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Hyphantria cunea ferritin heavy chain homologuecDNA sequence and mRNA expression.

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Archives of Insect Biochemistry and Physiology 56:21–33 (2004)
Hyphantria cunea Ferritin Heavy Chain Homologue:
cDNA Sequence and mRNA Expression
Hong Ja Kim,1 Chi Young Yun,2 Hyang Mi Cheon,1 Boa Chae,1 In Hee Lee,3
Seun Ja Park,1 Young Jin Kang,1 and Sook Jae Seo1*
We have sequenced a cDNA clone encoding a 26-kDa ferritin subunit, which was heavy chain homologue (HCH), in fall
webworm, Hyphantria cunea. The HCH cDNA was obtained from the screening of a cDNA library using a PCR product. H.
cunea ferritin is composed of 221 amino acid residues and their calculated mass is 26,160 Da. The protein contains the
conserved motifs for the ferroxidase center typical for heavy chains of vertebrate ferritin. The iron-responsive element sequence
with a predicted stem-loop structure is present in the 5¢-untranslated region of ferritin HCH mRNA. The sequence alignment of
ferritin HCH shows 68.9 and 68.7% identity with Galleria mellonella HCH (26 kDa ferritin) and Manduca sexta HCH, respectively. While G type insect ferritin vertebrate light chain homologue (LCH) is distantly related to H. cunea ferritin HCH (17.2–
20.8%), the Northern blot analysis revealed that H. cunea ferritin HCH was ubiquitously expressed in various tissues and all
developmental stages. The ferritin expression of midgut is more responsive to iron-fed, compared to fat body in H. cunea.
Arch. Insect Biochem. Physiol. 56:21–33, 2004. © 2004 Wiley-Liss, Inc.
KEYWORDS: Hyphantria cunea; ferritin; cDNA; cloning; expression
INTRODUCTION
Iron is an essential element for all living organisms and ferritin is an important iron storage
protein. Ferritin is a large hollow sphere with a
molecular mass of 440 kDa in vertebrates (Harrison and Arosio, 1996). In vertebrates, ferritin
polymers have 24 subunits of two types, heavy (H)
and light (L), which are encoded by separate genes
(Munro, 1993). The H subunit contains a ferroxidase center involved in rapid iron uptake and oxidation (Lawson et al., 1989). The L subunit is
responsible for the formation of the iron core (Levi
et al., 1992). In most animals, ferritin spheres are
found in the cytosol, where they serve to store ferric ions.
On the other hand, most insects store iron in
ferritin found in the vacuolar compartment and
can secrete it through the Golgi complex and secretory vesicles (Nichol et al., 2002). Insect ferritins
are larger (>600 kDa) than vertebrate ferritins, and
have larger subunits (24–32 kDa) (Huebers et al.,
1988; Nichol and Locke, 1989; Dunkov et al.,
1995; Winzerling et al., 1995; Capurro et al.,
1996). Insect ferritins were cloned and sequenced
from several species (Dunkov et al., 1995; Pham
1
Division of Life Science, Gyeongsang National University, Jinju, Korea
2
Department of Biology, Daejeon University, Daejeon, Korea 3Department of Life Science, Hoseo University, Asan, Korea
Kim and Yun contributed equally to this work.
Contract grant sponsor: Korea Science Engineering Foundation (ABRL Program) and Brain Korea 21 Project; Contract grant number: R14-2002-056-01001-0.
Abbreviations used: A. aegypti = Aedes aegypti; A. germari = Apriona germari; C. ethlius = Calpodes ethlius; D. melanogaster = Drosophila melanogaster;
G. mellonella = Galleria mellonella; H. cunea = Hyphantria cunea; M. sexta = Manduca sexta; PAGE = polyacrylamide gel electrophoresis; PCR =
polymerase chain reaction.
*Correspondence to: Sook Jae Seo, Division of Life Science, College of Natural Sciences, Gyeongsang National University, Jinju, 660-701, Korea.
E-mail : sookjae@gshp.gsnu.ac.kr
Received 18 April 2003; Accepted 24 December 2003
© 2004 Wiley-Liss, Inc.
DOI: 10.1002/arch.10141
Published online in Wiley InterScience (www.interscience.wiley.com)
22
Kim et al.
et al., 1996; Charlesworth et al., 1997; Nichol and
Locke, 1999; Du et al., 2000; Kim et al., 2001b,
2002). Insect ferritin subunits resemble those of
vertebrates and fall into two classes, according to
the overall sequence similarity to vertebrate H or
L chains. Although the vertebrate H chain has a
greater molecular mass than the L chain, the
smaller subunits of ferritins in insects are the homologues of the vertebrate H chains with the conservation of seven metal-binding residues found
in the H chains (Charlesworth et al., 1997). The
larger ferritin subunits (G) are homologues of the
vertebrate L chains. In D. melanogaster, a pair of subunits with apparent 25 and 26 kDa are products of
the same gene, and are homologues of the vertebrate H chain, while the 28-kDa subunit is a product of a different gene and this subunit likely serves
the role of the vertebrate L chain (Dunkov and
Georgieva, 1999). Ferritin from the tobacco hornworm M. sexta is composed of four bands (Pham
et al., 1996). Three, including the G subunit, share
the same N terminus, whereas the nonglycosylated
S subunit has a different N terminus.
The HCH and LCH of insect ferritin have been
cloned and sequenced for Aedes (Dunkov et al.,
1995), Drosophila (Charlesworth et al., 1997;
Georgieva et al., 2002), Manduca (Pham et al.,
1996), Calpodes (Nichol and Locke, 1999), Galleria (Kim et al., 2001b, 2002), and two tick species
(Kopá…ek et al., 2003).
The fall webworm, Hyphantria cunea, is a polyphagus pest that eats about 160 species of broadleaf trees, and damages the roadside and garden
trees around urban areas rather than forest on
mountains in Korea (Lee and Chung, 1998). Thus,
it can be good as a model of iron metabolism or
pest control research for polyphagus insects, according to insect diversity. We have also acquired
ferritin gene for further study on iron metabolism
since it was purified and some properties of ferritin from H. cunea characterized (Kim et al., 1996;
Yun et al., 1998).
Here we report that we have cloned and sequenced cDNA encoding ferritin HCH from H.
cunea, and compared their deduced amino acid sequences with other insect ferritins. This subunit is
a homologue of vertebrate H chain. H. cunea ferritin HCH has seven conserved amino acids common to vertebrate H chain that are required for
metal binding. We showed the presence of conserved
cysteine residues in the N-terminus and putative iron
response element from ferritin HCH of H. cunea.
3¢-Untranslated region (UTR) of this subunit cDNA
has several non-canonical putative polyadenylation
signals, besides one typical polyadenylation signals.
We also report that ferritin HCH of H. cunea is
expressed during the developmental stages from
larvae to adult and ubiquitously in all tissues tested
with the exception of testis.
MATERIALS AND METHODS
Animals
Fall webworm, H. cunea, was reared on an artificial diet (Ito and Tanaka, 1960) at 27°C and 75%
relative humidity with a photoperiod of 16 L:8 D.
For iron-feeding, larvae were fed on an artificial diet
containing 50 mM ferric chloride (FeCl3) for 16 h.
Collection and Processing of Hemolymph and Tissues
Hemolymph was collected into cold test tubes
by cutting forelegs of 4-day-old last instar larvae.
A few crystals of phenylthiourea were added to the
tubes to prevent melanization. Hemolymph was
centrifuged at 10,000g for 10 min to remove
hemocytes and cell debris, and the supernatant was
stored at –70°C until used.
Purification of Ferritin From H. cunea Hemolymph
For the improvement of purification yield of ferritin, the procedure by Kim et al. (1996) was modified. The purification of ferritin from H. cunea
hemolymph was accomplished by heat treatment
at 75°C, KBr density gradient ultracentrifugation
(TST 41.14 rotor, Kontron Co.), and TSK-gel DEAE650M anion exchange column (2.15 ´ 20 cm)
chromatography using FPLC system (Tosho Co.).
After ultracentrifugation at 200,000g for 20 h, ferritin fraction, the brownish precipitate at the bottom of test tube, was removed. The precipitate was
Archives of Insect Biochemistry and Physiology
H. cunea Ferritin HCH
resuspended in 0.02 M Tris-HCl buffer (pH 8.3),
and concentrated by removing ammonium sulfate
using desalting column (Bio-Rad, Richmond, CA).
The preparation in turn was subjected to anion exchange chromatography on TSK-gel DEAE-650 M
column (Toyopearl Co.) equilibrated with 0.02 M
Tris-HCl buffer (pH 8.3), and eluted between 0.02–
0.3 M buffer gradient at a flow rate of 5 ml/min
and with a fraction volume of 5 ml.
Electrophoresis
Native-polyacrylamide gel electrophoresis (PAGE)
was conducted on 4–24.5% gradient polyacrylamide
gel as described by Davis (1964). SDS-PAGE was
carried out on a 7.5–15.0% gradient separating gel
at 15 mA, according to the procedure of Laemmli
(1970). The gels were stained in Coomassie Brilliant Blue R-250 after electrophoresis.
Determination of Molecular Masses of Proteins
Native molecular mass of ferritin was measured
on 4–24.5% gradient polyacrylamide gel using thyroglobulin (669 kDa), horse ferritin (440 kDa),
catalase (232 kDa), lactate dehydrogenase (140
kDa), and albumin (67 kDa) as standard molecular mass marker proteins (Pharmacia Biotech. Co.).
The molecular masses of ferritin subunits were
measured on 7.5–15.0% gradient SDS gel as described by Lambin et al. (1976). Standard molecular mass marker proteins used were phosphorylase
b (97.4 kDa), bovine serum albumin (66.2 kDa),
ovalbumin (45 kDa), carbonic anhydrase (31 kDa),
soybean trypsin inhibitor (21.5 kDa), and lysozyme
(14.4 kDa) from Bio-Rad.
Primer Synthesis, PCR, and Subcloning of
PCR Product
Degenerate primers between 17 and 20 nucleotides
in length were designed from the N-terminal and consensus sequence from HCH insect ferritins. The sense
primer (17 mer) was 5¢-TGYCAYATHAAYCCNGT-3¢
(24–29 amino acids) and the antisense primer was
5¢-TGNCCYTTRTAYTGYTCYT-3¢ (181–187 amino acids). cDNA was amplified from cDNA library using
May 2004
23
degenerate primers and Taq DNA polymerase. The
PCR cycles were as follows; 3 min at 94°C; 35 cycles
of 30 s at 94°C/1 min at 40°C/1 min at 72°C; 3
min at 72°C. The amplified PCR product was separated on a 1% agarose gel and a 0.5-kb fragment
was obtained. This fragment was excised from the
agarose gel, purified, ligated into a T-vector, and
amplified in XL1 Blue competent cells.
Screening of H. cunea Ferritin cDNA From
cDNA Library
The cDNA library from the fat body of last instar larvae in H. cunea was kindly provided by Dr.
Ho Yong Park (Korea Research Institute of Bioscience and Biotechnology, Daejeon). For screening, 50,000 plaques were plated on 15-cm-diameter
agar plates. Nitrocellulose filters (Schleicher &
Schuell, Dassel, Germany) were taken from the
plates and hybridized at high stringency (65°C and
0.1 ´ SSC) with the DIG-labeled PCR product according to the manufacturer’s recommendation
(Boehringer Mannheim Corp., Indianapolis, IN).
The positive clones were rescreened at low density
on 9-cm-diameter agar plates. Colony hybridization yielded 5 positive clones from the library. Positive plaques were individually isolated in phage
buffer by the plate lysate method followed by
lambda DNA extraction (Sambrook et al., 1989).
The insert of the positive clone was subcloned into
an EcoRI site of pBluescript SK(+).
DNA Sequencing
The manual nucleotide sequence analysis of
both DNA strands was performed by the dideoxy
chain termination method (Sanger et al., 1977).
Six percent acrylamide gels, 35S-radiolabeled nucleotides, and sequenase (US Biochemical, Cleveland,
OH) were used, according to the recommendation
of the manufacturer.
Analysis of Sequence Data
The EMBL DataBank was searched with BLAST.
Editing and analysis of the DNA sequence data
24
Kim et al.
were performed with DNASTAR software (DNASTAR Inc., WI).
Northern Blot
Tissues were dissected from 4- and 5-day-old
last instar larvae (fat body, epidermis, midgut,
Malpighian tubule, and testis) and from 8-dayold pupae (ovary). Total RNA (15 mg) from tissues
were denatured and subjected to electrophoresis
in a 1.2% agarose gel containing 2.2 M formaldehyde. Following electrophoresis, gels were
rinsed in 10 ´ SSC and transferred to nitrocellulose (Schleicher & Schuell) in 10 ´ SSC. Blots were
prehybridized with 1.5 ´ SSPE, 7% SDS, 10%
PEG, 0.1 mg/ml sonicated denatured salmon
sperm DNA, and 0.25 mg/ml BSA for 4 h at 65°C.
Hybridization was performed for 18 h at 65°C in
the prehybridization buffer with 5 ´ 105 cpm/ml
of 32P-labeled probe (whole ferritin cDNA), which
was prepared by the method of random priming
(Feinberg and Vogelstein, 1983). The filter was
then washed twice with 1 ´ SSC, 0.1% SDS at
65°C for 15 min, and subsequently two more
times for 15 min with 0.1 ´ SSC, 0.1% SDS at
65°C before exposure to X-ray film at –70°C.
rRNA was used as an internal loading control.
RESULTS AND DISCUSSION
Purification of Ferritin HCH in H. cunea
On a 4–24.5% gradient polyacrylamide gel, H.
cunea ferritin purified from last instar larval hemolymph appears as a single band of about 660 kDa,
much larger than horse spleen ferritin, 440 kDa
(Fig. 1A). When the purified ferritin was denatured
and separated by 7.5–15.0% SDS-PAGE, it appears
to be composed of three subunits: 26, 31, and 32
kDa (Fig. 1B); 26 and 32 kDa subunits are major,
but the 31-kDa subunit is minor. Molecular mass
of ferritin in vertebrates is reported to be approximately 440 kDa and consists of 24 subunits with
the molecular mass range of 18–24 kDa. However,
insect ferritins and their subunits are reported to
be larger than those of vertebrate in molecular
mass. C. ethlius ferritin has a molecular mass of
Fig. 1. Shown are 4–24.5% native-PAGE (A) and 7.5–
15% SDS-PAGE (B) of ferritin purified from H. cunea
hemolymph. A: Lane 1: Horse spleen ferritin; lane 2: ferritin purified from H. cunea hemolymph (about 660 kDa).
B: Lane 1: Standard molecular mass marker proteins (BioRad); lane 2: H. cunea ferritin subunits (26, 31, and 32 kDa).
more than 600 kDa and is composed of two major subunits (24 and 31 kDa) and two minor subunits (26 and 28 kDa) (Nichol and Locke, 1989).
G. mellonella ferritin was determined to have a molecular mass of 630 kDa, and consists of two major polypeptides of 26 and 32 kDa and one minor
polypeptide of 30 kDa (Kim et al., 2001a, 2002).
Previously the N-terminal sequence of H. cunea ferritin HCH was determined as XPQXHINPV (Yun
et al., 1998), which was in agreement with sequenced data.
Unfortunately, attempts to perform N-terminal
sequencing of the two other ferritin subunits (31
and 32 kDa) failed to produce the result due to
almost overlapping of two bands. In an N-terminal sequence of ferritin HCH, the second X was
conserved cysteine residue in the N-termini of other
insect ferritins. Therefore, the sense primer was produced from the amino acid sequence of CHINPV
for the cloning of cDNA encoding ferritin HCH.
Archives of Insect Biochemistry and Physiology
H. cunea Ferritin HCH
Cloning and Sequencing of Ferritin HCH cDNA
in H. cunea
Lepidopteran hemolymph ferritin is composed
of several subunits (Nichol and Locke, 1989; Kim
et al., 2001b). We have sequenced the cDNA encoding the smaller hemolymph ferritin subunit 26
kDa of H. cunea, which was HCH. The cDNA library from fall webworm was screened with a PCRgenerated DIG-labeled probe of 0.5 kb. Degenerate
primers for the PCR-generated cDNA probe were
from the N-terminal and consensus sequence of
insect ferritins, which are homologues of the vertebrate H chain. From a total of 50,000 screened
25
plaques, five positive plaques were isolated. Insert
lengths were checked after plasmid isolation by digestion with EcoRI and XhoI. One insert was about
1.3 kb, consistent with a full-length cDNA clone
for H. cunea ferritin HCH. The length of cDNA for
H. cunea ferritin HCH was 1,293 bp (Fig. 2).
Within the cDNA sequences was an open reading
frame of 663 bp encoding a protein of 221 amino
acids with an estimated molecular mass of 25,160
Da. The remaining sequences were untranslated regions. The deduced amino acid sequence encoded
by the cDNA matching the N-terminal sequence
of ferritin was preceded by a sequence encoding
20 amino acids showing secreted-type ferritin. Ma-
Fig. 2. Nucleotide sequences
and deduced amino acid sequences of a cDNA encoding
ferritin HCH from H. cunea.
The iron-responsive element
(IRE) is bold-underlined in
which the consensus loop is
overlined. The signal peptide
sequence is in italic type and
the N-terminal sequence of
matured ferritin is underlined.
An asterisk marks the translation stop codon. The polyadenylation signal is boxed
and the potential phosphorylation sites are circled.
May 2004
26
Kim et al.
ture protein without signal peptide was an estimated molecular mass of 23,100 Da. The apparent molecular weight of small ferritin subunit in
H. cunea based on SDS-PAGE was approximately
26 kDa. The difference in its molecular weight
could be accounted for by posttranslational modifications such as glycosylation and phosphorylation. A computer analysis of our deduced amino
acid sequence does not reveal potential glycosylation sites, but phosphorylation sites (Fig. 2).
We observed the presence of cysteine residues
conserved in the N-terminus of H. cunea ferritin
(Fig. 3). In addition to the formation of disulfide
bonds, cysteine can be modified by post-translational fatty acid acylation by thioesterification
(Schultz et al., 1988; Nichol and Locke, 1999). This
kind of linkage acylation could assist in ferritin retention in the vacuolar system when iron levels are
low (Nichol and Locke, 1999). The N-terminal extension of the G subunit, or LCH, of insect ferritin
contains all three cysteine residues (positions 3,
11, and 23), while two conserved cysteines were
found around N-terminus in the S subunit, or
HCH, of insect ferritin including H. cunea ferritin
HCH. Since the proportion of S subunits increases
after iron loading at the same time as holoferritin
particles appear in the Golgi and secretory vesicles,
the delay of protein exit from ER should be related to G subunit in C. ethlius (Nichol and Locke,
1999). However, further studies are needed for the
role of two ferritin types against over-loaded iron.
Analysis of the 5¢-UTR of ferritin HCH cDNA
revealed the presence of a stem loop structure in
H. cunea (Figs. 2 and 4). This stem loop structure
appears to be a putative iron-responsive element
(IRE). A comparison of IRE in the H. cunea ferritin
HCH with IREs from other insect ferritins shows
that the consensus structure is well preserved, and
the bases of the loop and the c bulge are conserved
(Fig. 4). The synthesis of vertebrate ferritin is regulated at the translational level by means of a stem
loop in the 5¢-UTR having a consensus CAGUG
loop, which binds one of two iron regulatory proteins: IRP1 and IRP2 (Hentze and Kuhn, 1996;
Eisenstein and Blemings, 1998). The CAGUGN
bases of the loop and the bulge C are required for
IRP1 binding (Theil, 1994; Huang et al., 1996),
and in addition, the total length of the stem, as
well as the flanking regions, can influence the
IRP1/IRE interaction (Zhang et al., 2001). Zhang
et al. (2001) reported that the human IRP1 repressed translation of the M. sexta HCH and LCH.
Fig. 3. Cysteine residues (in box) are conserved in the
N-termini of S (top) and G (bottom) type subunit of insect ferritin. N-termini of insect ferritins were aligned using the PAUP program, respectively. The sequence sources:
Hc Fer HCH from Hyphantria cunea (26 kDa ferritin), Gm
Fer HCH (26 kDa ferritin, AAG41120) and LCH (32 kDa
ferritin, AF161709) from Galleria mellonella, Ms Fer HCH
(AAK39636) and Ms Fer LCH (AAF44717) from Manduca
sexta ferritins, Ce Fer HCH (AAD50238) and Ce Fer LCH
(AAD50240) from Calpodes ethlius ferritins S and G, Ag
Fer HCH (AAM44043) and Ag Fer LCH (AAM44044) from
Apriona germari ferritins 1 and 2, Dm Fer HCH (U91524)
from Drosophila melanogaster ferritin.
Archives of Insect Biochemistry and Physiology
H. cunea Ferritin HCH
27
Fig. 4. Comparison of the putative IREs of ferritin HCH from different insect species. See Figure 3 for abbreviations. Ir Fer HCH, Ixodes ricinus
ferritin S (AAC19131); Aa Fer HCH, Aedes agypti
ferritin (AY0641060).
These results suggest that the translational control
of ferritin synthesis by IRP/IRE interaction in insects is very similar to that of vertebrates. But the
transcriptional control, in addition to the translational control, has been reported from yellow fever mosquito cells (Aag2 clone), in which iron
treatment induces a threefold increase in ferritin
mRNA and protein by 16 h (Pham et al., 1999).
In the present work with H. cunea, one-typical
putative polyadenylation signal (AATAAA) (Fig. 2)
and six non-canonical putative polyadenylation signals—AATATA, AATTAA, ATTAAA, AAGAAA, AATATA,
and AATAAT—were found (data not shown), and,
among them, a last signal located 15 nucleotides
May 2004
upstream from poly(A) tail was considered to be
most functional. Typical (AATAAA) and modified
putative polyadenylation signals were found in
cDNA encoding HCH and/or LCH subunits from
M. sexta (Pham et al., 1996; Zhang et al., 2001), C.
ethlius (Nichol and Locke, 1999), N. lugens (Du et
al., 2000), A. aegypti (Dunkov et al., 2002), and G.
mellonella (Kim et al., 2001b). Ferritin HCH cDNA
in A. aegypti showed the presence of four overlapping non-canonical putative polyadenylation signals
proximally to the coding region (Dunkov et al.,
2002), and its alternative use of the two polyadenylation signals is confirmed by Northern blot
using the probe sequence between the proximal and
28
Kim et al.
distal polyadenylation signals (Dunkov et al., 2002).
D. melanogaster also produced HCH and LCH messages of different lengths by using two polyadenylation sites (Georgieva et al., 1999, 2002). Georgieva
et al. (1999, 2002) reported that Drosophila HCH
represented at least four different lengths of mRNAs.
That model consists of alternative splicing of the
5¢-UTR of ferritin mRNA containing IRE and the use
of two alternative polyadenylation sites. The shorter
messages in D. melanogaster lacking an IRE and/or
carrying a short 3¢-UTR were also predominant in
the midgut and their abundance increased after iron
supplementation of the diet. Dunkov et al. (2002)
also demonstrated that iron enrichment of the diet
results in increased abundance of HCH messages
in A. aegypti larvae of messages with short 3¢-UTR
particularly. Ferritin heavy chain messages of different lengths resulting from alternative polyadenylation have been described in human (Dhar and Joshi,
1993; Percy et al., 1998). Thus, alternative polyadenylation appears to be a common feature of ferritin mRNAs in vertebrates and insects. It has been
reported that differences in the length of the 3¢-UTR
could affect the stability, translational efficiency, or
localization of various mRNAs (Jackson, 1993). In
H. cunea, this kind of change has not been observed.
Alignment Sequence Comparison Among
Insect Ferritins
Figure 5 shows an alignment of the amino acid
sequences of H. cunea ferritin HCH. The degree of
similarity in the compared regions is quantified in
Table 1. To retrieve protein sequences similar to
that of H. cunea ferritin HCH, relevant databases
were searched using the BLAST (Altschul et al.,
1990) and FASTA programs. When the protein sequence data were compared to those of insect ferritins, significant identities were found to Galleria
ferritin HCH (26 kDa, 68.9%), M. sexta HCH
(68.7%), and other HCH of insect ferritins (40.7–
59.3%). G type insect ferritins, vertebrate L chain
homologues are somewhat distantly related to H.
cunea ferritin HCH (17.2–20.8%) (Table 1 and Fig.
6). Therefore, reconstruction of the phylogenetic
relationships based on amino acid distances revealed two subclades corresponding to the HCH
and LCH of insect ferritin, respectively (Fig. 6).
Since the percentages of similarity for the H.
cunea ferritin HCH to both human heavy (22.5%)
and light chains (20.6%) are close in values, no
conclusion researching the nature of the H. cunea
ferritin HCH can be drawn from this analysis. However, the seven ferroxidase center residues of H
chain homologue ferritins are well conserved in
the H. cunea ferritin HCH as well as other HCH of
insect ferritins from different orders (Fig. 5). This
putative ferroxidase center could enable the insect
ferritins to incorporate free iron rapidly, allowing
ferrous to ferric conversion for iron uptake (Harrison and Arosio, 1996). In non-iron-loaded larvae,
the S subunit of C. ethlius is a minor component
hemolymph ferritin produced by fat body. In the
midgut, however, the S subunit is much more abundant, which enables the midgut to take up iron more
rapidly (Nichol and Locke, 1999).
TABLE 1. Sequence Homology in the Insect Ferritin Members*
Percent identity
1
2
3
4
5
6
7
8
9
10
11
68.7
68.7
82.9
59.3
74.4
70.1
53.8
53.6
52.1
52.4
51.2
51.7
51.7
48.8
47.3
40.7
39.2
41.6
38.8
36.4
39.5
20.8
20.9
22.3
19.0
15.1
21.0
20.1
19.5
19.9
19.4
18.5
17.9
19.5
17.7
67.4
18.6
18.0
20.4
17.6
15.1
19.0
17.2
78.9
68.3
17.2
17.5
19.0
17.6
17.5
18.5
19.1
36.6
35.3
35.7
1
2
3
4
5
6
7
8
9
10
11
Hc
Gm
Ms
Ce
Ag
Dm
Aa
Gm
Ce
Ms
Ag
Fer HCH
Fer HCH
Fer HCH
Fer HCH
Fer HCH
Fer HCH
Fer HCH
Fer LCH
Fer LCH
Fer LCH
Fer LCH
*Identities were determined by pairwise alignment using MEGALIGN. See Figures 3 and 4 for abbreviations.
Archives of Insect Biochemistry and Physiology
H. cunea Ferritin HCH
Fig. 5. Multiple amino acid alignment of the insect ferritin subunits. Conserved residues for ferroxidase are
boxed. Circle above RGD/E residues indicates an integrin
attachment site. Tyr residue sites required for the Fe(III)-
May 2004
29
Tyr complex are marked by a triangle. Only one of three
Tyr residues is conserved in H. cunea ferritin HCH. Identical residues are marked by asterisk.
30
Kim et al.
Fig. 6. Distance-based phylogenetic analysis of H. cuena
ferritin HCH and other insect ferritins. The phenogram is
based on the alignment shown in Figure 5 and distances
are approximate. The letter designation for each of fer-
ritin is on the right. G, giga; S, small. A dense line and
arrows mark the division between G and S type ferritins.
Abbreviations are described in Figures 3 and 4.
Other common characteristics of both HCH and
LCH of insect ferritin is the conservation of an
integrin attachment site consisting of Arg (R), Gly
(G), and Asp (D), though aspartic acid is substituted by glutamic acid (E) in most HCH of insect
ferritin (marked by circle, Fig. 5). Slight variations
of the RGD sequence for cell adhesion have been
reported (Plow et al., 1985; Ghiso et al., 1992; Piali
et al., 1995; Underwood et al., 1995). The integrinbinding sequence motifs other than RGD share one
common amino acid, the aspartic acid (or sometimes the closely related glutamic acid) residue
(Ruoslahti, 1996). The aspartic acid may be important because of its potential to contribute to
divalent cation binding; one hypothesis regarding
the binding of ligands to integrins postulates that
the ligand provides a coordination site for divalent cation binding (Edwards et al., 1988). An
integrin attachment site could enable ferritin to
bind to the cell matrix. That means the characteristics may have a more important function in iron
uptake (Nichol and Locke, 1999).
Multiple sequence alignment indicates that the
H. cunea ferritin HCH like other insect HCH has
only one of three Tyr residues (Fig. 5, marked by
triangle) required for the formation of the Fe (III)Tyr complex in vertebrate H ferritins (Waldo et al.,
1993). In D. melanogaster HCH and most insect
LCH, two conserved Tyr residues are found. If this
residue is essential, possibly another subunit, LCH
of H. cunea ferritin, should be responsible for rapid
biomineralization of iron in H. cunea.
The pairwise identity between HCH and LCH
of insect ferritin is very low. Though they share
some identical residues (Fig. 5, marked by asterisk), more of the similarity between HCH and LCH
of insect ferritin is accounted for by their common
ferritin ancestry than by their insect ancestry
(Nichol and Locke, 1999).
Tissue Expression of Ferritin HCH in H. cunea
Total RNA from various tissues (Fig. 7A and C)
and developmental stages (Fig. 7B) of H. cunea
were analyzed by Northern blot. Various tissues including gut shows ubiquitous expression of ferritin
mRNA with the exception of testis in H. cunea. The
expression of ferritin cDNA in epidermis is interesting. Locke and Nichol (1992) reported that iron
was involved in several steps of cuticle formation;
hydroxylases for tanning, melanization, and wound
responses; peroxidases for protein cross linking;
and perhaps in hardening and reactions with melanin itself.
It was reported that mRNA expression of M.
sexta ferritin occurs in midgut, fat body, and
Archives of Insect Biochemistry and Physiology
H. cunea Ferritin HCH
Fig. 7. Northern blot analysis of ferritin HCH mRNA using cDNA coding region as probe in H. cunea. A,B: The
expression of H. cunea ferritin HCH in the various organs
and developmental stages, respectively. C: The comparative expression of fat body and midgut from control (–)
and larvae raised on FeCl3-supplemented diet (+). Te, testis; Mg, midgut; Fb, fat body; Ov, ovary; Ep, epidermis; Mt,
Malpighian tubule; L, 4-day-old last instar larvae; PP,
prepupae; P4, 4-day-old pupae; P8, 8-day-old pupae; Ad,
adult. Total RNA was extracted from fat body of different
developmental stage (B). rRNA was used as an internal loading control. For details see Materials and Methods.
hemocyte, and among these organs midgut is the
major expression site (Pham et al., 1996). In H.
cunea, we observed the slight degradation of gut
mRNA resulting in dispersed band by Northern
blot. Midgut, fat body, ovary, and Malpighian tubule show similar intensity by Northern blot. However, the gut is more responsive to being iron-fed.
In the iron-fed group, the prominent increase was
observed in gut rather than that of the fat body
May 2004
31
(Fig. 7C). Iron increased the amount of spliced and
shortened transcripts of Drosophila ferritin in a tissue-specific manner. In the larvae, this change takes
place in the gut, but not in the fat body. This is
particularly interesting because only increased synthesis and secretion of ferritin from the gut could
result in an increased iron excretion (Georgieva et
al., 1999). In H. cunea, no difference of mRNA depending on tissue or iron-fed was observed.
The expression of ferritin HCH in H. cunea occurs at all developmental stages with a similar intensity suggesting that iron is required at all
developmental stages. In C. ethlius, hemolymph
holoferritin is sequestered with storage proteins by
the premetamorphic fat body to form crystals in
granules that disappear in the pupa during adult
development (Locke et al., 1991). At the cessation
of larval feeding in M. sexta, Fe is rearranged from
all tissues to the fat body (Huebers et al., 1988).
In adult, rearrangement and formation of organs
might make cell metabolism requiring iron activated. Whether HCH and LCH of insect ferritin
show different kinds of roles with various tissues
or stages will require further study.
The cloning of another subunit of H. cunea ferritin is underway, which will help us understand the
functional differences between H. cunea HCH and
LCH for iron metabolism during insect development.
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