pH 7.0; PCR = polymerase chain reaction;
PMSF = phenylmethanesul onylfluoride; RACE = rapid amplification of cDNA ends; RT = room
temperature; SDS = sodium dodecyl sulfate; TBS = 0.02 M Tris, 0.1 M NaCI, pH 7.5; UTR =
untranslated region.
P
Mosquito Ferritin: Characterization and Cloning
295
Polytron homogenizer (PCU-2, Kinematica, Switzerland), 3 x 30 s at maximum speed. The homogenate was sonicated on a Branson Sonifier 200
(Branson Sonic Power Co., Danbury, CT) for 2 x 45 s, at 50%maximum power
to disrupt the membranes of the vacuolar system (Nichol and Locke, 1989)
and centrifuged for 15 min at 15,OOOg. The supernatant was filtered through
4 layers of filter paper (Whatman No. 1) and centrifuged at 180,OOOg for 1 h
at 15°C in a Beckman (Fullerton, CA) Ti 60 rotor. The resulting pellet was
homogenized in 1 ml buffer per g larvae or pupae and the homogenate was
heated at 75°C for 15 min (Nichol and Locke, 1989), cooled on ice, and the
heat-denatured proteins were removed by centrifugation (15 min at 15,OOOg).
The supernatant was layered over a KBr solution saturated at 15°C and centrifuged in a Beckman Ti 60 rotor at 180,OOOg for 22 h at 15°C (Winzerling et
al., 1995).Under these conditions some KBr crystals formed on the bottom of
the tube. The brown pellet that contained ferritin was suspended in a small
volume of 50 mM HEPES buffer, pH 7.4, layered on the saturated KBr solution and centrifuged as above. The final brown pellet was resuspended in 50
mm HEPES, pH 7.4, and the KBr was removed by dialysis.
Protein Gel Electrophoresis
Native PAGE was performed in 6.0% homogeneous slab gels using the
buffer system described by Davis (1964).Electrophoresis of ferritin under nonreducing conditions in SDS 5.0% homogeneous slab gels was done by solubilizing the protein in 80mh4 Tris, pH 6.8,1% SDS, 10%glycerol without boiling.
SDS-PAGE (reducing conditions) was performed according to Laemmli (1970)
using 12.5% homogeneous slab gels. Gels were stained with Coomassie blue
R-250. Native holoferritin also was visualized by Ferene S stain according to
Chung (1985). Relative molecular mass was determined from low molecular
mass standards (Bio-Rad, Richmond, CA), horse spleen ferritin (Sigma, St.
Louis, MO), and Munduca sextu ferritin (prepared as described by Winzerling
et al., 1995).
Western Blot
Fourth instar larvae, pupae, or adults were selected from mass cultures of
insects raised on either control diet or diet containing 0.005 M FeC13.Groups
of five insects were frozen immediately in liquid nitrogen and stored at -80°C.
These samples were homogenized in 25 pl of PBS containing a cocktail of
aqueous protein inhibitors (1 pl) (0.15 mg/ml aprotinin, 0.15 mg/ml
benzamidine, 0.2 mg/ml leupeptin) and 1 pl of protein inhibitors dissolved
in ethanol (100 mM PMSF, 0.2 mg/ml pepstatin A, 10 mg/ml TPCK). The
samples were diluted 1:l in SDS-PAGE sample buffer (2x) (Laemmli, 1970)
and centrifuged for 15 min at 12,OOOg. The supernatant was brought to 70 yl
with SDS-PAGE sample buffer (1x), boiled 5 min, and centrifuged at 12,0009
for 30 s. The supernatant (14 PI, the amount approximately equal to one
insect) and control purified ferritin (22 pg) were loaded on a 12.5%homogeneous slab gel and electrophoresis was conducted at 9 mA overnight (RT).
The polypeptides were transferred to a nitrocellulose membrane by semi-dry
blot (Idea Scientific, Minneapolis, MN). Blots were blocked with 1%NFDM
296
Dunkov et al.
in TBS for 30 min, allowed to react for 1 h with rabbit anti-A. negypti ferritin
serum (1:2,000) diluted in 1%NFDM-TBS, washed four times with TBS that
contained 0.05% Tween 20, and ferritin was visualized with alkaline phosphatase stain using goat anti-rabbit IgG alkaline phosphatase conjugate (BioRad, Inc.). Concanavalin A-binding glycoproteins were detected after
nitrocellulose blotting by probing with FITC-labeled concanavalin A (Sigma).
Production of Antibodies
Ferritin (200 pg) purified from A. aegypti pupae as described above was
subjected to SDS-PAGE on a 12.5% homogeneous slab gel, stained with Coomassie blue, and the bands corresponding to the three ferritin subunits were
cut from the gel. All bands were homogenized together in a glass-Teflon homogenizer, emulsified in 0.5 ml RIBI Adjuvant (RIBI Immunochem Research,
Inc., Hamilton, MT), and injected subcutaneously into a rabbit. Two additional injections were given, one at 2 weeks and one at 13 weeks. The antibody titers were determined by ELISA (Engvall and Perlmann, 1972) at 10
days after the second injection and followed thereafter. The final bleeding
was done at 16 weeks. Antibody specificity was determined by immunoblotting (Burnette, 1981) using the Vectastain ABC peroxidase immunodetection
kit (Vector Laboratories, Burlingame, CA). The antiserum reacted with 50 ng
of ferritin in 1:20,000 dilution.
Protein Sequence Determination
Mosquito ferritin (40 pg) was loaded on an SDS 12.5%homogeneous minigel and electrophoresis was conducted at 15 mA, RT, until the dye reached
the bottom of the gel. The polypeptides were transferred to a nitrocellulose
membrane (Applied Biosystems, Foster City, CA) by semi-dry blot. The blot
was stained for 1 min with Ponceau red or Coomassie blue R-250, destained
10 min with 1.0% acetic acid, and air-dried. The protein bands were cut from
the membrane and sent for N-terminal sequencing; the 24 and 26 kDa bands
were sequenced by Harvard Microchemistry Facility (Cambridge, MA), and
the 28 kDa band was sequenced by the Macromolecular Facility, Arizona Research Laboratories-Biotechnology Program, University of Arizona, Tucson, AZ.
Screening and Isolation of the Recombinant Phage and Subcloning
of the Inserts
Rabbit anti-A. aegypti ferritin serum was used to screen a female A.
aegypti (Bahama strain) cDNA hgtll library kindly provided by Dr. A. A.
James (University of California, Irvine, CA). Positive plaques were detected with goat anti-rabbit IgG conjugated to alkaline phosphatase (BioRad). Approximately 140,000 plaques were screened and eleven putative
positive clones were rescreened to homogeneity. The inserts from nine positive clones were amplified by PCR using single plaque extracts as a DNA
template with primers flanking the EcoRI site in hgtll vector. The PCR
products were cloned into pCR I1 vector using the “TA cloning” kit
(Invitrogen, Inc., San Diego, CA). Plasmid DNA was purified using the
Magic Minipreps kit (Promega Corp., Madison, WI). DNA was sequenced
Mosquito Ferritin: Characterization and Cloning
297
using Sequenase (version 2.0) according to manufacturer’s instructions
(United States Biochemicals, Cleveland, OH).
Rapid Amplification of cDNA Ends (RACE)
The 5VTR and part of the coding region of the ferritin cDNA were obtained by a modified RACE procedure (Edwards et al., 19911, using the 5’AmpliFINDER RACE kit (Clontech, Palo Alto, CA).An antisense primer with
the sequence, 5’-TGGTGACGGTTGGCACCT-3’ was designed from cDNA
sequence (see Fig. 6, nt 568-585). Using this primer, we synthesized cDNA
from A. aegypti adult female mRNA. The RNA was hydrolyzed with 6 N
NaOH and a single-stranded oligonucleotide anchor was ligated to the 5’
end of the single strand cDNA by T4 RNA ligase. A second pair of primers,
one complementary to the anchor and the other with the sequence 5’TGGTCGTAGTCCAGCTTGAAGTGCTTG-3’ (see Fig. 6, nt 539-5651, were
used for PCR. The PCR products were subcloned and sequenced as described
above. Messenger RNA was purified from adult female mosquitoes using
the Micro-Fast Track system (Invitrogen, Inc.).
DNA and Protein Sequence Analysis
Analysis of DNA and protein sequences was performed using the DNA
and protein sequence analysis programs from the GCG Package (Genetics
Computer Group, 1991).
RESULTS
Purification of Ferritin
Two properties of the ferritins, the high density of the iron-loaded
holoprotein and its thermostability, were employed to purify ferritin from
A . aegypti larvae and pupae. Although the high pH of the growth medium caused the formation of insoluble ferric hydroxides, the larvae
showed a significant increase in ferritin yield when animals were raised
on the culture containing FeC13. This is probably because the iron salts
were precipitated on food particles ingested by the larvae. Ferritin resistance to thermal denaturation at 75°C is well documented (Nichol and
Locke, 1989). Heat treatment precipitated other proteins from the
homogenate and centrifugation produced a clear extract that contained
ferritin. Following the first ultracentrifugation through a KBr solution,
we obtained a sticky brown pellet containing primarily ferritin. An additional ultracentrifugation resulted in homogeneous ferritin. Native gel
electrophoresis of ferritin purified from control larvae showed a single
protein band while the preparation from iron-loaded larvae showed two
bands of apparent high molecular weight (Fig. la). Staining with Ferene
S (not shown) indicated that the protein contained iron. A . aegypti ferritin did not dissociate into subunits when subjected to non-reducing SDSPAGE (not shown). However, SDS-PAGE under reducing conditions
revealed that this ferritin consists of 3 major polypeptides of about 28,26,
and 24 kDa (Fig. lb) and a minor amount of a polypeptide of about 30
kDa. The 28 kDa and the minor 30 kDa subunits bound FITC labeled con-
Dunkov et al.
298
a
b
kDa
k Da
669
94
440
67
330
43
140
30
a
b
d
c
e
f
c
20
kDa
14
28,
26 24/
a
b
c
d
e
r
g
a
b
c
d
e
f
Fig. 1. a: Native PAGE of A. aegypti larval ferritin. Mosquitoes were fed a control diet or a diet
that contained 5 mM FeCI3. Ferritin was isolated, loaded onto a 6% homogeneous native slab
gel, and electrophoresis conducted as described in Materials and Methods. The gel was stained
with Coomassie blue R-250. Lane a: high molecular mass standards; lane b: horse spleen ferritin;
lane c: M. sexta ferritin; lanes d and e: A. aegypti ferritin from control larvae, and lane f: A.
aegypti ferritin from iron-fed larvae. b: SDS-PAGE of A. aegypti larval ferritin. Mosquitoes were
fed a diet that contained 5 mM FeC13. Ferritin was isolated, prepared and run on an SDS-PAGE
12.5% homogeneous slab gel a5 described in Materials and Methods. The gel was stained with
Coomassie blue R-250. lane a: low molecular mass standards; lanes b,c, and d: A. aegypti
ferritin 33, 16, 9 pg, respectively; lane e: adult M. sexta ferritin; lane f: horse spleen ferritin. c:
Western blot of the differential expression of ferritin subunits in various A. aegypti life stages.
Mosquitoes were raised on a control diet or on a diet containing 5 mM FeCI3 and protein extracts prepared as described in Materials and Methods. Samples were loaded onto a 12.5%
homogeneous slab gel and electrophoresis was conducted at 9 mA, overnight, at RT. The polypeptides were transferred to a nitrocellulose membrane by semi-dry blot. The blot was blocked,
washed, and visualized with alkaline phosphatase stain as described in Materials and Methods.
Lane a: purified pupal ferritin; lane b: control larvae; lane c: iron-fed larvae; lane d: control
pupae; lane e: pupae raised on iron-supplemented diet; lane f: control female adult; lane g:
female adult mosquitoes raised on iron-supplement diet.
canavalin A indicating that they were glycosylated with a mannose-rich
oligosaccharide (data not shown).
Presence of Ferritin in Adult Mosquitoes
Western blotting showed that the rabbit antiserum reacted specificaIly with
all three major subunits, if present, in the extracts from larval, pupal, and
Mosquito Ferritin: Characterization and Cloning
299
adult animals (Fig. lc). Western blotting also confirmed the presence of ferritin in extracts from adult mosquitoes and showed that adult ferritin had
the same subunit composition as the larval and pupal proteins (Fig. lc). Only
two of the three subunits were seen in the control larvae and control adult
extracts, while iron feeding stimulated synthesis of all three subunits in both
of these stages. The 26 kDa protein was the major band in all samples. Rabbit anti-A. aegypti ferritin antiserum did not cross-react with horse spleen or
M . sexta ferritins (data not shown).
N-Terminal Sequence of the A. aegypti Ferritin Subunits
The sequences of the first 20-11 amino acids of the A. aegypti ferritin subunits are shown in Figure 2. The first 30 amino acids from the N-terminal
sequences of the 26 and 24 kDa subunits are identical. Comparison of these
mosquito ferritin sequences with those of other organisms showed no similarities.
Cloning of A. aegypti Ferritin cDNA
Figure 3 shows the strategies used to sequence the clones obtained from
the cDNA library and via RACE. Partial sequences of all nine cDNA clones
differed only in a 27 bp insertion immediately preceding the poly A tail. RACE
revealed that all cDNA clones were truncated at an EcoRI site at the 5’ end;
the truncation probably occurred during the construction of the cDNA library. One of the clones (F) was completely sequenced using nested sequencing primers. This cDNA sequence and the deduced amino acid sequence are
shown in Figure 4. The RACE procedure also yielded two groups of clones,
one group that contained the remaining coding sequence and 203 bp of the
5’ untranslated region (nt 1-203, Fig. 4). The N-terminal sequence obtained
from the 26 and 24 kDa subunits matched the deduced amino acid sequence
(Fig. 4, underlined sequence). Further comparison of the deduced amino acid
sequence with the N-terminal sequence also revealed the presence of a signal
peptide. Analysis of the 5’UTR of the A. aegypti ferritin cDNA revealed the
presence of a stem loop structure (bases 81-115, Fig. 4). This stem loop structure appears to be a putative iron-responsive element (IRE), since its sequence
matches the consensus sequence derived from vertebrate IRES (Fig. 5). Multiple sequence alignment with eleven other ferritin sequences (Fig. 6) showed
regions of similarity and conservation of the putative ferrooxidase center residues. Pairwise comparison to other ferritin sequences showed that the A .
28 kDa
D
1
N N CS TV N FTA Q F S S I A H I G
5
10
15
20
26 kDa
E QT V G A T D N Y Q W D S V D D Q[C] L A A L H R Q I N K E F D A - I 1 Y L (K) (Y) (A)
1
5
10
15
20
25
30
35
40
24 kDa
E Q T V C A T D N Y Q W D S V D D Q[C] L A A L H R QI N K E
1
5
10
15
20
25
30
Fig. 2 . N-terminal sequences of A. aegypti ferritin subunits.
300
Dunkov et al.
1
0
I
200
I
I
400
600
I
800
1
I
I
1000
1200
clone 4
clone 3
clone F
ATG
t
Ec9R I
--203
POlYA
TAA
I
627
v/l
k
426
20
7
f------------
____)
Fig. 3. Sequencing strategy of the ferritin cDNA clones. The scale denotes nucleotides in base
pairs. Clone F represents a cDNA clone obtained by screening an expression library (see Materials and Methods), while clones 3 and 4 were obtained by the RACE technique (see Materials and
Methods). The center diagram shows the coding region (627 bpi, part of the 5’ UTR (203 bp),
and the 3‘UTR (427 bp). Both strands of the cDNA were sequenced throughout the coding region
and 3’ untranslated region. The terminal region of the 5’ untranslated region was sequenced
unidirectionally in two independent RACE clones. The horizontal arrows indicate the length and
direction of the sequenced fragments.
aegypti sequence has greatest similarity to the Limnaea stagnnlis soma ferritin
subunit (Fig. 7).
DISCUSSION
While ferritins have been isolated and characterized from a variety of organisms ranging from plants and bacteria to mammals, relatively little is
known about ferritins from invertebrate animals. Sequences are available only
for a snail (von Darl and Bottke, 1992), a schistosome (Dietzel et al., 1992),
and a crustacean (ferritin-like protein, artemin) (De Graaf et al., 1990). Up to
this point, ferritins have been isolated from only two insects, both lepidopterans (Heubers et al., 1988; Nichol and Locke, 1989; Winzerling et al., 1995),
but their sequences have not been determined. Insect ferritins have some interesting differences from most other ferritins. They are larger than those reported from other animals in terms of the apparent masses of the subunits,
although the size of the ferritin sphere appears to be about the same as ferritins of other organisms (Locke and Nichol, 1992). Furthermore, in many
insects, ferritins are glycosylated and they appear in the endoplasmic reticulum of the cells (rather than in the cytoplasm) and in relatively large quantities in hemolymph (Locke and Nichol, 1992). This may indicate some
fundamentally different functions for the insect ferritins. One of the snail ferritins, that which occurs in the oocyte, is also a secreted protein that can be
found in the hemolymph (Bottke, 1982).
Mosquito Ferritin: Characterization and Cloning
1
301
60
GGACTAGTACCGGACACCGATAGAGGTGGAAUTACGACATAGAGGTGGAAGCAATACGA
61
120
T l ' A T K C A T C G C A G C C c T K l G T W ~ A T ~ G T
121
180
TpGRARAcTAGTGAAGcrc;cTAcGAAAATcTAGITcn;T
181
240
T I C G G A A A C C ~ T G G T A G C C A T C A
M M K S V F F G V V A I T
241
300
C C G P A C C C A ' P C A ~ A C t A G G A G A C G G C C C A G G
V
A
I
L
S
I
Y
Q
E
T
A
Q
A
Q
E
Q
T
V
G
A
301
360
C A A C C G A T A A ' I T A C C G A % A T C A G % C ~ C ! G C C
T
D
N
Y
Q
W
D
S
V
D
D
Q
C
L
A
A
L
H
R
O
361
420
AGA~'ITCGAlGCGXGA'KATCTACCTGUGTASCCGCCTA~
I
N
K
E
F
D
A
S
I
I
Y
L
K
Y
A
A
Y
F
A
Q
421
480
481
540
541
600
601
660
661
720
721
780
781
840
841
900
901
960
961
1020
1021
1080
1081
1140
1141
1201
1200
1260
1261
Fig. 4. Nucleotide sequence of the cDNA for A. aegypti ferritin and the deduced amino acid
sequence for the protein. The underlined protein sequence matches the N-terminal sequence
obtained from the purified 24 and 26 kDa ferritin subunits. The underlined 27 bp nucleotide
sequence near the 3'-end of the cDNA was not present in all clones. The putative polyadenylation signal is underlined.
We have undertaken a study of iron metabolism in mosquitoes because of
the differences in the sources of dietary iron between the male and female
adult animals, and between the larvae and adult animals. Larvae are aquatic,
and their diet can be expected to contain mainly inorganic iron. Adult males
Dunkov et al.
302
G U
A
G
G
c
U
G
A
u
C
N
N
N
N
N
C-G
G-U
U - A
G-U
U - A
C
-
N
N
N
N
N
N
C
N-N
N-N
N-N
N-N
N-N
U - A
U - A
C-G
C-G
A - U
c-c
5'
U
3'
U - A
G-C
5'
3'
A. aegypti
Consensus
IRE
Fig. 5. Sequence and structure of the putative IRE of A. aegypti ferritin mRNA and consensus
IRE (according to Harford and Klausner, 1990).
1
%ail-S
RaM-L
m - L
%his-1
%his-2
Soy
SMil-Y
Aaeqypci
Artemin
*
100
50
......................................
. . . . . . . . WVRCWFWROCE MIblRWWC3
......................................
. . . . . . . .W V W W I = C E M ......................................
. . . ..TpIsTFQ vRouuMIcGB MImQMLE
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .....nsvsQm@mmEsE Y i l t m ~ I m ~
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M E Q VRpmqOcB
...........................................................................
IS- II(QIysDvE MVNSLVNLY
..........................................................................
KSL
.uma.Q. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . KSSSR -PALRIIOIQ-SUVSl'FSGFSPKPN
S VSLSFGauyLRlffAs "PLTGVIFK P F r m K K S U IIVPl%WSL W A C E C E SAIWQTHVE
....................
........................... bSWJ LELTULYCSS UY-T
VrcOmwlN Q W I ....................
. . . . . . . . . . . . . F-.
GWAITVATL SIYQECAQAQ m T L N f PmmraqCL
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . AT DXFlNICQSAP-P
SRIDWPECE -1200
RaM-H
RaM-n
h - H
Snail4
.........................
KPDR
.......................... KPm
.......................... KPAE
.......................... Mm?
. . . . . . . . . . . . . . . . . . . . . . . . . GPIX
.......................... NnPS
LBB FRauIpcICo NATKTIW
..................... Nt
............................. P
Fig. 6. Alignment of known ferritin subunit amino acid sequences. Abbreviations and sources:
Rana = Kana catesbeiana heavy (H), medium (M),
or light (L) ferritin chain (Dickey et al., 1987;
Didsbury et al., 1986); Hum = human light (L) or heavy (H) ferritin chain (Boyd et al., 1985;
Dorner et al., 1985); Snail = Linmaea stagnalis soma (S) and yolk (Y) ferritin (von Darl and
Bottke, unpublished data); Schis = Schistosorna mansoni ferritin chains 1 and 2 (Dietzel et al.,
1992); Soy = soyabean (Lescure et al., 1991; Ragland et al., 1990); Aaegypti = A. aegypti ferritin
(this paper); and Artemin = artemin from Arfemia salina cysts (De Craaf et al., 1990). The Cterminal portion of Artemin is not shown. * = conserved putative ferrooxidase residues.
Mosquito Ferritin: Characterization and Cloning
303
Fig. 7. Pairwise identities of amino acid sequences aligned in Figure 6. Identities were calculated using the program DISTANCES (GCG Package). For each pairwise comparison the sum of
identities were divided by the length of the shorter sequence without gaps.
feed mainly on nectar, which is probably iron deficient. In contrast, the adult
female feeds on vertebrate blood, a diet rich in iron-containing hemoglobin.
In order to isolate ferritin from larvae and pupae, we homogenized whole
animals that had been fed an iron-rich diet. Sonication was used to release
cellular ferritin. While these procedures of protein isolation can be risky because both digestive and cellular proteases are released, the known resistance
of many ferritins to proteolysis provided optimism that intact ferritin could
be isolated by this method. The isolation procedure presented here varied
slightly from another we recently published (Winzerling et al., 1995). This
technique takes advantage of the fact that iron-loaded ferritin has greater
density than most cellular material, thus it pellets with ultracentrifugal force
in a KBr solution, while other proteins float. The resulting product is essentially homogeneous.
A. aegypti native ferritin behaves in polyacrylamide gels as a very large
molecule, although its exact molecular mass has not been determined. On
dissociation under reducing conditions in the presence of SDS, ferritin from
iron-fed larvae dissociates into four subunits. Those of masses 24,26, and 28
kDa are almost equally abundant, while that of mass 30 kDa is a minor component. The 28 and 30 kDa subunits are glycosylated, while the 24 and 26
kDa polypeptides apparently are not. The second protein band observed on
the native PAGE of preparation from iron-fed larvae probably represents ferritin with different subunit composition, produced under the conditions of
iron excess.
Both the snail oocyte ferritin and mosquito ferritins are known to be secreted from cells, and both are synthesized with leader sequences. According to von Heijne (1983, 1987), the predicted site of cleavage of the leader
peptide for the mosquito subunit would be after the AQA sequence at residues 24-26, while the sequence for the mature subunit begins at residue 28
304
Dunkov et al.
(Fig. 4). If this prediction is correct, there must be a subsequent processing
step to remove the intervening glutamine residue.
The amino acid sequence deduced from the cDNA sequence (209 residues)
corresponds to the molecular weight of 23,758. If the leader peptide and the
intervening glutamine residue (residues 1-27) are removed, then the mature
subunit sequence (182 residues) corresponds to a molecular weight of 20,566;
this is smaller than that estimated by SDS-PAGE (24,000). Since the 24 and 26
kDa subunits share the same N-terminal sequence, it is likely that they are
products of the same gene. The difference between the molecular weight calculated from the deduced sequence and that predicted from the SDS-PAGE
suggests that both subunits may be posttranslationally modified. At present,
we have no idea what such a modification might be. However, glycosylation
is unlikely, both because neither subunit binds concanavalin A and because
the deduced sequence has no predicted glycosylation sites. The 28 kDa subunit is clearly the product of a different gene. We did not have sufficient 30
kDa subunit to determine its N-terminal sequence.
Vertebrate ferritins can consist of any combination of three subunits; two
very similar subunits designated H or H’ (heavy) and a related, but less similar
L (light) subunit. Of the invertebrate ferritins sequenced to date, two have
been shown to have two different subunits. The schistosome has sex specificity of subunits, with one predominating in males, and the other in females
(Dietzel et al., 1992).The snail has tissue specific ferritins, one is found in the
somatic cells and the other is vitellogenic, secreted by the midgut gland and
sequestered in the ovary (Bottke, 1982). Whether similar specificity exists in
insects is not yet clear. The subunits of the snail somatic ferritin (von Darl and
Bottke, 1992)and the schistosome ferritins (Dietzel et al., 1992)show greater similarity to the H chains of vertebrates than to vertebrate L chains. The mosquito
ferritin has greater similarity to the human H chain than to the human L chain,
but the similaritiesto the three chains of the frog are not very different.
Vertebrate H chain subunits have a catalytic site (ferroxidase site) that is
responsible for the oxidation of cytoplasmic ferrous ion to ferric ion (Balla et
al., 1992; Wade et al., 1991).Thus cytoplasmic iron is converted to the proper
form for mineralized storage as it enters the ferritin sphere. With respect to a
ferroxidase site, the mosquito ferritin has retained all of the putative binding site residues, marked by asterisks in Figure 6.
Waldo et al. (1993) have recently suggested that an iron-tyrosinate complex formed only by vertebrate H chains (not L chains) is involved in rapid
biomineralization of iron. The specific tyrosine (or tyrosines) responsible for
formation of this complex has not been determined, but these authors indicated that the residue 102 (shown boxed in Fig. 6) might be responsible for
complex formation. This residue is present in all vertebrate heavy chains, as
well as in the snail somatic ferritin and in the Scm 1 (female predominant)
ferritin of the schistosome. However, it is lacking in the snail vitellogenic
ferritin, the male predominant schistosome ferritin, and the mosquito ferritin. If this residue is essential, possibly another of the mosquito subunits is
responsible for rapid biomineralization in mosquitoes.
The fact that insect ferritins are secreted from cells, and also presumably
taken up as well, suggests that they function differently from the cytoplas-
Mosquito Ferritin: Characterization and Cloning
305
mic ferritins of other species. Locke and Nichol (1992) have speculated that
insect ferritins might function in the excretion of iron (and possibly other
cations) by exocytosis from midgut cells into the midgut lumen. They also
postulate that lumenal ferritin could function in transport of iron through midgut cells, as well as from hemolymph into cells. The availability of molecular
tools should facilitatethe experimental testing of these interesting hypotheses.
As demonstrated by Western blotting, the amount of ferritin polypeptides
is increased by administration of iron in the diet (Fig. lc). In vertebrates, synthesis of ferritin in response to iron is primarily controlled at translation. The
ferritin message contains an IRE in the 5'-untranslated region, where an ironregulatory protein (IRP, formerly called IRE-BP) is bound under conditions
of iron depletion (Eisenstein and Munro, 1990; Theil, 1990b).IRP blocks translation of the ferritin message, so that ferritin is not synthesized. When iron is
present, an iron-sulfur cluster in IRP is assembled, and IRP loses affinity for
the IRE and gains aconitase enzymatic activity (Haile et al., 1992). Cytoplasmic aconitase thus functions as an iron sensor for the cell. Several other proteins of iron metabolism are controlled in a similar or reciprocal fashion
(OHalloran, 1993). The A. aegypti ferritin cDNA contains a putative IRE 88
bases upstream of the initiation codon with sequence homology to the IRES
of the vertebrates (Fig. 5). This strongly suggests that the induction of ferritin
synthesis by iron in mosquitoes is also regulated at the translational level.
The molecular tools developed in this investigation will now make possible an examination of the response of the adult female mosquito to the iron
resources of a blood meal. They will also permit us to investigate the control
of iron absorption by the midgut and how iron is provided to the oocyte. It is
likely that they will also facilitate the study of iron metabolism in other insects and enrich our understanding of the comparative biochemistry of iron.
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