ln~ect Biochem. Molec. Biol. Vol. 26. Nos 8 ~, pp. 755 765, 1996
Pergamon
PIh S0965-1748(96)00035-5
Cop2rright ~ 1()96 Elsevier Science Ltd
Printed in Great Britain. All rights reserved
~)965-1748/96 ~,15.00 + 0.00
Mini-Review
Insect Storage Proteins: Gene Families and
Receptors
N. H. HAUNERLAND*+
Received 17 Jatzuarv 1996: revised and accepted I M a y 1996
The accumulation and utilization of storage proteins are prominent events linked to the metamorphosis of holometabolous insects. Storage proteins are synthesized in fat body, secreted
into the larval hemolymph and taken up by fat body shortly before pupation. Within the
pupal fat body, these proteins are initially stored in protein granules, and later proteolytically
broken down to supply amino acid resources necessary for the completion of adult development. Most, but not all storage proteins belong to a superfamily of hexameric larval serum
proteins that are evolutionarily related to hemocyanin. This article reviews the classification
of these proteins, based on their amino acid sequences, and the current knowledge of the
receptors that mediate their selective uptake into pupal fat body. Copyright © 1996 Elsevier
Science Ltd
Storage protein
Hexamerin
Arylphorin
VHDL
Receptor
INTRODUCTION
Munn et al. ( 1971) described an abundant serum protein
from Calliphora ervthrocephala larvae which, shortly
before pupation, was taken up by fat body tissue. Since
the protein accumulated in dense protein granules, it was
called storage protein, indicating its proposed function as
an amino acid reserve for the production of adult proteins. Subsequently, similar proteins were found in other
dipteran and many lepidopteran species. Because of their
high content of aromatic amino acids these storage proteins were classified as arylphorins.
Over the years much information has been gained on
the structure and distribution of these proteins. Arylphorins are large hexameric proteins composed of 80 kDa
subunits. They were also discovered in other insect
orders, including hemi-metabolous species. Furthermore,
structurally similar hexameric proteins, collectively
called hexamerins, were found that lacked the high content of aromatic amino acids. Several excellent reviews
have been written about the composition, distribution and
properties of hexameric storage proteins (Telfer and Kun-
kel, 1991; Kanost et al., 1990), which summarize the
knowledge accumulated up to 1990. Since then, dramatic
progress has been made both on structural and functional
aspects of storage proteins. Several of these proteins have
been sequenced, shedding light on their structural
relationships. In addition, structurally unrelated proteins
have been discovered in some insecl species which may
be functionally equivalent to hexameric storage proteins.
In the first part of this review, the different families of
storage proteins are discussed and compared with other
related proteins. The term "storage protein" implies
uptake from the hemolymph and storage in fat body tissue. There, storage proteins appear to serve as a storage
pool for the amino acid resources needed later in development. Whatever the ultimate fate of the proteins or
their amino acid constituents may be,, the uptake into fat
body is common to all true storage proteins and therefore
central to their function. This process has been studied
intensively in recent years. The second part of tiffs review
is focused on the current advances in the elucidation of
the uptake process of storage proteins.
STORAGE PROTEIN FAMILIES
*Department of Biological Sciences, Simon Fraser University, Burnaby, B.C. V5A IS6, Canada.
+Author for correspondence: Tel. (604) 291-3734; Fax (604) 2913496; c- mail; haunerla(a;sfu.ca
The different classes of storage hexamers were comprehensively described in the review of Telfer and Kunkel
(1991). These authors covered only hexameric proteins,
755
756
N.H. HAUNERLAND
including, but not limited to storage proteins. Such proteins have mostly been studied in Diptera and Lepidoptera, but more recently also in other insect orders. All
dipteran species possess a protein rich in aromatic amino
acids (>15%) and methionine (>4%). While different
names have been used in the past for this protein, e.g.
calliphorin (Munn and Greville, 1969) or larval serum
protein 1 (LSP-1, Wolfe et al., 1977) it is now called
arylphorin, to describe its perceived function as carrier
of aromatic amino acids. The second storage hexamer
common to Diptera has a much lower content of aromatic
amino acids and methionine; an example is LSP-2 from
Drosophila (Roberts and Brock, 1981). Two common
lepidopteran storage proteins, in contrast, are rich in
either aromatic amino acids or methionine, but not both.
Again, the name arylphorin is used for proteins with
more than 15% tyrosine and phenylalanine content, while
the second protein is referred to as "methionine-rich storage protein". Additional hexamerins have been described
in some lepidopteran species, including several juvenile
hormone suppressible proteins from Trichoplusia ni
(Jones et al., 1990, 1993) and Galleria mellonella
(Memmel et al., 1994), and a riboflavin binding hexamer
(Silhacek et al., 1994; Magee et at., 1994). While all
these proteins share similar subunit structures and developmental profiles in larvae, their uptake, storage and utilization has not always been established, and they may,
therefore, not all be "storage proteins". On the other
hand, at least one lepidopteran family possesses a storage
protein that is structurally unrelated to hexamerins
(Haunerland and Bowers, 1986; Jones et al., 1988: Greenstone et al,, 1991); this very high density lipoprotein
(VHDL) of Noctuids is, like arylphorin, synthesized
mostly in last instar larvae, taken up rapidly by fat body
prior to pupation, deposited in protein granules and later
hydrolyzed (Wang and Haunerland, 1992). The hexameric structure is therefore neither sufficient nor necessary
for proteins that function as storage proteins.
Storage proteins have also been found in other insect
orders; arylphorins as well as other hexamerins were
described in bees (Shipman et al., 1987), ants (Wheeler
and Martinez, 1995), beetles (DeKort and Koopmanschap, 1994), but also in the hemimetabolous locusts
(DeKort and Koopmanschap, 1987) and cockroaches
(Duhamel and Kunkel, 1983; Jamroz et al., 1996). In
most cases, little is known about their utilization during
development, but evidence exists that at least arylphorin
is taken up by pupal fat body in all holometabolous species. On the other hand, a non-hexameric protein has been
found in pupal fat body of some coleopteran species that
store aromatic amino acids (Delobel et al., 1992).
While the early classification of storage proteins was
based on their amino acid composition, many proteins
have now been sequenced, giving a more reliable basis
for their classification. All hexamerins show a clear homology to arthropod hemocyanins, and it is obvious that
these proteins are evolutionarily related (Willot et al.,
1989). Hemocyanins are copper-containing, oxygen-
binding proteins found in chelicerates and crustaceans.
which also form multimeric structures from 80 kDa subunits. Beintema e l al. (1994) have recently analyzed this
protein superfamily in detail and proposed, based on multiple sequence alignments, that the insect hexamerins
evolved from a common hemocyanin precursor. Since
the tracheated insects generally have no need for oxygen
transporting hemolymph proteins, the oxygen binding
function may have been lost and the protein may have
assumed different functions within the insecl. By c o m
paring the sequence identities within the insect hexamerin group, it was found that the hexamerins form four
distinct groups which have evolved separately, namely
(1) lepidopteran methionine rich proteins, (2) lepidopteran arylphorins, (3) certain lepidopteran juvenile hormone-suppressible proteins and (4) the dipteran storage
proteins. This grouping does not correlate well with the
classification that was based on amino acid compositions
and it does not include several recently sequenced proteins. For a better understanding of the differenl storage
proteins described above, it is helpful to correlate both
functional aspects and structural data. However, it is dif
ficult to compare the reported sequence homologies
directly because different methods were used for
sequence alignment. For the structural comparison given
in this review, all currently known storage protein
sequences were aligned with the ClustalW multiple alignment program (Fig. I). To determine the homologies
between individual proteins, their sequences were pairwise aligned using the ALIGN algorithm and their amino
acid identity determined (Table 1 !, Sequence similarities
were also calculated; these may be more important parameters for a classifications of the proteins since conservative substitutions tend to maintain the overall protein
structure.
Arvlphorins
Diptera Numerous studies have been camed out to
elucidate the physiological functions of the dipteran arylphorin (for reviews, see Scheller, 1983). The protein is
synthesized in fat body of larvae in the penultimate and
ultimate larval stage, and secreted into the hemolymph
where it accumulates in extremely high concentrations.
When the insect begins wandering, arylphorin biosynthesis stops; instead, the protein is gradually taken up into
pupal fat body, where it is stored in protein granules~
Although detailed studies on different regions of fat body
have not been carried out, there is evidence that storage
protein granules are more abundant in the posterior
region of the fat body. During the development of the
pharate adult, much of the arylphorin is proteolytically
destroyed and its constituent amino acids can be found
in the newly formed cuticle. The arylphorin found in dipteran species has been sequenced from Calliphora vicina
(Naumann and Scheller, 1991). The protein shows similar homologies to all other hexameric storage protein,~,
forming a separate hexamerin (Table 1).
STORAGE PROTEINS AND RECEPTORS
1
t s 16
a0
~n-A
m ~ ' V Z 1 I . a G L W U ~ o - - - - s ~ n ~ . ' l ~ l t s l ,x
BOm-A
I~I~VAVAL
S----SAVPKP~I K
GaI-A
MQTVLIPLA~VSSAA A - - - - G T I ~
Man-M
I~A~V~AAILAVAKA S T I N V C , ~ V V I G ~
TTI-JI ~ V b V S V A S I ~ R G S
---gX~,DDTT~I(k~
BOm-M M~R%~VLLACLAAASA S AISGGEGTMVIW~
~llJ2 ~
S
~
MARPEID D
T r * aa ~ v l m ~ M m v ~ a
O----L~VI'Vm,INV~
Gal-82 MGRbVLCVLALLVGG o - - - I S ~ - - L
Lep A
..........................
BIa-A
M~TALVLI~ATATLA
.
VATI, S S ~ D T
CaI-A
I~IAIVLLAIVGLAA s
--ASITDI~X
.
H~o
.
.
.
.
.
46
ao
5DU~DVI~Vm~ND~
LgII~DV~I~'rDDE
~LLI~4KQV~I~E
LKLL~I~PTIT~
~IDQ~TIflI~
I~RI~II~I~I~
~ p L M T K D
~ I E ~ .
LTL/~pREPIRIKE
L!~LI~TI~pSTTp E
LRLLVRI~TTTAD
~EI'VTRVEDpIA~EE
3~
as
vlmvDammr~Q~
SKMV~AVI~Z~QI~ I
TRKLDpSL~IQTIC4
ET~gNVI)VKVR3~CI
IA~M%TM~IKI~R~LC£
EPMVNLI~I
TTLVT~IKQRQLVI
~XP~m~¢IVAI~'IVt,
Q ~ Q T K L
RWADKT~TRQRD~
VKIAD~QI~
. . . . . . . . . . . . . . . . . . . . . .
~1
7s
~Z~IO~ZSI~A~-TIIKI~I)TI)I~
TTKIGKETNVEAN-IREVARET%'LE~
IREVARI~/I~q-IKELKKET~I~-LEEL~K~KI~-l~xvx~laaea-QQEIAASWOL4~-RVEIG~SCI~CI~CS
QT~ATDIEKN-WI]~GKSk~--
~sl
375 376
~ge x g l
,~as 406
-I~ETNREVVTI~L
-TDKTLKTD%"~I
-MSDK'II~Y/~V%~I
- C D I ~
- T D ~ I
-IEI~S~'4TAVTTEI
-IGLT~TAVHLTI
L ~ D K % ~
I
~
~vaD1wmm~mm/
-LFEKLSVAAT- - -GEPVpADQID~RLR
MTI Ld4DKQVQALK -
z~l
~5 ~
2~0 a n
225 2~6
2~0 2~I
25s
~n A
. . . . . . . . . . .
Lm'~L K V ~ T ~ I ~
...........................
~vs
Bom A
. . . . . . . . . .
~ V L Q KITVTI~Q . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
RGLIN
Gel A
. . . . . . . . . . . . .
SDVLS KITRIQ(4Q
......................
KGLII
M~- M
. . . . . . . . . . .
S ~ I N !~tl~11~(1,1T. . . . . . . . . . . . . . . . . . . . . . . . . .
K A V TD
Tri-jl
. . . . . . . . . .
SHII N K A ~
. . . . . . . . . . . . . . . . .
KAARE
so~ s
..............
suvxs ~
.........................
r r t a~ . . . . . . . . . . . . . . . .
~vi~ x ~
.........................
maLLD
T r i aa
..............
G E I M T TAARI . . . . . . . . . . . . . . . . . . . . . . . . . . .
NTRG
C~t 82 . . . . . . . . . . . . . . . .
~rD
DSm~ ............................
bep A
...................
~'VIN K A ~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Qv
Bla A
....................
S,,~VIQ I~,'Elg"I~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
N
CaI-A
S R D I ~ 7 ~ W
SK'flI~EYI~ILI
KDXATWyK~D~'Rs~ ~
MGDa~'S~Rlfl~
~ro ~
. . . . . . . . . . . . .
SQAL~ ~
.............................
aw.~
:4~
. . . . . . . . . .
~DSL~ ~
. . . . . . . . . . . . . . . . . . . . . . . .
4~0 421
256
......
EU~-p~DuW~W
NI~FL~--p.-NE~IW
~
v
SC~DRI2gQAKK-~
~tat1~au~m'rl~m/v~a~bln~
~
S
L
A K Q ~ A ~
285 286
~-~n.vlr/&m~
] 0 0 301
~AVL~-Z~RLTZ
283
I R E D IN
~ST'~DpA-FI~K
VKI~GI~IpT~
N
22]
-A~
m
.........
'115 136
D~.gDIIVD,VKDT GNILDP - - ~ ' ~ l
450 451
a65 466
480 4111
onrmcT~,Ql-.~Im~
~m'Im~a~GIL~
IOa~UTXI~
~ 4 ~ f I R E ~ I L T C , K IERRDGTVISLIa~s-
~i-aJ
r I-A82
~epBIa-A
Cal-A
EIPELG-HE'/VKI~"
EIpEL~-SNEVEEGT
DIEI'LDgDSpIQTPT
EVEp%~n~DRp~TET
EIpAIP~IDSIETGT
TRSI~.M~SSW
VSGbL%'H.NGIPIE~/
Np~fg~P-CC, A ~ p S
TPRLR~S-I~QE~p
NPQLVI~-NGIa~ST
~
............................
RP~LVLI~
R P K / ~
K ........................
RpTETSRRSLTTSNG N
~
I~£G~pSTE
ItI(~eFEVE . . . . . . . . . . . . . . . . . . . . . . . . . . .
Redo
P.MIpI~q/NEpLGGT
AAffLTMVASGRRTAQ
RPDGLA~ .........................
s~l
sss ss~
GeI-A
I~pSLRDpA/TQLTA
Man-M
ITTC[A%DPVI~fIM~
Tri-Jl ~ I ~ ' I I % ~ L ~
Bom-M
T~fCLRDF4/~II~K
Tri-J2 I~TALRDpVIP~I~QK
Tri-aJ T~TAI~RDPAIP~MIWK
Gal-82 T~TASRDpA~EI~MII~
Lep-A
TETOIRDp~IAK
BIa-A
ITTADRDpA~MILK
CaI-A
~ I ~ F M I ~ K
Pro-2
~ p I / ~ % ~ K
R~
TATSLRDpI/TRTMR
s~o s v l
KILDTIMETKETLEp
~ L L p K
RVTD,f/WL/I~pK
RVCNIFIR21~II~
R~IDL91~4PKLRLPS
RVLGLI~QWQ~.LpR
R ~ L p H
~MII~K/QRTL~DLPp
HINSIn~RTKG'~ff~pR
K
I
~
TIVS~T~L~I'~E
/I[AW91~ET~TLpV
721
7~5 736
750 751
-SVFI,t ' I E L ~ Q
H . . . . . R1~HDMSE D I ~ p F ~ p R ~
C.aI-A
--SVSLIKITELLSN
TTI-J2 ~
i
r
TTI-aj W I T S T Q ~ A S
CaI~82 I
I
~
LeD-A
K
~
.
s~s s~a
T S Q ~ I N D
~EELDIFI~"~%'EM
~ P G V K I ~
TI~/S~PGVKI/~
TTI~DL~'~PGgKIIA~
TKpEELAMPQVAI~
~FrK~SgKI~
TTEE~LIPPGVSV~
TITDELVIPPGgKI~
TT~SREL~PGVTIKE
TER~d~TAIPEG~IDA
T S ~ I T D
Man-A
.
.
.
.
.
.
.
.
.
.
DVREVDVQ~M
soo ~oz
VKVDK.L~I~IF
v
ITTDK-LVTIPI~ETD
~FI~K-LTT~ZDETD
I%'I'DE-LV'/'MgDETD
9"4V~K.LgTTII~DTL
VDV~-5gTTIPETST
VEV~-~,~.zr~rl~
LI~Dp-LITT~WST
",/~/GK-L%-zx~swu
VKVSE-LWI'EI~LVD
VR~F.~LyL~pEI~0
VKgT~KI~I
Q p ~ t
Q,/,afffA~I~i
KWSWSR/GTI~p[~
~-'TSTE~D3~F~ED L
SI~a~#~EV
.
~s
~ls
~o
WNATNA91LS __E . . . . . . . . . .
~D
VDIT~LTLDQ
NE .......
I~
I~ITNA~I~3DoVE .........
~
I~DISNAMTL~-TE . . . . . . . . .
MQ~
I~gTNAV~T~.DE ..........
IKKT
MRI/TS~LI"~%'V-EE . . . . . . . . . .
AKEL
FNVTNHLhLNE-IE . . . . . . . . . . . .
C~
S D L ~
..............
~QED
VDII~N~K
...............
VAED
~
. . . . . . . . . . . . DSQp
SDISNAV~E!~AV~
SADPIPTT~RNSRTK
R--E~ELELSH . . . . . . . . . . . .
CLM/
rEAr-~XIDr~D~-
--~RG~pL~Lrv'VVT
pT~.-
D MRDLMIKDL4%WE
R --.EI~LSTSTRR
Q G - - - I ~ Q
E- -GDG~fKI[3ERQ
--VG~TVIVT
S%RfALRPDGLMIAR~
--VGG~I~gI~4VTIS
- - ~ I V T
pbRLVIX~DII~-ILD
RT~----GCVR-CRT
EI,'d~---A!~q~-VHR
pTE----~-G.K
R'I~g/pTRLLLPKG~
QHAQV~SHLLLLK~/
N I ~ K G R
NTI~RINI~PRGS
ApV ........
I~E-~QTI
INRKJDL~R~'R~'~
QPRASEagLEPLTRQL
GTVETSTIIA~'TIRLT
T ~ T ~ G I p ~
49S 496
510 511
ago
aa~
~
S25 526
T E V U ~
2sa
282
243
5le
-KA1~W~U~
r & ~ - s
--~KI'FI, I ~
41V
-l~vmua~la~
-~II~%'LGGL
~vuD~lu~-- ~raoaua.sTm~
EI~DOkKIgIH .... L T I ~ T N
--.~*'*~I'TAt~L
- ~ L E ~
4t~
419
TDTI~%RVRDAII~
EVI~IRLIMIEA~V
~ I D ~ G ~
QIIDRRVL~IDTGT
LI~N~IIT~GV
VI~III~RIDIS/~TITN'I~RVSINSPVIH~K(~IELDTp~ L T J ~ T
T~'~TIDLRKp-
-~IEILC, R V I F ~
-D~II~/LGRLIEa~V
-EG~N%'SG~LIQ~
TKGIDTL~D~I~G~G
-ESIEEIGSIM~g
I ~ I ~ I S - V
D S ~ I S - I
W~VU~SLVSZSVA
DSP~K/TC~&L~S . . . . . ~
DTgI~RI~I~IT . . . . . R/TRQLA~I~%~p
~
-~AI~I-~AT~T~
~l~-Ip~vv~la~
--VMNTRE~SALI~
--TNDS~pAPSAL~
.... TLQVI~PHI~LI ~
41~
3~4
442
456
~WTI~RIM~AIDLRR
VISPTGETIpLDE~-
-RGADILGALIESST
ESI~RGTTGSLM---
- - D G ~
352
~3I
~45 s ~
TVSpSTIVRQpRIA~
HSI~MI~VARMRRL~
RSD~fVARI~IA%~
TS~ITI~MAR~RI~
KS[IMVIPMVRI~a~
TDQVSVLV~pRL~H
IN'/I~/LV~RTRL~
AKT~IRVRQTRL~
G~aY&'RARQTRL~
V~KTLLAR~RI~
~SS~IKARQQRL~
AKPC, S V R A R T ~ L D ~
a~o s a l
KpI~%'NIDIK--SD~
HPI~/NID~--S~K
H ~ - - S ~ K
HpI~VSI~'M--SI~K
QpYKVTLDIL--SI~
I~I~VRVNVK--TEV
KVIW/~RVMVK.-SGV
KPIPTTKII~/T--SDK
KPTf~IEVN--SEQ
KP/E~I~VIE--S~
K!~D~T--S~
ES~ST~ISAQNNS~
7~5 76~
?8O 7gl
795 796
818 811
825 826
--EGG~pI~IvI~VI~/T plaN----ADS . . . . . . . . KD ~ E A I ~ I
QIA~U~pLGTPPD~I~'V D A T W K - - - O ~
G ..... Q,/~STMVD RTITLPRIRLILPRGT
a86
lt4~zt,lmza~l~
LIARTI~SMGLG
LLART~Ik~RLS~-.LG
~
~
TI~3~LI~Q ~
.
~u~,,r~Tman
~m~im~r~lm,~
I~HRRGIgI~@I~Q
KI~R-GELII~%~Q
KIM~RG'ELT~AI~Q
T~n~nmop~L
.
s~tt,i.im~Tsll-rm~ r ~ l m . ~ u ~
ls~
123
3~0
vm~m~wa~-m~K
r~'L]~-A~PV
LATPIWLRSD-ACAI
LT~IPI~aSE-E/G~
.................................
............................
.
345 346
~nmi~&wnm~g
LHSRL~I~LILLQRS
Y/'E~t~L%'ENIK
FLEDIII~ISLTTI~N
mermam~ziDm~T
D~T4m.~-~a~L~T
,I,TE,
WE~A*F,;VIRW~pH LALLQQ..P~ITTSL
NICL~E~CCIRI~ET ~
PG-SIPI~-'ITZ~- ~
H~4~L~.MEQSLST
~ 1 I V
I ~ A
.
a ~ - ~ z J ~ t a ~ D l ~ - v Vl,pAIm~Im~WSTIm
ZU~VALL~REDCRGV IVPpVQEV~ADIU~Ip
330 331
172
176
17~
17S
169
173
171
134
171
LKSmmm~I--IARLVDI~Tp S . . . .
~ p I
.....
~'SRDVHQ N . . . . .
Rpm~-~La . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
.
VLpAJ~II~IMPEYI~I
TLPAF~ITI~'FF4D
TLPAPTEI~V/~D
TLpAPTEITIWfI~VD
ILPpFEEITI~TII~R
TZpRI/~I~pSTI~M
RIPPITI)~pSIff~N
I~PPlTEIKPRI'I~N
ILPPPTEITFIIAm/D
t.=am~l~r~w~.m~
l,~am~c~-l~zlmmn-r~ ~ . I m c
r~t~LT--KIPTPrAQ
.
315 116
~nmi~rrn~
W~KIR~-~D~
WPKIRLp-SGDEMPV
.
AA~ARVI~2~EG~I'VT AI~U.~VI~%~DTRGI
A C W L R ~
ALTAA%'IPI~TI~IGI
ACWIAR~ING~%'T
ALTAC%'IPI~RTDCRGI
AC~4RI~R~'VE
A~TAA~GL
I T [ / ~ T
RLTVAVRI~t~GI
A%~%~DRir~DLI~ST VLSAVI~/I~I~Y/~GI
A ~ R Q ~
VLS%~II4{RSDT~DI
A
~
A~S%'AII~CRI~
VCMa%R~II~Ly
~J~STAV/~RI~DT
~ T i m z ~ ¢ a ~ , l ~ z a len't,mwr~g~-xman
rrlmIlxarnncaTta ~ - z ~ n r l ' ~
KIpOSWrSPIKTGT
.
15~ 151
1-{;5 166
180
ATTIAVlQRKI~I~GF 9~/PApTEVTI~F~AN 172
pwrAl~r*u~ . . . . . t'v1'l~ul,vvil~w~m~o v p a , . A m - r - - ~ t ~
m,1~.a:,rmal ...... xv'rzx~u,-/iI~,mlt~a ~ a m r r l * T - - ~ i ~
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90 91I 0 5 106
120 121
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AKT~VIaI~LAPE'~
~
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AQK~VIRT~GI~TD
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FIGURE I. Multiple alignment of hexamerin sequences. Hexamerin sequences were aligned using the ClustalW algorithm.
Phcnylalanine and tyrosine residues are bold-printed. Sequences were downloaded from Genbank files. Man-A: Arylphorin
from Manduca se,rla (Genbank sequence [D 159491); Born-A: Arylphorin from Bombyx mori (Genbank sequence ID 134926):
Gal A: Arylphorin t¥om Galleria mellonella (Genbank sequence 1D 449954); Man-M: methionine-rich protein from Manduca
.~'e*la {Genbank sequence ID 159526); Tri JHI: basic juvenile hormone suppressible protein I from Trichoplu~'ia ni (Genbank
sequence 11) 729863): Born-M: methionine-rich protein from Bombyx mori (Genbank sequence ID 134925),: Tri-JH2: basic
juvenile hormone suppressible protein 2 from Trichoplusia ni (Genbank sequence ID 729864); Tri-aJH: acidic jm,'enile hormone
suppressible protein from Trichoplusia ni (Genbank sequence ID 125066); Gal-82: juvenile hormone suppressible protein from
(;alleria melh,zella (Genbank sequence ID 156154); Lep-A.: Arylphorin from Leptinotarsa decemlineata (Genbank sequence
ID 556786): Bla-A: Arylphorin from Blaberus discoidalis (Genbank sequence ID 951139); Cal-A: Arylphorin from Callil)hora
ricina {Genbank sequence ID 288282); Dro-2: larval serum protein 2 from D. meMnogaster; sequence obtained from Naumann
and Scheller, 1991; Hem: hemocyanin from Eurypelma cal(fi)rnicum (Genbank sequence ID 122'799.
T A B L E 1. Sequence similarities in the hexamerin superfamily. Sequence identities and similarities were determined by pairwisc alignment
using ALIGN. Identities are shown below, similarities above the diagonal line. Abbreviations and sequence sources as in Fig. 1
Man-A
Man A
Bom-A
GaI-A
Man-M
Tri J I
Bom-M
Tri-J2
Tri-aJ
Gal-82
Lep-A
Bla-A
CaI-A
Dro-2
Hemo
Bom-A
90.0
66.6
54.2
3 t.8
2~L0
3F.4
33.8
26.7
26.2
30.5
32.3
27.8
30.5
24.7
49.9
3/I.3
28.9
28.1)
32.2
26.1
27.4
29.1
29.8
25.7
28.1
25. I
GaI-A
85.5
83.3
29.7
27.9
30.0
31.7
27.(/
28.6
31.0
30.8
29.8
28.5
24.8
Man-M
68.8
68.5
69.7
71.2
69.5
44.9
26.1)
26.2
28.5
29.3
25.3
22.5
23.6
Tri-J1
68.3
66.7
67. I
93.6
91.8
44. I
23.8
25.0
27.4
28.1)
23. I
24.3
23.7
Bom-M
68.3
67.7
67.1
87.0
66.2
47.5
25.1
25.7
28.2
29.0
27.6
24.3
25.0
Tri-J2
69.7
69.5
70.0
78.1
78.5
78.5
27.1
29.5
30.4
29.2
27,5
26.3
27.2
Tri-aJ
69.5
68.0
66.6
65.1
67.5
65.9
67.8
45.0
26.8
21.6
22.1
21.7
21.6
Gal-82
69.4
67.1
69.2
64.5
66.0
67.0
68.0
78.4
Lep-A
66.6
67.2
68.0
61.0
61.0
60.5
63.7
65.4
66.8
28.7
22.9
25.8
25.3
27.2
34.9
28.6
30.7
26.8
BIa-A
64.8
63.2
63.0
62.2
59.5
60.(/
60.5
64.1
63.8
67.0
27.1
31.0
27.4
('aI-A
64.4
65.4
63.8,
66.4
65.2
65,2
65.3
63.6
64.2
62.6
65.0
31.0
23,4
Dro-2
65.9
656
65.8
62.5
62.4
64.5
63.3
64.0
65.4
66.9
62.4
62.6
25.3
Hemo
63.5
61.8
62.3
58.5
59.2
58.6
58.6
62.2
62.5
63.7
63.0
56.9
60.5
758
N. H. t:AtlNERLANI)
Lepidoptera The overall characteristics in lepidopreran arylphorins resemble those of the dipteran proteins,
but several important differences exist. Lepidopteran
arylphorins have a similar percentage of aromatic amino
acids, but are relatively low in methionine. The sequence
identities between those lepidopteran arylphorins
sequenced (Manduca sexta, Willot et al., 1989; G. mellonella, Memmel et al., 1992; Bombyx mori, Fujii et al..
1989) is between 50 and 70%, but they are much less
homologous to the dipteran arylphorin. While arylphorin
is synthesized at high rates in fat body of last instar larvae and released into the hemolymph, evidence exists
that the protein is expressed at a low rate throughout larval life (Ray et al., 1987; Webb and Riddiford, 1988),
and also in the gonads of adult insects (Miller et al.,
1990; Kumaran et al., 1993). Nevertheless, high hemolymph concentrations are found only during the last larval instar and re-absorption into fat body begins in prepupae. In most cases, arylphorin is not completely removed
from the hemolymph, possibly due to its high concentration (Haunerland et al., 1990). In detailed studies the
uptake and accumulation into fat body has been biochemically and electron microscopically documented in
Hyalophora cecropia (Tojo et al., 1978), B. mori (To.jo
et al., 1980) and Helicovetpa zea (Wang and Haunerland,
1991, 1992). Arylphorin has been positively identified in
crystalline protein granules in fat body and it appears that
these granules are gradually, but not completely broken
down during the pupal stage; in fact, many granules have
been detected in adult fat body and it has been suggested
that arylphorin may also serve as an amino acid source
for yolk protein production (Wang and Haunerland,
1991).
As for the place of synthesis and storage, most studies
have not attempted to distinguish between different
regions of fat body. Detailed studies have only been performed in H. zea, where, aided by a colored storage protein (VHDL, see below), a clear distinction could be
made between the place of synthesis and storage (for a
review, see Haunerland and Shirk, 1994). Arylphorin is
synthesized in the larval fat body that is found peripherally, between the outer muscle layer and the cuticle of
5th instar larvae. This white tissue, however, does not
sequester arylphorin, instead the protein is actively taken
up by newly formed fat body tissue that is located centrally, within the body cavity, surrounding the gut. From
electron micrographic studies it is now clear that this perivisceral fat body persists through the pupal stage and
develops into the adult/'at body; the peripheral fat body,
however, decays.
Coleoptera Arylphorin-like proteins have been identified in a number of coleopteran species, including the
mealworm Tenebrio molitor (Delobel et al., 1992) and
the Colorado potato beetle, Leptinotarsa decemlineata.
The latter protein was recently sequenced (DeKort and
Koopmanschap, 1994). It is rich in aromatic amino acids,
as other arylphorins, but structural similarities with arylphorin Dom other families are not greater than with the
remaining hexamerin classes (Table l). Relatively little
is known about its developmental profile, but it appears
that the protein is also stored in granules of pupal fat
body.
Dico,optera In Periplaneta americam~, tv,.o arytphorin-like molecules have been identified (Duhamel and
Kunkel, 1983). One of these is present throughout all life
stages, while the other one is found mostly in larvae. No
evidence exists that points towards the uptake and stof
age in fat body, and the physiological role of these proteins has remained unclear. Recently, a related protein
has been cloned and sequenced from Bhtberus discoidali.s
(Jamroz et al., 1996). The protein is similarly homologous to all other hexamerins, including the arylphorins
from other families [Table 1).
LSP-2 j)'om l)iptera
The second storage protein known in Diptera is present
in smaller amounts than arylphorin, but it is similar in
its developmental profile. Apparently, this protein is also
taken up into fat body, but its ultimate fate is not known.
This protein may play an important role in the adult
insect, as suggested by the fact that its expression
resumes alter adult eclosion (Benes et al.. 1990). I,SP-2
from D. mehmogaster is the only protein of this group
that has been sequenced (S. Mousseron: cited in Naumann and Scheller, 1991): its sequence homology to dipteran arylphorin and to all other hexamerins is similar
and relatively low, indicating no close evolutionmy
relationships between LSP-2 and any other hexamerin
(Table 1).
Methionine-rich storage proteins
./tom LeFidopwra
Methionine-rich proteins contain more than 4~/~ methionine. In contrast to arylphorin, these proteins are not glycosylated. They have been found in several, but not all
lepidopteran species investigated. In some species, e.g.
M. sexta and H. cecropia, two or more isoforms of methionine-rich proteins have been found (Wang cl al., 1992:
Tojo et al., 1978). Their biosynthesis in fat body appears
to commence later than the synthesis of arylphorins,
towards the end of the last larval stage. Then, methionine-rich protein disappears completely from the hemolymph and is taken up by pupal fat body. While its deposition in granules has not been studied. Pan and Telfer
(1992) demonstrated that its concentration m the fat body
dc,es not significantly decrease during the pupal stage. It
is interesting to note, however, that, in contrast to all
other classes of storage proteins and hexamerins, methionine-rich proteins, at least in M. sexta, are much more
abundant in female than in male larvae (Ryan et al..
1985). Sequence comparison indicates that two basic juvenile hormone-suppressible hexamerins described in E
ni (Jones et al., 1993) also belong into this group
(Beintema et al., 1994). The identity belween the methionine-rich hexamerins is relatively high, between 44 and
70% (Table 1). In contrast, identity with other lepidopteran and dipteran hexamerins is less than 34%. Since the
STORAGE PROTEINS AND RECEPTORS
two basic juvenile hormone suppressible storage proteins
from T. ni appear to be the equivalent to the methioninerich storage proteins in other lepidopteran species, it is
likely that the latter proteins are suppressed by juvenile
hormone in other species as well. Indeed, it has been
shown that the methionine-rich proteins of M. sexta
appear only alter the juvenile hormone titer declines in
the final larval instar (Webb and Riddiford, 1988; Corpuz
et al., 1991 ).
Juvenile hormone-suppressible proteins
The expression of storage proteins is mostly confined to
the last larval instar, a period where juvenile hormone
titers are low, and it has long been suspected that the
hormone prevents the production of storage proteins.
Indeed, juvenile hormone has been shown to suppress the
expression of some, but not all storage proteins. In
addition to the above mentioned basic juvenile hormonesuppressible proteins, an acidic hexamerin has been
characterized in T. ni (Jones et al., 1990); amino acid
composition, sequence homology and juvenile hormonesuppression suggest that a recently cloned hexamerin
from G. mellonella (LSP-82, Memmel et al., 1994) is an
analogous protein. These acidic juvenile hormone-suppressible proteins are highly homologous to each other,
but much less to all other hexamerins (Table 1). At this
point, little is known about their developmental profiles
and fate and whether they function as storage proteins.
Riboflavin-binding proteins
In spite of structural similarities to other hexameric storage proteins, it is known that the riboflavin-binding hexamerins found in H. cecropia (Magee et al., 1994), G.
mellonella (Silhacek et al., 1994) and Heliothis virescens
(Miller and Silhacek, 1992) are not true storage proteins.
Although they are expressed strongly only in the last larval stage, they are not actively sequestered by fat body
before pupation and do not accumulate in pupal fat body.
Their hemolymph concentration diminishes rapidly during the pupal-adult eclosion, but it is not known whether
these proteins are simply hydrolyzed or taken up by fat
body, ovaries, or any other tissue. These proteins are glycosylated and appear to contain a high concentration of
histidine and arginine. To date, no sequence information
is available for these proteins.
VHDL
A non-hexameric storage protein has been characterized
in the corn earworm, Helicoverpa zea (Haunerland and
Bowers, 1986) and other Noctuid species (Jones et al.,
1988; Greenstone et al., 1991). This protein, a dimeric
or tetrameric complex of 150 kDa subunits, is characterized by two unusual properties: it is colored blue, due
to non-covalently bound biliverdin, and it is a very high
density lipoprotein (VHDL) with approx. 10% lipid. It is
synthesized in peripheral fat body of last instar larvae,
and accumulates in high concentrations in hemolymph,
before it is specifically taken up by perivisceral fat body
759
prior to pupation. Within the fat body, it can be found
in protein granules, but it is apparently proteolytically
digested within a few days. Its amino acid composition
is not different from average proteins and thus it appears
that this protein bears no relation to the hexameric storage proteins described above; however, sequence information is not yet available. VHDL is the only non-hexameric larval serum protein known so far that clearly
functions as storage protein. In the hymenopteran species
Apis mellifera (Shipman et al., 1987) and Camponotus
festinatus (Wheeler and Martinez, 1995) a VHDL w.ith
similar lipid and apoprotein structure, without the colored
chromophore has been reported, but the developmental
profile has not been established.
Tyrosine-Rich proteins
Tyrostaurins and related tyrosine-rich storage proteins
are proteins with up to 27% tyrosine found in coleopteran
fat body (Delobel et al., 1992). Their synthesis and
accumulation in fat body occurs shortly before pupation,
and these proteins are deposited in protein granules of
pupal fat body. Later in development these granules are
partially broken down, suggesting thai the tyrosine residues of tyrostaurins are used for the biosynthesis of
cuticular structures of the pharate adult. While they
apparently fulfil similar functions as other storage proteins, tyrostaurins are fundamentally different not only in
their smaller but highly variable size (30--65 kDa) but
also because they are never released into the hemolymph.
Instead, these poorly soluble proteins appear to be
deposited into protein granules immediately after being
synthesized. No structural or sequence data are available
for these interesting proteins, which may represent an
alternative mechanism for amino acid storage that has
evolved independently. In fact, some coleopteran species
possess arylphorin only, but no tyrostaurins, others only
tyrostaurins and some express both proteins.
Conclusions
All hexamerins are clearly related to each other and to
their proposed evolutionary ancestor hemocyanin, but
nothing suggests close relationships between hexamerin
families from different insect orders, even if they are
similar in their amino acid composition. The structure
of the hemocyanin molecule contains many hydrophobic
amino acids. Conservative substitution with other hydrophobic amino acids, including aromatic residues, is
unlikely to lead to major changes in the overall structure,
and therefore an "arylphorin" may have evolved independently in different orders, in response to the need to store
aromatic amino acids. This hypothesis is supported by
the fact that the position of aromatic: amino acid residues
are not more conserved in arylphorins than in the other
hexamerins; in fact, when all insect hexamerins are
aligned with the Clustal W multiple alignment program,
phenylalanine and tyrosine residues are common in many
positions in all hexamerins (Fig. 1). Not a single aromatic
amino acid residue is conserved in all arylphorins but not
760
N.H. HAUNERLAND
in any other hexamerin. Some coleopteran species, on
the other hand, may have used a different strategy to
store aromatic amino acids, in the form of insoluble tyrostaurins.
Storage proteins other than arylphorins may also have
evolved independently in response to the need to store
amino acids during the pupal stage, taking advantage of
the strongly expressed hemocyanin gene, but other proteins, serving completely different functions, have also
evolved from the hemocyanin ancestor. These include the
hexamerins from hemimetabolous insects and the above
mentioned riboflavin binding hexamerins that are not
taken up into fat body. Moreover, other non hexameric
insect proteins have been discovered that have a clear
sequence homology to hemocyanin: prophenoloxidases,
abundant hemolymph proteins, were sequenced from D.
melanogaster (Fujimoto et al., 1995), B. mori (Kawabata
et al., 1995) and M. sexta (Hall et al., 1995) (sequence
homology 29-39%) and a somewhat weaker homology
was also found for the arylphorin receptor from Dipteran
species (Burmester and Scheller, 1995b; Chung et al.,
1995; see below).
PROTEIN STORAGE AND UTILIZATION
Some hexamerins are not taken up and stored by fat body
and hence are not storage proteins, while proteins of different evolutionary origin can serve as storage proteins.
Essential for a storage protein is its specific recognition
and uptake by the fat body. Ever since their discovery it
has been questioned why storage proteins are released
into the hemolymph after their synthesis in fat body, only
to be taken up by fat body just a few days later. Several
hypotheses have been offered to explain this seemingly
complex pattern, but no uniformly convincing explanation has arisen. It may just be an evolutionary consequence of the availability of ubiquitous, water-soluble
hemolymph proteins (hemocyanins). Their storage in fat
body required the evolution of an efficient uptake mechanism. Storage protein uptake occurs only during a brief
time period shortly before and after pupation, and this
uptake is an endocytotic process. Since storage proteins
are normally present in large concentrations in hemolymph, non-selective endocytosis alone would assure the
import of large amounts of storage proteins into fat body,
and initial experiments with horseradish peroxidase demonstrated this (Locke and Collins, 1968). However, the
clearing of proteins from hemolymph and the accumulation in fat body is not a function of their original concentration, indicating that the uptake occurs in a selective, receptor-mediated process (Pan and Telfer, 1992).
Such a process would not exclude the unspecific import
of other abundant hemolymph proteins, since the lumen
of endocytotic vesicles would always enclose a small
volume of hemolymph. Indeed, when fat body of H. zea
was incubated with equal amounts of labeled arylphorin
and a foreign protein (IgG) in vitro, a small amount of
IgG accumulated in the tissue, but a tenfold excess of
arylphorin was taken up (Wang and Haunerland, 1994b).
To achieve the observed selectivity, the uptake must be
mediated by specific endocytotic receptors. Potential candidates for such storage protein receptors have been
found in both dipteran and lepidopteran species.
Dipteran receptors
Evidence for an arylphorin receptor in dipteran fat body
dates back to 1983, when Ueno et al. (1983) reported that
fat body membrane fractions of the fleshfly, Sarcophaga
peregrina, bind radiolabeled arylphorin in a specific and
saturable manner, with a Kd=4XI0 '~ M. Various sugars
did not seem to influence binding, in contrast to most
known endocytotic processes in other animals. The binding of arylphorin requires Ca 2+ ions and is pH dependent,
occurring optimally at pH 6.5, the pH of the hemolymph.
In a subsequent study, these authors found that a flavin,
lumichrome, prevented binding of arylphorin to its receptor (Ueno and Natori, 1987). In the presence of Ca 2+,
lumichrome bound to arylphorin in stoichiometric ratios
(1 molecule per subunit), suggesting that the lumichrome-arylphorin complex could no longer be bound by
the receptor. The authors proposed that a lumichromelike molecule may be part of the arylphorin binding site
of the receptor, and that this molecule can participate in
the recognition of arylphorin. With ligand blotting techniques it was demonstrated that the putative arylphorin
receptor has a molecular weight of 120 000 Da. Binding
of arylphorin was detectable only in pupal fat body, but
not in larval fat body, unless the insects had been treated
with ecdysteroids. Other evidence indicated that the
receptor is present in larvae as an inactive precursor
(M~=125 kDa), and that this "cryptic receptor" is proteolytically cleaved to give rise to the active 120 kDa protein (Ueno and Natori, 1984). Chung et al. (1995) have
recently cloned the putative arylphorin receptor and have
re-assessed their earlier conclusions. It now appears that
the 125 kDa protein, previously called the cryptic receptor, is an unrelated protein. Moreover, since it was shown
that the fat body membrane preparations contained
numerous protein granules, it is now questioned whether
the 120 kDa receptor is actually a membrane protein, or
instead an intracellular protein that binds arylphorin
within the granule. The latter possibility was favored by
the authors since they failed to detect the 120 kDa protein, or crossreacting material, with affinity-purified antibodies in fat body cell membranes, but saw strong fluorescence
in
protein
granules.
Interestingly,
immunoreactive proteins were detected in both larval and
pupal fat body, although only the latter can bind arylphotin. In western blots it was demonstrated that both fat
bodies contain three immunoreactive proteins, of 120, 76
and 53 kDa, but that the larval tissue has only traces of
the 120 kDa protein, which was shown to bind arylphorin. Amino terminal sequence analysis provided conclusive evidence that the smaller proteins are fragments of
the 120 kDa protein. Therefore, it was assumed that in
larval fat body the 120 kDa arylphorin binding protein
STORAGE PROTEINS AND RECEPTORS
is rapidly cleaved into the two fragments, possibly by a
specific protease. The presence of 20-hydroxyecdysone
before pupation may inactivate the protease, so that the
intact arylphorin binding protein prevails. However, 20hydroxyecdysone also induces the expression of the
120 kDa protein itself.
Its cDNA, which codes for 1163 amino acids, starts
with an endoplasmic reticulum-targeting sequence, followed by the N-terminal sequence of the 120 kDa protein. This is also the amino-terminus of the 76 kDa fragment, which is 695 residues long. The 53 kDa fragment
starts at amino acid 713. The sequence shows clear similarity (29.1% identity, 66.2% homology) to a previously
sequenced protein from Drosophila fat body with
unknown function (fbpl protein, Maschat et al., 1990),
and to the independently cloned and characterized arylphorin receptor from Calliphora (Burmester and
Scheller, 1995a; 46.1% identity) (Fig. 2).
In Calliphora fat body membranes, three arylphorin
binding proteins had been found in 1992 by Scheller and
co-workers (Burmester and Scheller, 1992), with molecular weights of 130, 96 and 65 kDa. In a direct ligand
blot with radioiodinated arylphorin only the 96 kDa protein was labeled, but all three proteins became visible
when blotted proteins were first incubated with the ligand
arylphorin and then with anti-arylphorin antibodies, a
more sensitive procedure (combined ligand immuno15 16
1
30 31
45 46
60 61
blotting). In vitro translated mRNA however produced
only the 130 kDa protein. All three proteins crossreacted with specific antibodies raised against each protein and therefore the 96 and 65 kDa proteins appear to
be fragments of the 130 kDa protein. Both the complete
130 kDa protein and the 96 kDa protein were prominent
in larval fat body before arylphorin uptake takes place;
the 65 kDa fragment is absent earlier in larval life, but
rises with the initiation of arylphorin uptake. Since both
the uptake of arylpborin and the appearance of the
65 kDa arylphorin-binding protein (as well as another
30 kDa protein) is induced by 20-hydroxyecdysone, it
was concluded that the 96 kDa protein must be modified
before arylphorin uptake can take place, possibly by
cleavage to the 65 kDa protein, which may be the active
arylphorin receptor (Burmester and Scheller, 1995a). The
96 kDa protein itself is a fragment of the initial 130 kDa
gene product. In addition to a clear sequence homology
with the Sarcophaga receptor and the Drosophila fbpl
gene (27.8% identity), the receptor is also related to its
ligand arylphorin (17.8% identity).
As mentioned above, the sequences of the arylphorin
binding proteins from Sarcophaga and Calliphora are
very similar and, hence, we must assume that these are
the equivalent proteins in the two species, although their
reported function and activation appear, at a first glance,
contradictory. However, when considering the aligned
75 76
90
FILDVLQQV~(PIQN
FILDVLHQVYMPLQQ
FILDIVQNIRQPDQQ
S a t M ~ R I % q J u L L G M A S L A ASGIITDRIRGGLDM VAGLGIGLQGOQGLQ GSVHHQGIGPE~4GS INVNTLNQQQLLRQK
GVVNTLTRDELLRQK
Cal ~ I A I
ILVGLASLV ASGVIMDRG-GRVGL SANIGLGVQTMQGIR ..... ~ I E G S
D r o b~IIRII~LLVAPAAGVV AAGRITI~Q . . . . . . . . DR ...... VQGID . . . . . . . . . . . . . . . . . . . RMSLEDSQLQK
271
106
120
DLYTRPLTEE~K~L
DLYQRPLT~EM~4VL
QRYRGGII~"a~VI
121
135
DLIRQQQIL~QQIC
~SQ~(LGKQTIC
DLDRQRRLLDEHQVY
135
129
101
226
240
S~VDVVGLGRQ
SVRP~LMDVIG~RQ
T~RPSLMDIVGMR--
241
255 256
270
~ E Q Q N T
G~LGIL~P~
Q~J~NSRMQ-EQQ-G G L L G V L N R S Q L ~ P W
Q ~
. . . . . . . . . . . . . . . . ILMPQ
270
262
217
181
195
ALl L A L A E R Q D T E ~
ALVLAI~ERSDT~
ALALAIRDREInV~
196
210
VVPALHEVLPQLYHH
IVPALYEILAQIYHK
IVPAVQELLpELYLD
211
225
QSIIQQVQHLDTGVS
DQVIDEIAHVDTGVS
~"VIQ(~RSVLR-EQ
285 286
300 301
315
VVQLPGQGLLTDDIG
-NVIQQTDR~K-L
VIFLPC.WGLLSEDVG
QNVLGQKRVVVRAm~ Q ~ L L T D D I
E
316
330
5KAYVNVLVDELIVK
LKAYVNLLIDELIVN
5QNFVQNL IQELALL
331
345
QDVVG-RSGDR~
QDVIPQREI~4AL
ED~LEQSQLINND
346
360 361
375 376
390
GTLRGVEGIRG . . . . . . . . . . . . . . . . . . . . . . . . . LRNRIRNTL
RKQQNIQNVLGQKRV V V R A ~ V E G T S L
LTDPIELQNFVRDSN
QRE---EQWVG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . RQR-V
Sat RELHRQM~IRRMAGG
Cal R E P t ~ W a 3 ~ ] ~ G G
Dro REI]~QQNI
406
91
I05
RELLVLVSRND%"I~E
Q
~
SELI~-LIADS
166
180
V V Y A R O ~
VV~ARQNINT~4FVN
VV~7~RNINmr~LVN
136
150
Sat T I S ~ Y
Cal R I~EIHL~Y
Dro
SVGRLEHVQQLRGIY
151
165
~LLVC4U~FEAF~L
RLLVTARDFDTFKRL
RLLARAQDSDTLRRN
761
420 421
435 436
450 451
480 481
495
465 466
DD ..... DIFGGRRT G L ~ D Q D - R T S ~ R
DDDQLNRDSI~GRRL GLDVDELNRNRDINA
DE . . . . . . . . . . . . . . . . . . YQ ........
S a t IX). . . . . . . . . D Q D N ...... ~ I G W N - - - Ib~QR ...... RRI V N T G R ~ L F N V E R S
Cal E R G I W ( ~ G R I N Q D D
NLGGLERNRGVNGED YLNNQQG~ILGGRQG INSDRLLFVGRRQRV
D r o R E . . . . . . . . . . . . . . . . . . . R D L G N D . . . . VD'I~R . . . . . . . . . . P T P C E S A A G L L E Q Q
368
396
310
496
510 511
525 526
548
M~RVGLNR-DDIN . . . . . . . . . . . . . . . . . . . . V Q E R L R ~ Q - - R N
GRRLGLDNEDDLTGN RDINAGRRHVi~IDI ~ % @ ~ G G R R
GQNIYGNQ .... K . . . . . . . . . . . . . . . . . . . . . DRFQRIJ~R -RD
450
531
336
646
660
TD---R~QQNSWQ
V ~ ~ N
~M~LVRGDRLS~GRR
661
675
99TERGDDD---ILG~
KQ~DD{)~TLYDI
VGQLIX3~R .......
566
656
456
765 766
780 781
795
TN~RGR[AMQG ..... G Q R G - D S ~
- F L N V V T R ~ L U Q R Q YIq)QGQDKRFNHG~ GQY~%ESLD~{LFTT
ERLVH INR ..... RR LNQDNQDIQQ . . . . . . . . . . Q~4NFPRRIN
796
810
SIDD~LLY%~RRKL
SINE~%LLHI~%MEL
S IG~GRRRHG -RAPI
651
790
540
856
870 871
885 886
~
.... NRR~4W N~- - -DEENIHVG . . . . . . . . . GRRYRRSL
R~D~NS~YRYRRSL
~W~RNSW~G'Y0
QGLRSRRS5
Q K H Q N .... DDDDDD b i D - - -DDVNVVHR . . . . . . . .
901
915
DQQQQNWRQN~RDE
LN~WQ ...... QLDN
PNLRQ ....... QNN
916
930
TTGINGKLLLHTL4~Q
~SISGKLLLHTLQQ
R--LS-E~Q
931
945
LVARINVERIALGLP
L~IALGQP
5VARLNQESIAQG--
759
917
638
976
990
TNI DITSRVSGQTLN
QGVDV ...... QTAN
NSQRSR ..... QVLA
991
1005
KIEEIMQRID~VLQQ
KIEE I IQRII]~q~Q
QIGQIEQRIQEVIGQ
1006
1020
QVQQGQQ -QTLSATT
RIG ....... DISVM
VLSQVNVNSLRGQVI
1021
1035
NY~DILNQIGLLL~
SYN~VN~IG~
DQRQVESLIADVLI~
1036
1050
QVEDINLVDLMGEVL
QIQDIGLIQILAEIQ
RLGQVGIMTIIRQVV
1051
1065
QHS--ENQLRK~
QQGSL~RKGQVA
Q~SEQIDRNG-I,G
1066
1080
NII~IQILLAGI
889
NIL~ILLAGI
1039
IRLSDPVVQYTLRRI
767
1096
1110
QHITGQ~DININ
EEFIST~RG--VTIN
RQEQLQMRG--VSIN
1111
1125
NVQ IDKLQTYLEQTE
~
~
DVRVDKLRTR I ~ H E
1126
1140
VDLSNL~ENIDLSM
IDLSNLLmaUm~%K
LDLSNLVEQQVQG IQ
1141
1155
~ I V G ~ R I ~
..... EVVGRVPRLN
Q .... EIVGRQRRLN
1156
1170
H][~HIDIEVTSQRQ
HE~FNI~Q
~ % P T II]~DI TSD~D
1171
1185
Q~VPKVDG
QQV%g~RNLLVI~KVDG
QDAIIRIFLGpAEDQ
1186
1200
RG-NTLP~RR~
LG-NIIPLQQRR~
QGRQC.ASLD~RRRDF
1201
1215
ILLDITTVDLH~
1023
IVLDI~PGE{
1166
VLLDAIQVQLENGRN
896
1216
1230 1231
1245
Sat LVKLHS~DITLTGCD T T P Y T K I Y ~
Cal L L ~ I T M T A R D
TTPLT~IYQH~
Dro
R IQRRS ID I P%FFI'SD VTPLVEIYRQVMLQ~.
1246
1260
m~QI~-Q~Q~IC
NGRT~M---QQE~SV
KGQQAQQV%~IQQLV
1261
1275
GgnH~HRLLVR~
G ~ P R G
GENG~HLLLPRG
1276
1290
RVNGLPMQLVTVITP
RINGLRM~LITVITP
RPEGLPMQLL%~VSP
1320
1291
1305 1306
VQNTLNTMGRP- I ~ --LDVLLLDRLPLNY
--LDTLLLDRLPLNY
VQRSAG ~ R G D G
LVELQVQDIVPAITI GIGSASLRDARPLGY
1321
1335
pLHSDITD~
pLRCDITDLDRVV~M
pLDRPI}~TEQELLQL
1336
1350
PNVLVEEVQZYHDDN 1154
FBLMVKDVKII~DON 1296
TNVLLQDVVIIQ~1030
541
555 556
570 571
585
TG . . . . . . . . . . . . . . . . . . . . GRTIMGRN
GGYNNDQLISGRGLR NRLDLD~GQGVGGRN
MG . . . . . . . . . . . . . . RVQLQRGISQGGLR
S a t NI~NDDDDDD-DED~Y
Cal QGI~AESD-IHNIY
D r o D~%~D~)DDDDQDIR
676
690 691
705 706
586
600
--EHQRGRNTVSV~D
QQ~GQNGINTVS%~D
-IGC~&RNLPTVSVNS
720 721
601
615
PRLLFVGRRRINP~
ARLLFVGRRR~I%~]
DRLLHVSRRR---LN
391
405
NVN ......... D~D
RNQ~RDTN
QYQ ......... D~W
735 736
750 751
Sat ~
. . . . . . . . . . . . R R - - M Q N D ~ F U ~ D DD ..... MIQG . . . . . . . . Q ~ D Q Q R - -MG QG .... I G G G H G R ~
Cal R R L ~ T N S E R Y I
N V N R R G ~
EDNLy SNLNRGRNI3M N V N S R Y ~ S D V Y G
~LGRGSU~GRDD
D r o - - - Q E R . . . . . . . . . . . . . . . . . . Q N N W Q K QD-- -YILTQG . . . . . . . . RTFGVQQ - - ER QN-yADQLESVSRDD
811
825
S ~ Z ~
Ca/ SNVE--EGRRVNSRQ
Dro QDEDILKLIRG~k~L
Sar
946
826
840 841
855
DD~DVRRQ
R ....... TRN ....
GDF~DDED~SQTG~Q R ~ n g ~ % G S R L D E Q S
K[/~2DDEIL~(LQRN R ....... QQR---L
960 961
975
KQGILGRIAERRNAN
FNNLRGQISLNRNDI
TQS~RYAL~IRI
S a t QLIDOQS--LNRQQQ
Cal QLIA~RGQL
Dro QLIEEQQQLI~PRL
1081
1095
Sat VKVIDDHVQDLIQNR
Cal VKVI~VYKQR
D r o VRIVDEQREQIDGGY
1351
Sar I QLNLM~LN
Cal IKMPLSHLY
Dro
.........
616
630 631
645
V I ~ g G S N R N Y N T R N YRK)RD{3N~MISGGRR
V%~TSGRN-N~N . . . . . . . . D~DEISGGRR
AIQQDQQQ . . . . . . . . . . . . . . DQLNGRQR
-FLK~HMIRD
1163
1305
1030
FIGURE 2. Multiple sequence alignment of putative arylphorin receptors from Diptera. Sequences were aligned using the
ClustalW algorithm. The arrow indicates the proteolytic cleavage site found in Sarcophaga bullata. Sequences were downloaded from Genbank files: Arylphorin receptor from Calliphora vicina (Genbank sequence ID 630903); storage protein binding
protein from Sarcophaga bullata (Genbank sequence ID 984655); fat body PI protein from Drosophila melanogaster (Genbank
sequence ID 544281).
762
N.H. HAUNERLAND
sequences, the results from both studies become more
congruent. The sequence of both cDNAs contain an
endoplasmic reticulum-targeting sequence at their 5'-end.
Burmester and Scheller (1995b) postulate a single membrane spanning alpha-helix (I 936-L953); in the aligned
sequences of the Calliphora and Sareophaga receptors
(Fig. 2), this region is very well conserved (61% identity,
89% similarity) indicating that the Sarcophaga protein
could also reside in the plasma membrane. The total protein is 90 amino acid residues longer in Calliphora and
the calculated molecular weights correspond fairly well
with the sizes found in SDS electrophoresis. (Table 2).
The cleavage site was determined for Sarcophaga only;
it occurs between R713 and $714. In the aligned
sequences, Calliphora has an analogous sequence at this
site (R824/$825), with a conserved stretch of 6 amino
acids (RYRRSL) present in both sequences (Fig. 2). This
could indeed represent a unique cleavage site for a specific protease. If cleavage occurred here in Calliphora
as well, the resulting fragments would have a molecular
weight of 92 000 and 48 400, respectively (Table l ) and
be equivalent to the 76 and 53 kDa proteins found in
Sarcophaga. In both species, the receptor is inactive
unless 20-hydroxyecdysone is present. Different in the
interpretation of the uptake events is that Chung et al.
(1995) suggested that the cleavage of the 120 kDa protein is prevented by 20-hydroxyecdysone, keeping the
active arylphorin binding protein intact, while Burmester
and Scheller provided evidence that the 96 kDa protein
is activated by 20-hydroxyecdysone, probably through
further cleavage to the active 65 kDa receptor. However,
it should be noted that the postulated membrane-spanning
helix would be located in the smaller fragment (840-859
or 937-953), if cleavage occurs in both species at the
same position, which would not be consistent with a
receptor function of the 96 kDa protein or its 65 kDa
fragment. Moreover, the smaller fragment is much more
conserved than the larger one (Table 2). Since it is rather
unlikely that clearly homologous proteins are subject to
fundamentally different regulation in two closely related
dipteran species, it appears that different experimental
methodologies used in the two studies may be responsible for the contradictory results. While convincing evidence is presented by these and other authors that 20-
hydroxyecdysone is regulating the uptake of arylphorin
in Diptera, it remains unclear how this is actually
accomplished, and whether the arylphorin binding proteins cloned represent membrane spanning endocytotic
receptors. Future work will undoubtedly address these
important questions.
Lepidopteran receptor
To date, storage protein receptors have been studied only
in one lepidopteran species, the corn earworm, Helicoverpa zea (Wang and Haunerland, 1993, 1994a). The
characterization of the receptor and its localization was
directly linked to the two different fat body types
observed in this species (see above). Early observations
had shown that arylphorin as well as the nonhexameric
storage protein VHDL accumulate only in perivisceral
(pupal) but not in peripheral (larval) fat body, indicating
that specific uptake takes place only in the former tissue
(Haunerland et al., 1990). To investigate the possibility
of a receptor for these storage proteins, VHDL and arylphorin were radioiodinated and incubated with a fat body
cell suspension. Both proteins interacted with fat body
cells in a specific, saturable manner; moreover, the binding constants calculated were similar, 7.8 10 ~ for VHDL
and 9.2×10 s for arylphorin (Wang and Haunerland,
1993, 1994a). Bovine serum albumin, in contrast, did not
interact with the membranes and had no influence on the
binding of either VHDL or arylphorin. When membranes
were incubated with labeled arylphorin in the presence
of an excess of unlabeled arylphorin, less radiolabel was
bound by the membrane; similarly, cold VHDL reduced
the binding of labeled VHDL, as expected for a saturable
receptor. In addition, unlabeled arylphorin also reduced
the binding of labeled VHDL and unlabeled VHDL
reduced arylphorin binding, suggesting that a single
receptor mediates the uptake of these two structurally different storage proteins. The binding to both proteins
occurs between pH 6.5 and 7.5 and requires at least
4 mM Ca 2÷, similar to the binding data observed in Calliphora, but the dipteran proteins appear to bind arylphorin more tightly.
In order to purify the receptor protein for VHDL,
membrane proteins extracted from perivisceral fat body
were separated by SDS gel electropfioresis. When the
TABLE 2. Comparison of storage protein receptor fragments
Sarcophaga peregina
Reported Mra
120000
76 000
53 000
Calculated M,~
132 346
81 964
5(/400
Calliphora vicina
Reported M?
Calculated Mfl
130 000
96 000
not detected
"Reported by Chung et al. (19951.
bCalculated from the sequence published by Chung et al. (1995).
~Reported by Burmesterand Scheller (1992).
dCalculated from the sequenced published by Burmester and Schetler (1995b).
~Determined by pairwise alignment with the ALIGN program (Myers and Miller, 19881.
148 255
99 986
48 443
Identityc
46%
40%
55~
STORAGE PROTEINS AND RECEPTORS
sample had been treated only with mild conditions, the
separated membrane proteins retained their affinity to
VHDL. After blotting membrane proteins to nitrocellulose, one protein band could be seen at a molecular
weight of 80 000 that bound labeled VHDL. This band
constituted a major protein band of the membrane
extract. This ligand blot was used as an assay during the
isolation of the VHDL receptor (Wang and Haunerland,
1993). Through as series of chromatographic steps this
receptor protein could be purified to homogeneity. The
receptor is a basic, glycosylated protein of 80 kDa with
a high isolelectric point (pH 8.2). Binding of both storage
proteins in ligand blots was also competitively reduced
by excess of either unlabeled protein, but not by albumin,
confirming that a single storage protein receptor binds
arylphorin and VHDL. While no conclusive evidence
exists for a motif that recognizes two apparently different
storage proteins, it has been proposed that the high mannose carbohydrate structures of both proteins could be
involved, as well as ionic interactions between the highly
positively charged receptor and its negatively charged
ligands. If indeed one storage protein receptor was generally responsible for the uptake of all storage proteins,
then proteins lacking carbohydrates should not be incorporated. However, methionine-rich protein is taken up by
fat body, although it is not glycosylated. It is interesting
to note that the glycosylated riboflavin-binding hexamerin characterized in several Lepidoptera is not
sequestered by fat body; it may be possible that the flavin
prevents binding to the receptor, as it was observed in a
dipteran species by Ueno and Natori (1987; see above).
Antibodies generated against this receptor were used
to analyze its distribution within the insect. The receptor
protein could be found only in perivisceral fat body and
only during the latter half of the last larval instar. With
electron microscopy and immunogold labeling techniques, it was shown that the VHDL receptor mostly
lines the fat body plasma membrane, but is also visible
within protein granules (Wang and Haunerland, 1992).
This observation is consistent with its role as endocytotic
receptor. The receptor appears to be present only briefly,
but at very high concentrations. Antibodies detected
receptor protein in fat body membrane extracts only
between day 4 and 8 of last instar larvae (Wang and
Haunerland, 1993). During this period the receptor seems
to be the most prominent membrane protein, suggesting
that, in contrast to other endocytotic receptors, the lepidopteran storage protein receptor is not recycled, but
incorporated into the protein granules. Indeed, the presence of the receptor in protein granules supports this
notion.
Through a series of in vivo and in vitro uptake experiments with gold-labeled VHDL and arylphorin the
uptake process could be elucidated (Wang and Haunerland, 1994b). The uptake of storage proteins occurs as
illustrated in Fig. 3: storage protein receptor, an integral
membrane protein, is synthesized towards the end of the
last larval instar and positioned in the plasma membrane,
763
"he'm"oly'mph Diptera
Lepidoptera
5L1-5/~
.a
v.<.
•
",11;.
•
-
peripheral FB
,"i;,
P6-10
perivisceral FB
FIGURE 3. Proposed models of storage protein uptake and utilization.
Diptera: model for Calliphora vicina according to Burmester et al.
(1995); Lepidoptera: model for Helicoverpa ~ea according to Wang
and Haunerland (1994). L3-5, L4-8, L6-8: Calliphora larvae 3-5, 48, 6-8 days after hatching, respectively. 5LI-5, 5L5 7, 7: 5th instar
larvae of Helicoverpa zea, 1-5, 5-7, 7 days after the final larval molting; Pt-3, P6-10: pupa I-3, 6-10 days folk)wing the larval-pupal molting. A: arylphorin; R: receptor.
with its storage protein binding site: facing the hemolymph, and possibly a clathrin binding site facing the
cytosol. Once storage proteins have bound adjacent
receptors, they aggregate and, possibly through the action
of intracellular clathrin, form a coated pit. Eventually the
curvature increases until a vesicle containing receptorbound storage proteins buds off the plasma membrane.
Coated vesicles may lose their clathrin coat and fuse with
each other to form multivesicular bodies, which contain
receptor-bound VHDL and arylphorin, receptors, membrane components, as well as a certain proportion of
hemolymph proteins that were enclosed in the vesicle
lumen. Soon these multivesicular bodies fuse with primary lysosomes, whose enzymes will digest membrane
components and the receptor, so that electron dense protein granules can form. These granules are processed
over the next few days, resulting in a gradual digestion
of VHDL, leaving behind only arylphorin that can now
crystallize and is largely protected from proteolytic
digestion. During development of the pharate adult, proteinases partially hydrolyze arylphorin granules, thus
764
N.H. HAUNERLAND
p r o v i d i n g the n e e d e d a m i n o acids; large a m o u n t s of arylp h o r i n r e m a i n in partially digested g r a n u l e s until well
into the adult stage, w h e n a r y l p h o r i n m a y serve as an
a m i n o acid reserve for y o l k protein production.
Certainly, there are m a n y u n a n s w e r e d q u e s t i o n s with
regard to the receptor in H. zea. W h a t is the c o m m o n
m o t i f that is r e c o g n i z e d by the receptor? W i l l other storage proteins, for e x a m p l e the m e t h i o n i n e - r i c h proteins,
b i n d to the s a m e receptor? Is this receptor c o m m o n to
other lepidopteran species as well? A n d is there any
relationship b e t w e e n the lepidopteran a n d d i p t e r a n storage protein receptors? T h e latter appears rather u n l i k e l y ,
in view of the different size, processing, control mecha n i s m s and life history, as illustrated in Fig. 3. C o n s i d e r ing that storage proteins m a y have e v o l v e d i n d e p e n d e n t l y
after L e p i d o p t e r a a n d D i p t e r a had diverged, it m a y well
be p o s s i b l e that different a d v a n t a g e o u s uptake m e c h a n i s m s have also d e v e l o p e d i n d e p e n d e n t l y . E v i d e n c e
s u p p o r t i n g or c o n t r a d i c t i n g this hypothesis, h o w e v e r , can
be o b t a i n e d o n l y after c l o n i n g a n d s e q u e n c i n g o f the lepid o p t e r a n receptor a n d the characterization of storage protein receptors in other insect species.
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Aeknowledgements--I am indebted to Professor Dr Klaus Scheller and
Dr Thorsten Burmester for providing preprints of their papers. Work
from my laboratory cited in this review was funded in part by grants
from the Natural Science and Engineering Research Council of Canada.
Note added in proof--Burmester and Scheller (1996) have recently
published a detailed plylogenetic analysis of hexamerins and arylphofin receptor. J. Mol. Evol. 42, 713.