cDNA Cloning of a Developmentally
Regulated Hemocyanin Subunit in the
Crustacean Cancer magister and Phylogenetic Analysis of the Hemocyanin
Gene Family
Gregor Durstewitz
and Nora Barclay Terwilliger
Oregon Institute of Marine Biology, Charleston;
and Department
of Biology, University
of Oregon
The complete cDNA sequence and protein reading frame of a developmentally
regulated hemocyanin subunit in
the Dungeness crab (Cancer magister) is presented. The protein sequence is aligned with 18 potentially homologous
hemocyanin-type
proteins displaying apparent sequence similarities. Functional domains are identified, and a comparison of predicted hydrophilicities,
surface probabilities, and regional backbone flexibilities provides evidence for
a remarkable degree of structural conservation
among the proteins surveyed. Parsimony analysis of the protein
sequence alignment identifies four monophyletic
groups on the arthropodan branch of the hemocyanin gene tree:
crustacean hemocyanins,
insect hexamerins, chelicerate hemocyanins,
and arthropodan prophenoloxidases.
They
form a monophyletic
group relative to molluscan hemocyanins and nonarthropodan
tyrosinases. Arthropodan prophenoloxidases,
although functionally similar to tyrosinases, appear to belong to the arthropodan hexamer-type
hemolymph proteins as opposed to molluscan hemocyanins and tyrosinases.
Introduction
Hemocyanins
and related copper proteins are ancient molecules. They probably arose about 1.6 billion
years ago (BYA) when the earth’s atmosphere changed
from a reducing to an oxidizing environment.
Prior to
that time, most of the earth’s available copper (Cu) was
precipitated in insoluble sulfides, CuS and Cu,S (Ochiai
1983), and was therefore virtually inaccessible to living
organisms. When oxygen-producing
photosynthesis
began to increase about 2 BYA, significant amounts of Cu
were oxidized to Cu2+. In this form, it was readily dissolved in aquatic systems, distributed in the biosphere
and therefore became available to living organisms.
Hemocyanin
is the oxygen transport protein of
many arthropods
and molluscs.
It occurs freely dissolved in the hemolymph.
Although similar in function
to molluscan hemocyanins,
arthropodan hemocyanin
is
radically different in molecular architecture. Molluscan
hemocyanin
subunits are multidomain
polypeptides
of
about 350-450
kDa, containing
7-8 functional
units,
each of which contains two Cu-binding
sites and combines reversibly with oxygen. The subunits form cylindrical macromolecules
of 3,500-4,500
kDa and higher
multiples depending
on the species. Arthropodan
hemocyanin is composed of heterogeneous
subunits with
molecular weights of about 75 kDa. These subunits selfassemble into 450~kDa hexamers or multiples thereof.
Each subunit contains two Cu-binding
sites, CuA and
CUB, that together reversibly bind one molecule of oxygen
The evolutionary
relationship
between the hemocyanins has long been the topic of speculation:
are arthropodan
and molluscan
hemocyanins
homologous
gene products or the result of convergent
evolution?
They are very different in sequence as well as in subunit
Key words: hemocyanin,
respiratory proteins,
magister,
parsimony analysis,
prophenoloxidases.
phylogeny,
Cancer
Address for correspondence
and reprints: Dr. Nora B. Terwilliger,
Oregon Institute of Marine Biology, 4619 Boat Basin Drive, Charleston, Oregon 97420. E-mail: [email protected].
Mol. Biol. Evol. 14(3):26&276.
1997
0 1997 by the Society for Molecular Biology and Evolution.
266
ISSN: 0737-4038
structure and composition (van Holde and Miller 1995),
but there is good evidence for a common origin of at
least part of their active site (Drexel et al. 1987). On the
basis of sequence comparisons, other potential members
of a putative hemocyanin
gene family have recently
been identified. These include tyrosinases (Lerch et al.
1986), prophenoloxidases
(Aspan et al. 1995), and insect
storage proteins or hexamerins
(Munn and Greville
1969; Telfer and Massey 1987). The latter do not bind
Cu. Other Cu proteins like the plastocyanins,
Cu-dependent cytochrome c oxidase, ceruloplasmin,
azurins, laccase, ascorbate oxidase, or Cu-dependent
superoxide
dismutase show no structural or sequence similarity with
the hemocyanin
family (Markl and Decker 1992).
In this paper we present the complete cDNA and
protein sequence of a developmentally
regulated hemocyanin
subunit from a brachyuran
crustacean,
the
Dungeness
crab Cancer magister. In brachyurans,
hemocyanin occurs in the hemolymph predominantly
as a
two-hexamer
molecule. Although subunit sequences of
chelicerate hemocyanins
and hexameric crustacean hemocyanins have been reported (Linzen et al. 1985; Beintema et al. 1994), subunit sequences of multihexameric
crustacean
hemocyanins
have been unavailable
up to
now. This protein, Cmag6, is the first Cu-based respiratory protein described whose expression appears to be
developmentally
regulated. In adult C. magister, onehexamer Hc is composed of subunits 1, 2, 4, 5, and 6,
while the two-hexamer
Hc contains subunits 1, 2, 3, 4,
5 and 6. The megalopa and early juvenile crab Hcs lack
subunit 6 until about the sixth juvenile instar (Terwilliger and Terwilliger
1982). This is the same time at
which Cmag6 mRNA is first detectable in hepatopancress tissue (Durstewitz and Terwilliger 1997). The appearance of subunit Cmag6 correlates with a change in
oxygen-binding
properties; under the same experimental
conditions, the oxygen affinity of adult purified two-hexamer Hc is about 50% higher than that of juvenile twohexamer Hc lacking Cmag6 (Terwilliger
and Brown
1993). Once expression of subunit Cmag6 is initiated,
it persists for the rest of the crab’s life.
Phylogeny
In this study, we also aligned 18 potentially
homologous proteins with Cmag6 for investigation
of potential conserved structural features and for parsimony
analysis. The combination
of both structural and sequence comparisons is used to shed light on the evolutionary relationships
among hemocyanin-type
proteins.
Materials and Methods
Isolation of Total RNA from Cancer magister
Hepatopancreas
Fresh tissue samples (100 mg) from adult male C.
magister hepatopancreas
tissue were rinsed with C. magister hemolymph
buffer (50 mM Tris-HCl, 454 mM
NaCl, 11.5 mM KCl, 13.5 mM CaC12, 18 mM MgC12,
23.5 mM Na2S04, pH 7.6; Terwilliger and Brown 1993),
frozen in liquid nitrogen, and ground to a fine powder
with mortar and pestle. Total RNA was isolated with the
guanidinium
isothiocyanate
method using a RAPID Total RNA Isolation Kit (5 Prime + 3 Prime, Inc.). Total
RNA yield was quantified by measuring absorbance at
260 nm.
Reverse Transcription
(PCR) Amplification
Sequences
and Polymerase Chain Reaction
of Hemocyanin
Coding
In an eppendorf tube, 1 l.~l total RNA (1 pg/bl)
was diluted with 10.65 pl autoclaved water, and 0.75 ~1
oligo dT-primer (0.27 pg/pl) was added. The mixture
was incubated for 3 min at 65°C and then was allowed
to cool down to room temperature. Next were added (in
this order): 4 pl 5 X reverse transcription
buffer (250
mM Ti-is-HCl, pH 8.5; 200 mM KCl; 30 mM MgC12),
1 ~1 20 mM dithiothreiotol
(DTT), 1 l.~l 25 mM dNTPs,
1 ~1 RNAsin (10 U/p,l), and 0.6 I_L~AMV reverse transcriptase (17 II/@). The reaction was incubated at 42°C
for 90 min and then diluted to a total volume of 500 l~11.
A lo-p1 aliquot of this reverse transcription reaction was
added to 18.5 pl water, 5 pl 10 X PCR-buffer (670 mM
Tris-HCl), 4 ~12.5 mM dNTPs, 5 ~1 10 X bovine serum
albumin (1 Fg/pl), 1 ~1 of each primer (0.2 p&l),
0.5
~1 Taq polymerase (5 U/pi), and 5 ~1 40 mM MgC12.
PCR was carried out using the following protocol: (denature: 94°C for 40 s; anneal: 55°C for 40 s; polymerize:
72°C for 1 min) * 35 cycles, then 5 min at 72°C and
hold at 4°C. Ten-microliter
aliquots of each reaction
were analyzed
on 1.2% agarose Tris-acetate/EDTA
(TAE) minigels.
Cloning
cDNA
and Sequencing
of PCR-Amplified
Cmag6
Unless indicated otherwise, the following procedures were performed in accordance with Sambrook,
Fritsch, and Maniatis (1989). PCR products (40 pl total)
were separated by size through electrophoresis
on 1.2%
agarose TAE maxigels. Bands of interest were excised
under UV-light and purified in a glassmilk procedure
(GENECLEAN
II kit, Bio 101, Inc.). Ends were repaired
with Klenow polymerase and 5’ ends were phosphorylated with T4 polynucleotide
kinase. A Bluescript II
SK+ vector (Stratagene) was cut with restriction endonuclease Sma I and dephosphorylated
using calf intes-
of Hemocyanin
Gene Family
267
tinal phosphatase (CIP). In a total volume of 20 ~1, the
inserts were blunt-end-ligated
into 50 ng vector DNA in
a molar ratio insert/vector of 3: 1, using 1 Weiss unit T4
DNA ligase. Ligation occurred overnight at 16°C. Competent Escherichia
coli XL-l Blue cells were transformed with 50 ng ligated DNA and plated out on
LB-Amp plates. Positive clones were selected and DNA
was isolated in alkaline lysis minipreps. When desired,
inserts were excised and analyzed using restriction enzymes EcoRV and Xba I. cDNA inserts were sequenced
manually by standard walking, using the dideoxy method with a SEQUENASE
2.0 kit (U.S. Biochemical) and
radioactive 35S-labeled nucleotides (NEN-Du Pont). T3
and T7 were used as initial sequencing primers. To provide generous overlap between the sequenced parts, further primers were designed as 17mers based on stretches
of cDNA sequence located approximately
at the -50 bp
position relative to the end of the known region.
Screening a cDNA Library of Cancer magister
Hepatopancreas
Tissue and Sequencing of CmagX
A cDNA library of adult C. magister hepatopancress tissue was created as described before (Terwilliger
and Durstewitz 1996) and screened with a 32P randomprime-labeled
783-bp C. magister hemocyanin-specific
probe (Durstewitz and Terwilliger 1997). Positive clones
were analyzed for insert size and partially sequenced to
identify them as hemocyanin
coding sequences.
An
l,SOO-bp insert (CmagX) was sequenced by creating
overlapping
nested deletions: the clone containing
the
CmagX cDNA fragment was digested with DNase I in
the presence of Mn2+ (Lin, Lei, and Wilcox 1985; Terwilliger and Durstewitz 1996). The resulting population
of plasmids contained overlapping nested deletions and
was sequenced with the dideoxy method.
Results
The complete cDNA sequence coding for C. masubunit 6 (Cmag6) was amplified in
gister hemocyanin
two overlapping
fragments by PCR. The template was
first-strand
cDNA derived from hepatopancreas
total
RNA. The four primers used to amplify hemocyanin
coding sequences were (1) a degenerate primer based on
the unique N-terminal amino acid sequence of C. magister hemocyanin
subunit 6 (5’ TCT-GCA-GGC-GGAGCG-TIC-GAC-GCG-CA
3’, “5’ sub 6 primer”), (2)
an antisense primer based on the 3’ PCR product (5’
CAC-TGC-CTG-GGG-ATC-GAA-GCC-CTC-ATG
3’)
“CuA II primer”), (3) a degenerate primer based on a
conserved sequence within the copper A site (CuA) of
arthropodan hemocyanin (5 ’GAA-C’IT-TIT-TIT-TGGGTT-CAT-CAT-CAA-CTT-AC
3’) “CuA I primer”;
Bak and Beintema 1987), and (4) a universal oligo-dT
primer (see Terwilliger and Durstewitz 1996, fig. 2). Using the primer combinations
1 and 2 in one PCR reaction plus 3 and 4 in another, the experiments generated
two overlapping
cDNA fragments. Each fragment was
blunt-end-cloned
into a Bluescript II SK+ vector and
sequenced. One fragment coded for the 5’ end, the other
one for the 3’ end of hemocyanin
subunit 6. The iden-
268
Durstewitz
and Terwilliger
GCAGCACGATGTCAACAGCGCTCTGTGGAAGGTCTACGAG
1 ---------+---------+---------f------------~---_-_---~---------~-________~_________~
AGACGTCCGCCTCGCAAGCTGCGCGTCTTCGTCGTGCTACAGTTGTCGCGAGACACCTTCCAGATGCTCCTATAGGTCCT
TAGGAFDAQ
KQHDVNSALWKVYEDIQD
TCCCCACCTAATACAACTTTCCCAGAACTTCGACCCGCTCGACCCGCTCTCCGGCCACTATGACGACGATGGTGTCGCCGCC~GCGCC
81 ---------+---------+-------__+_________+---------+---------+---__---_+_________+
AGGGGTGGATTATGTTGAAAGGGGTCTTG~GCTGGGCGAGAGGCCGGTGATACTGCTGCTACCACAGCGGCGGTTCGCGG
P
H
L
I
Q
L
S
Q
N
F
DPLSGHYDDDGVAAKRL
TCATG~GGAGCTCAACGAAAACCGCTTGCTTGCTG~GCAG~CCACTGGTTCTCACTGTTC~CACCCGCCAGCGCGAGGAG
161 _---_-_--+---------+---------+------------~_________~_--------~---------~_____-___~
AGTACTTCCTCGAGTTGCTTTTGGCGAACGACTTCGTCTTGTTGTGGGCGGTCGCGCTCCTC
M
K
E
LNENRLLKQNHWF
SLFNTRQREE
GCTCTCATGCTCTACGACGTCCTCGAACACTCCACTCCACAGACTGGAGCACCTTCGCCGGC~CGCTGCCTTCTTCCGCGTTAG
241 -------_-+---------+---------+--_______+----_----+---------+---------+---------+
CGAGAGTACGAGATGCTGCAGGAGCTTGTGAGGTGTCTGACCTCGTGG~GCGGCCGTTGCGACGG~G~GGCGC~TC
ALMLYDVLEHSTDWSTFAGNAAFFRVS
CATGAACGAGGGCGAGTTCGTTTACGCACTGTACGCTGCCGTTATCCACTCTGAGCTGACAC~CACGTGGTGCTACCAC
321 _________+________-+---------+------------~---------~---------~-------__~_________~
GTACTTGCTCCCGCTCAAGCAAATGCGTGCGTGACATGCGACGGC~TAGGTGAGACTCGACTGTGTTGTGCACCACGATGGTG
MN
E
G
E
F
VYA
L
YAAV
I H
S
EL
T
Q
Hl7VL
P
P
CCCTCTACGAGGTCACTCCTCACCTCTTCACCAACAGCGAATGACCCAGACT
401 ---------+__-----_-+--_-_---_+---_--------~_________~_________~_________~-----_---~
GGGAGATGCTCCAGTGAGGAGTGGAGAAGTGGTGGTTGTCGCTCCACTAGGTTCTTCGGATGTTTCGGTTCTACTGGGTCTGA
LYEVTPHLFTNSEVIQ
EAYKAKMTQT
GCCGCCAAGATTGAGTCCCACTTCACCGGCAGCAAGAGTACATCGG
481 ---------+---------+----_-_-_+---------+_________+_________+_________+_________+
CGGCGGTTCTAACTCAGGGTGAAGTGGCCGTGGCCGTCGTTCTCATTGGGCCTTGTCGCACACCGGATG~GCCGCTCCTG_TAG~C
AA
K
I
E
S
H
F
T
G
SK
S N
P
E
Q
RVAYF
G
EIY
*
*
EB
CATGAATACCCATCACGTCACCTGGCATTTGGAGTTCCCCTTCTGGTGGGACGACGCCCATGAG~CCACCACATCGAGC
561 _______--+---------+---------+-----------_~---------~---------+---------~_-_______~
GTACTTATGGGTAGTGCAGTGGACCGTAAACCTCAAGGGGG~GA~~A~~~~T~C‘T~C~~~TA~TCTTGG~TG~~~TAG~~CG
H E N Hx H? . I
&&<
\T,, Fr,_+g V ,%l W ,li -L 43 E: Et"'i?l W 'VP D D> A
Primer 3 +
E
$i
*
GCAAGGGC~AGAGCTGTTCTTCTTGGGTCCACCACCAGC?CTACTTG
641 _________+_________+_--------+------______~---------~---------~---------~---------+
CGTTCCCGCTCTCGACAAGAAGAACCAGGTGGTGGTCGAC
K
G
E ':s e& "S
5: W
V 'a H'"Q
721
L
$s'"*V :R
F, D
,A
E
-kc &,
S
N‘ y
L
---------+---------+_________+--_-CTAGGGCAGCTGCTTGAGGTGACCCTGCTACAGTA
DPVDELHWDDVIHEGFDPQAVYKYGGY
TTTCCCCTCCCGCCCTGACATATCCACTTTGAAGATGTGTGGATGGTGTTGCTGATGTTCGTGACATGCTTTTGTATG~G
801 _________+________-+---------+---------+---------+---------+---------+-----____+
AllAGGGGAGGGCGGGACTGTTATAGGTGAAACTTCTACACCTACCACAACGACTACAAGCACTGTACGAAAACATACTTC
FPSRPDNIHFEDVDGVADVRDMLLYEE
AACGTATTCTTGACGCTACTGCTCATGGCTACGTGCGGATCAACGGTCAGATCAGATCGTTGACCTGAGAAACAATGATGGCATC
881 ---------+---------+--------_+-----___-+---------+---------+---------+---------+
TTGCATAAGAACTGCGATGACGAGTACCGATGCACGCCTAGTTGCCAGTCTAGC~CTGGACTCTTTGTTACTACCGTAG
IVDLRNNDGI
RILDATAHGYVRINGQ
*
GATCTCCTTGGAGACGTGATTGAATCTTCTTCCTTATACAGCCCC~TCCTCAGTACTACGGCGCCCTGCAC~CACAGCTCA
961 ---_-----+---------+_--______+-------_-+---------~---------~---------~---------~
CTAGAGGAACCTCTGCACTACTTAGAAGGAATATGTCGGT
S
L
Y
S
P N
P
Q
Y
Y
G
A
L
Bt.N
DLLGDVIES
*
.%&A
B'x
FIG. I.-Cancer
magisfer hemocyanin subunit 6; cDNA sequence and correct protein reading frame. Shaded areas, Cu-binding sites CuA
and CUB. Asterisk indicates conserved histidine, presumably acting as copper ligand. Boxes indicate PCR primers. Boxed region labeled “primer
3” marks annealing site for universal crustacean CuA primer based on CuA site of Punulirus interruptus hemocyanin subunit a.
tical overlap between both clones was 133 bp. The complete cDNA sequence of C. magister hemocyanin
subunit 6 (GenBank accession number U48881) with the
correct protein reading frame (Cmag6) is shown in figure 1.
The subunit is composed of 650 amino acid residues. The six histidines marked by an asterisk in figure
1 have been implicated in Cu binding and are highly
conserved
among other arthropodan
hemocyanin
subunits (Linzen et al. 1985; Beintema et al. 1994). The
molecular weight of subunit 6, calculated from the amino acid residues, was determined to be 74,903 Da, as
opposed to an estimate of 67,300 Da, based on its mo-
bility on SDS-PAGE gels, by Larson et al. (1981). The
subunit is acidic (isoelectric point [PI] = 5.02), and a
glycosylation
site (Asn-Thr-Ser)
occurs at residue 600.
The sequence of another putative hemocyanin subunit, CmagX, was obtained from a C. magister hepatopancreas cDNA library. We call it a “putative hemocyanin subunit” for three reasons: (1) it was obtained
through a cDNA library screen with a C. magister hemocyanin-specific
probe, (2) it shows an extremely high
degree of sequence similarity with Cmag6 (85% sequence identity), and (3) all of its potential Cu ligands
are conserved.
It is unknown, however, which hemocyanin subunit, if any, this clone represents, whether it
Phylogeny
of Hemocyanin
Gene Family
269
TATGATGCTTGGCCGCCAGGGTGACCCTCATGGAAAGTTCGACCTTCCTCCCGGTGTTCTGGAGCACTTCGAGACCGCAA
--_____--+---______+_____--__+_________+_________+_________+_____--__+____-__--+
GTGCACTAGGGCG~G~GGCAGATGTGTTCATGTACCTATTGTAG~GTCTTTTGTGTTCCTGTCGGACGGTGGGAT~
R :;b fi+ A‘,>E ,F .R
L 8
X
Y
M;,?b“‘N
I,\~$?:'k K
H
K
D
S
1281
1361
1441
1521
1601
1681
1761
1841
1921
L
p
CTTCGAGTACAGTCTTGTGAATGCTGCTGTTGACGACACAG~GATGTCGATGACGTGGATATCTTCACGTATATTTCACGCT
________-+------___+____-----+--_______+--------_+__-------+____-----+_________+
GAAGCTCATGTCAGAACACTTACGACAACTGCTGTGTCTTCTACAGCTACTGCACCTATAGAAGTGCATATAAAGTGCGA
F
E
Y
S
LVNAVDDTEDVDDVDIFTYI
P
S
Y
R
L
TGAATCATAAGGAATTTTCATTTGTTGGTGATGTCACCAAGCCACTGTGCGCATCTTT
________-+------___+_________+-----____+________-~-________~_________~_________~
ACTTAGTATTCCTTAAAAGTAAACAACCACTACACTACAGTGGTTACTTG~CTAGTACTACATGATCGGTGACACGCGTAG~
NHKEFSFVGDVTNELDHDVLATVRIF
GCCTGGCCGCACGAGGACAACAATGGAGTGGAGTGGCTGTT
---------+__-___-_-+_________+____---__+_________+-________+-----__--+---------+
CGGACCGGCGTGCTCCTGTTGTTACCTCACCGCAAGTCGA
AWPHEDNNGVAFSFNDGRWNAIEMDKF
CTGGGTTATGTTGCATCCCGGCCACAACCACACATCGAGCGATCGTCTCATGACTCCTCCGCGACCGTTCCTGATATACCCA
-_______-+------___+_________+__--_____+____----_+_________+-_--_____+____-----+
GACCCAATACAACGTAGGGCCGGTGTTGGTGTAGCTCGCTAGCAGAGTACTGAGGAGGCGCTGGC~GGACTATATGGGT
WVMLHPGHNHIERSSHDSSATVPDIPS
GCTTCCAAATCATTAAGGACAGGACCAATGAAGCGATAGCGATAGCTCAG~C~GG~CTCCATATTG~G~TTTG~GCGGT
--______-+------___+_________+---______+_______-_~_________~----____-~---------~
CGAAGGTTTAGTAATTCCTGTCCTGGTTACTTCGCTATCGAGTCTTGTTCCTTGAGGTAT~CTTCTT~CTTTCGCCA
EEFESG
F
Q 1 IKDRTNEAIAQNKELHI
CTTGGCCTGCC~CAGGTTCCTCATTCCCAAGGGGC~TGTG~GGGCCTTGACATGGATGT~TGGTGGCCATCACGAG
--------_+--___-___+_________+---__-___+--------_~______---~----____-~---------~
GAACCGGACGGTTTGTCCAGAGTAAGGGGTTCCCGTTACACTTCCCGG~CTGTACCTACATTACCACCGGTAGTGCTC
LGLPNRFLIPKGNVKGLDMDVMVAITS
CGGAGAGGCGGATGCTGCCGTTGAAGGGGTTGCACG~CACTTCCTTC~CCACTACGGCTGTCCTGACGGCACCTACC
________-+------___+_________+-----____+_________~________-~----____-~---------~
GCCTCTCCGCCTACGACGGCAACTTCCCC~CGTGCTTTTGTG~GG~GTTGGTGATGCCGACAGGACTGCCGTGGATGG
GEADAAVEGLHENTSFNHYGCPDGTYP
CAGACAAGAGGCCCCACGGTTACCCACTGGACCGCCACGTCGACGATGAGCGCATCATC~TGACTTGCAC~CTTC~G
-----___-+------___+_--------+---------+-------__+---------+---_____-+---------+
GTCTGTTCTCCGGGGTGCCATGGGTGACCTGGCGGTGCAGCTGCTACTCGCGTAGTAGTTACTG~CGTGTTG~GTTC
I ND
L
H
DKRPHGYPLD
RHVDDERI
N
F
K
CACATTCAGGTCAAGGTGTTCCATCATGCG
------_--+---------+-_____--_+
GTGTAAGTCCAGTTCCACAAGGTAGTACGC
HIQVKVFHHA
FIG. 1 (Continued)
might even code for a crustacean storage protein or prophenoloxidase,
or whether it reflects an error in reverse
transcription
or second-strand
cDNA synthesis. Aligned
with Cmag6, its 484-amino-acid
open reading frame
shows a 191-residue deletion between Cmag6 residues
4 10 and 601. This extensive deletion extends from the
C-terminal
part of domain 2, beyond the second Cubinding site (CUB), well into domain 3. However, all
putative Cu-binding
histidine residues are preserved. In
addition to that, it shows a typical signal peptide of 21
hydrophobic
residues at the N-terminal end, indicating
that the gene product is targeted for secretion. This
CmagX sequence and the as yet unpublished
sequence
of Penaeus vannamei hemocyanin
subunit 1 (GenBank
accession number X82502) are the first examples of hemocyanins
with leader sequences (fig. 2). The hemocyanin e gene in the spider Eurypelma contains no sequence coding for a signal peptide (Voll and Voit 1990);
whether other arthropodan hemocyanin subunits (including Cmag6) contain a signal peptide is not known.
A protein sequence alignment of C. magister hemocyanin subunit Cmag6 as well as CmagX with other
members of the hemocyanin
family is shown in figure
2. Alignment
was done manually. It was our goal to
include in our analysis representatives
of all major
groups thought to be within the hemocyanin
family of
proteins. Among these, the 02-transporting
hemocyanins
of arthropods and molluscs are respiratory proteins. Tyrosinases and prophenoloxidases
(Lerch et al. 1986),
both binuclear copper proteins, are enzymes involved in
dopa and melanin biosynthesis,
catalyzing the hydroxylation of monophenols
and the oxidation of diphenols.
Recent studies (Aspan et al. 1995) assign prophenoloxidases a key role in the arthropodan immune system.
Another group of proteins, the hexamerins, is found in
insect hemolymph. One of several functions assigned to
270
Durstewitz
Cmag6
CmagX
Pintc
Pinta
Penvl
LimII
Euryd
Eurye
Anda
BombA
MsexA
Tni_M
BombM
PapPO
DrpPO
Octoe
Hpomd
NeuTy
HumTy
Cmag6
CmagX
Pintc
Pinta
Penvl
LimII
Euryd
Eurye
Anda
BombA
MsexA
Tni_M
BombM
PapPO
DrpPO
Octoe
Hpomd
NeuTy
HumTy
and Terwilliger
. . . .. . . . . . . . . . . . . . . . . . . .ADCQAGDSADKLLAQKQHD~L~KLYGDIRDDHLKELGETFNPQGDLLLYHDNGAS~TL~DFK~RLLQKKH
MRvLwL:;;LvAA::::::::::::::.
..DALGTGNAQKQQDINHLLDKIYEPTKYPDLKEIAENFNLLEQRH
.AAFQVASADVQQQKDVLYLLNKIYGDIQDGDIQ~DLLAT~SFDPVG~GSYSDGG~VQKLVQD~DGKLLEQKH
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .TLHDKQIRVCHLFEQLSSATVIGD...........GD..........KHKHSDRL~GKLQPGA
. . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . .TIADHQARILPLFKKLTSLSP...........DPLP...........EAERDPRLKGVGFLPRGT
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .PDKQKQLRVISLFEHMTSIN............TPLP...........RDQID~LHHLGRLPQGE
. . . . . . . . . . . . . . . . . . . . . . . . . . ..I.... TVADKQARLMPLFKHLTALTR...........EKLP...........LDQRDERLKGVGILPRGT
MKSVLILAGLVAVALSSAVPKP.. .STIKSKNvDAvFvEKQKKILSFFQDVSQLNTDDEWKIGK
MKTWILAGLVALALSSAVPPPKYQHHYKTSPVDAIFVEKQK~SLF~QLDY~EYYKIGKDYD~~.ID~S~~DFLLL~TG.FMPKGF
MRVLVLVASLGLR..GSVVKDDTTWIGKDNMVTMDIKMKELCILKLLNHILKL~ILQPT~DDIREV~E~IE~.MDKYLKTD~KFIDTF~G.~PRGE
MRVLVLLACLAAASASAISGGYGTMVFTKEPMVNLDMKMKELCIMKLLDHILQPTMFEDIKEIAKEYNIEKS.CDKYMNVDVVKQFMEMYKMG.MLPRGE
MQVTQKLLRRDTE....................
.MADAQKQL..LYLFERPYDPINAPRADGSFLYAVAGAXTLLG
MTNTDLKALELMFQRPLEPAFT..........
..TRDSGKTVLELPDSFYTDRYRNDTEEVGNRFSKDVDLKQFSLFN
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . .. . . . . . .. . . . . . .. . . . . . . . . . .. . . .. . . . . . . . . . . . . . . . . . . . .
......................................
......................................
......................................
WFSLFNTRQREEALMLYDVLEHSTDWSTFAGNAAFFRVSM
........ ..NEGEFVYALYAAVIHSELTQHVVLPPLYEVTPHLFTNSEVI....QEAY.
WFSLFNTRQRKEALMLYDVLEHSTDWSTFAGNAAFFRVHM
........ ..NEGEFVYALYAAVIHSELTQHWLPPLYEVTPHLFTNSEVI....QEAY.
WFSLFNTRQREEALMMHRVLMNCKNWHAFVSNAAYFRTNM
........ ..NEGEYLYALYVSLIHSGLGEGVVLPPLYEVl'PHMFTNSEVI....HEAY.
WYSLFNTRQRKEALMLFAVLNQCKEWYCFRSNAAYFRERM
........ ..NEGEFVYALYVSVIHSKLGDGIVLPPLYQITPHMFTNSEVI....DKAY.
WFSLFNTRHRNEALMLFDVLIHCKDWASFVGNAAYFRQKM
........ ..NEGEFVYALYVAVIHSSLAEQWLPPLYEVTPHLFTNSEVI....EEAY.
IFSCFHPDHLEEARHLYEVFWEAGDFNDFIEIAKEARTFV
........ ..NEGLFAFAAEVAVLHRDDCKGLYVPPVQEIFPDKFIPSAAI....NEA
F
LFGSFHEEHLAEAIVFIEIIHDAKNFDDFLALATNARAW
........ ..NEGLYAFAMSVALLSRDDCNGWIPPIQEVFPDRFVPAETI....N'RAL.
LFSCFHEEDLEEATELYKILYTAKDFDEVINLAKQSRTFV
........ ..NEGLFVYAVSVALLHRDDCKGIWPAIQEIFPDRFVPTETI....NLAV.
LFSCFHARHLAEATELYVALYGAKDFNDFIHLCEQARQIV
........ ..NEGMFVYAVSVAVLHREDCKGITVPPIQEVFPDRFVPAETI....NRAN.
EFSVFYDKMRDEAIALLDLFYYAKDFETFYKSACFARVHL
........ ..NQGQFLYAFYIAVIQRPDCHGFWPAPYEVYPKMFMNMEVL....QKIY.
EFSIFYERMREEAIALFELFYYAKDFETFYKTASFARVHV
........ ..NEGMFLYAYYIAVIQRMDTNGLVLPAPYEVYPQYFTNMEVL....FKVD.
VFVHTNELHLEQAVKVFKIMYSAKDFDVFIRTACWLRERI
........ ..NGGMFVYALTACVFHRTDCRGITLPAPYEIYPYVFVDSHII....NKAF.
TFVHTNELQMEEAVKVFRVLYYAKDFDVFMRTACWMRERI
........ ..NGGMFVYAFTAACFHRTDCKGLYLPAPYEIYPYFFVDSHVI....SKAF.
RAPSVPRGAVFSFFIRSHREDLCD~~TQNSTDLMQ~S~R~.NENLFIYALSFTILRKQELRG~LPPILE~PHKFIPMEDLTSMQ~~
NRHREIASELITLFMSAPNLRQFVSLSVYTKDRVNPVL
.............. ..FQYAYAVAVAHRPDTREVPITNISQIFPSNFVEPSAFRDARQEAS
V
.........................................................................
EGNEYLVRKNVERLSLSEMNSLIHAFR
.......................................................................
DAVTVASHVRKDLDTLTAGEIESLRSAFL
.STDIKFAITGVPTTPSSNGAVP.LRRELRDLQQNYPEQFNLYLLGLRDF
APL~~~F~~T~~DDRESWP~~~~~~~~~~~~~~~~~~~~~~~~~~~~~TE~LL~IFDLSAPEKDKFFAYLT~KHTISSD~IPIGTYGQM~G
230
Cmag6
CmagX
Pintc
Pinta
Penvl
LimII
Euryd
Eurye
Anda
BonibA
MsexA
Tni_M
BombM
PapPO
DrpPO
Octoe
Hpomd
NeuTy
HumTy
-Cmag6
CmagX
Pintc
Pinta
Penvl
LimII
Euryd
Eurye
Anda
BombA
MsexA
Tni_M
BombM
PapPO
DrpPO
Octoe
Hpomd
NeuTy
HumTy
I
FDAERLSNYLDPVDELHW.DDVIHEGFDPQAWK.YGCYFPD....
FDAERLSNYLDPVDELHW.DDVIHEGFAPHTMYK.YGGYFPS.RPDNVHFEDVDGVARVRDMLILESRIRDAIAHGYVTGRT....GSIISISDSH....
YDAERLSNHLDPVEELSW.NIDEGFAPHTAYK.YGGYFPS.RPD~FSD~GV~~DMSMTEDRIRDAIAHGYID~D....GSHIDI~SH....
FDFERLSNWLDPVDELHW.DRIIREGFAPLTSYK.YGGEFPV.RPDNIHFEDVDGVA~DLEITESRIHEAIDHGYITDSD....GHTIDIRQPK....
FDAERLSNYLDPVGELQW.NKPIVDGFAPHTTYK.YGGQFPA.RPD~FEDVDDV~IRD~I~SRIRDAIAHGYIVDSE....GKHIDISNEK..~~
YDCERLSNGMHRMLPFNN.FDEPLAGYAPHLTHV.ASGKYERILDSIHLG~ISED....GSHKTLDELH....
YDCERLSVGLQRMLPFQN.IDDELEGYSPHLSSL.VSGLSYGSRPAG~LRD.INDCSVQ.MER~ERILDAIHTGL~DSH....GKEIKITEEN....
YDCERLSNGMRRMIPFSN.FDEKLEGYSAHLTSL.VSGLPYAFRPDGLCLHD.LKDIDLKEMFR~ERILDAIDSG~IDNE....GHQ~LDIVD....
YDSERLSNGLQRMIPFHN.FDEPLEGYAPHLTSL.VSGLQYASRPEGYSIHD.LSDVDVQD~~ERILDAI~YI~KD....~KIPLDIEH....
YYFERLTNGLGKIPEFSW.YSPIKTGWPLMLTK..FTPFAF.....GQKIDFHDPK....
WLERLTNGLGEIPEFSW.YSP~TG~P.MLYG.SYYPFAQ.RP~YDI~D~EQIRFLDMFE~FLQYLQKGHF~F.....DKEINFHD~....
LRLERLSHEMCDIKSIMW.NEPLKTGYWPKIRLH.TGDEMPV.RSNNKIIVTKENVKVKRMLDDVERMLRDGILTGKIERRD....GTIINLKKAE....
MRLERLSHKMCDVKPMMW.NEPLETGYWPKIRLP.SGDEMPV.RQ~ATKDNL~KQ~DDVE~IREGILTGKIE~D....GTVISL~SE....
YDWERLSVNLNR VEKLENWRVPIPDGYFSKLTANNSGRPWGT.RQDNTFIKDIHQG
YMLNRNGERVPLSDNVTT
YNVERFCNNLKKVQPLNNLRVEVPEGYFPKILSSTNNRTY.RVTNQKLRDVDRHDGRVE...
ISDVERWRDRVLAAIDQGYVEDSSGNRIPL.DEV..
. . .VPYWDWTRPISKIPDFIASEKYSDPFTKIEVYNPFNHGHIEQTDYCDF...........
. . .VPYFDWISPIQKLPDLISKATYYNSREQRFDPNPFFSGKVA..GEDA~TRDPQPELF~.......YFYEQALY~EQDNFDDF...........
DFRAPYFDWASQPPKGTLAFPESLSSRTIQWDVDGKTKSI~PLHRFTFHP~PSPGDFS~WSRYPST~YP~LTGASRDERIAPI~E~SL~
.FTIPYWDWRDAEKCDICTDE~GGQHPTNPNLLSPASFFSSWQIVCSRLEE~SHQSLCNGTPEGPL~PGNHDKSRTPRLPSSAD~FCLSLTQYES
3 la
FIG. 2.-Sequence
alignment of hemocyanin-type
proteins. Residue numbers refer to Cmag6. Other numbers indicate protein domain.
Shaded areas, Cu binding sites CuA and CUB. Cmag6, Pintc, Pinta, LimII, and Anda
have not yet been examined for presence of leader
sequence. Carboxy-terminal
regions of Tni_M, BombM, and NeuTy not shown. Cmag6 = C. magister hemocyanin subunit 6, GenBank (GB)
accession number U48881; CmagX = Possible C. magister hemocyanin subunit with deletion between residues 410 and 601; Pintc = Punulirus
interruptus hemocyanin subunit c, SwissProt (SP) accession number P80096; Pinta = Punulirus interruptus hemocyanin subunit a, SP number
P04254; Penvl = Penueus vunnumei hemocyanin
subunit 1, GB number X82502; LimII = Limulus polyphemus hemocyanin subunit II, SP
number P04253; Euryd = Eurypelmu culifornicu hemocyanin
subunit d, SP number P02241; Eurye = Eurypelmu culifornicu hemocyanin
Phylogeny
Cmag6
CmagX
Pintc
Pinta
Penvl
LimII
Euryd
Eurye
Anda
BombA
MsexA
Tni_M
BombM
PapPO
DrpPO
Octoe
Hpomd
NeuTy
HumTy
of Hemocyanm
Gene Family
271
. . .GIDLLGDVIESSLYSP.N...
. . . GIDVLGDVIESSLYSP.N...
. . . GIEFLGDIIESSGYSA.N...
. . .GIELLGDIIESSKYSS.N...
. . .GIDILGDIIESSLYSP.N...
.GTDILGALVESSYESV.N...
1:.GINVIGALIESSHDSV.N...
.GINVLGALIESSFETK.N...
::.GTDILGDIIESSDESK.N...
. . .AINFVGNYWQDNADLY.G...
. . .AVNFVGNYWQANADLY.N...
.DVEHLARLLLGGMGLV.G...
::.DIENLARLVLGGLEIV.G...
GKRGIDILGDAFEADAQLSPN...
..RGIDILGNMIEASPVLSIN...
. .... ... .. .... . .... .... .
;sLLLLb;KDFD..FS;NRWDPtiN
GSMDKAANFSFRNTLEGFASPLTG
Cmag6
CmagX
Pintc
Pinta
Penvl
LimII
Euryd
Eurye
Anda
BombA
MsexA
Tni_M
BombM
PapPO
DrpPO
Octoe
Hpomd
NeuTy
HumTy
3+
PPYTKEELNFEGVNIDNFYIKGNLETYFETFEYSLVNAVDDTED-VDD....
.VDIFTYISRLNHKEFSFVGDVTNELDHDVLATVRIFAWPHEDNNGVA
TP;TRDELEFNGVs;Dd;A;~~~~~~~~~~~~~~~~~~~~~:~~~::::..................................*.............
.VEILTYIERLNHKKFSFLILVTNNNNTEVLATVRIFAWPLRDNNGIE
PPYTHDNLEFSGMWNGVAIDGELITFFDEFQYSLINAVDSGEN.IED....
.VEINARVHRLNHKEFTYKITMSNNNDGERLATFRIFLCPIEDNNGIT
PPYTKADLEFSGVSVTELAWGELETYFEDFEYSLINAVDDAEG.IPD....
.VEISTYVPRLNHKEFTFRIDVENGGA.ERLATVRIFAWPHKDNNGIE
KPYDHDVLNFPDIQVQDVTLHARVDNWHFTMREQELELKHGINPGNA....
.RSIKARYYHLDHEPFSYAVNVQNNSASDKHATVRIFLAPKYDELGNE
PSYTHQQLDFPGVRISRVTVSKVPNILHTYSKDSLLELSHGLNIKGH.....
IQVKYNYEHLDHEPYNYEIEVDNRTGEARETCVRIFLAPKYDELGNR
PHYTPEDLTCPGVHVVNVT VNAKVPNVVTTFMKEAELELSYGIDFGSD....
.HSVKVLYRHLDHEPFTYNISVENSSGGAKDVI'~IFLGPKYDELGNR
HPYTKEELSFPGVEWGVSINSKTANVITTLIKESLLELSHGINFGTD....
.QSVKVKYHHLDHEPFTYNIVVENNSGAEKHSTVRIFLAPKYDELNNK
KPYTQDKLYFDGVKITDVKVD.KLTTFFENFEFDASNSVYFSKEEIKN..
.NHVHELRCATRLNHSPFNVNIEVD..SNVASDAVVKMLLAPKYDDNGIP
QPYNQNDLHFVGVKISDVKVD.KLATYFEYYDFDVSNSVFVSKKDIKN..
.FPYGYKVRQPRLNHKPFSVSIGVK..SDVAVDAVFKIFLGPKYDSNGFP
PKYTREDFDFPGVKIEKFTTD.KLTTFIDEYDMDITNAMFDCLGRL
PKYTREQFSFPGVKVEKITTD.EL~FVDEYDMDISN~LDATEMQ~T.SDMTF~RLNHHPFQVSID~..SDKT~A~IFLGPKYDCMGRL
PPYTMEDLSLPGVVLDKVG~DQ~TLTTGWS~EF~SRGLDFNSPNP~AHYPSRPCTLHLPSPD~QHRKPKS.....~~I~PK~ERGLE
NPYNAGELNFDGITVDYIEAKIGKSNT~TLLT~QKSSAD~GLDFGPTTD~IFASFTHLQNAPFTYTF~~G~TGTCRIFICP~E~QA
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . .. .ELQKLRGLNAYESHCALELMKVPLKPFSFGAPYNLNDLTTD
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .ELQRYRGLPYNEADCAINLMRKPLQPFQDKKL.NPRNITNIYSRPADTFDYRN
NSFMTPRPAPYSTFVAQ..............................
..EGESQSKSTPLEPFWDKSAANFWTSEQVKDSITFGYAYPETQKWKYSSVKE
PLQEVYPEANAPIGHNR..............................
..ESYMVPFI.PLYRNGDFFISSKDLGYDYSYLQDSDPDSFQDYIKSYLEQAS
Cmag6
CmagX
Pintc
Pinta
Penvl
LimII
Euryd
Eurye
Anda
BombA
MsexA
Tni_M
BombM
PapPO
DrpPO
Octoe
Hpomd
NeuTy
HumTy
FSFNDGRWNAIEMDKFWVMLHPGHNHIERSSHDSSHDSSATVPDIPSFQIIKDRTN~IAQ.. .NKELHIEEFESGLG.LPNRFLIPKGNVKGLDMDVMVAITS
587
;6FNEG~~ELDRFW;I;;I;HGHHQ;TRQS~~~::...............................
.FHLVVFVSD
.GCALHLEDYESALG.LPNRFLLPKGQAQGMEFNLWAVTD
LTLDEARWFCIELDKFFQKKGPETIERSSKDSSVTVPD..
.GHDLDLSAYERSCG.IPDRMLLPKSKPEGMEFNLYVAVTD
YTFDEGRWNAIELDKWSLKGGKTSIERKSTESSVTVPD......
..LAKFESATG.LPNRFLLPKGNDRGLEFDLWAVTD
IKADELRRTAIELDKFKTDLHPGKNTVVRHSLDSSVTLSHEH........
..KSEYCSCG.WPSHLLVPKGNIKGMEYHLFVMLTD
LLLEEQRRLYIELDKFHRRLEPGKNVLVRASGDSSVTLSK...........
EYCSDG.KPEHMLVPRGKERGMDFYLFVMLTD
LQPEQQRTLNIELDKFKATLDPGKNWTRDHRNSTVTVEQSG............
.EYCSCG.WPEHMLIPKGNHRGMDFELFVIVTD
LEPDEQRRLFIELDKFFYTLTPGKNTIVRNHQDSSVTISKEGVSEDST........
..EYCSCG.WPEHMLIPRGSHKGMEFELFVMLTD
LTLEDNWMKFFELDWFTTKLTAGQNKIIRNSNEFVIFKEDLD..
..EGKVPFDMSEEFCY.MPKRLMLPRGTEGGFPFQLFVFVYP
IPLAKNWNKFYELDWFVHKVMPGQNHIVRQSSDFLFFKED....SLPMSEIYKLLD..
..EGKIPSDMSNSSDT.LPQRLMLPRGTKDGYPFQLFVFVYP
MSVNDKRMDMIEMDTFLYKLETG~TI~SLE~GVIEQRP~~I~IGTVGTISKT~~S~K.RHR.LPH~LPLG~GGMPMQMFVI~P
MSVNDKRLDMFELDSFMYKL~G~I~SSMDMQGFIPEYLSTRR~ESE~PSG..DGQT~D~CKS~G.FPQRL~PLGTIGGLEMQ~IVSP
MGFMEQRLLWAEMDKFTQDLKPGQNQIVRASNLSSITNPSETNFCGCG....
..WPEHLLLPRGKPEGMTYQLFFMLTD
LNLEEQRLLAIEMDKFTVDLVPGENTIRRQSTESSVAIPFFK.FCGCG....
..WPQHLLLPKGNAQGMLFDLFVMISD
NFHYEYDILDINSMSINQIESSYIRHQKDHDRVFAGFLLSFEICIEGGEC....
..HEGSHFAVLGGSTEMPWAFDRLYKIEITDVLSDMH
HFHYEYDTLELNHQTVPQLELL~Q.EYGR~AGFLI~GLSAD~~CVPSGPKG~DC~~G~S~GGELEMPFTFDRLYKLQITDTIKQLG
YQAAIRKSVTALYGSNVFANFVENVADRTPALKKPQATGE
QHAEEKAQKPWPVKDTKAESSTAAGMMIG
RIWSWLLGAAMVGAVLTALLLVSLLCRHKRKQLPEEKQLPEEKQPLLMEKEDYHSLYQSHL...........................................
Cmag6
CmagX
Pintc
Pinta
Penvl
LimII
Euryd
Eurye
Anda
BombA
MsexA
Tni_M
BombM
PapPO
DrpPO
Octoe
Hpomd
NeuTy
HumTy
..............................
GEADAAV.EGLHENTSFNHYGCP .. .DGT..YPDKRRHGYPLDRHDERIINDLH.NFKHIQVK
............................
GAKDAAI.DGLLENTSFNHYG .. .AHSGK..YPDKQPHPYPLDRRVDDKRIITGVT.NFKGMDEQ
.........................
GRTDAAL.DDLHENTKFIHYGY .. ..DRQ..YPDKRPHGYPLDRRVDDERIFEALP.NFKQRTVKLYSHEG
..........................
GDKDTEG.HNGGHDYGGTHAQCGV.HGEA..YPDNRPLGYHLEHHD
..........................
.. .YPDKRPHGYPLDRKVPDERVFEDLP.NFKHIQVKVFNHGEHIH
GDADSAV.PNLHENTEYNHYG...SHGV
..............................
WDKDKVD.GSESVACVDAVSYCGA.RDHK..YPDKKPMGFPFDRPIHTEHISDFLT~FIKDIKIKFHE
...........................
YEEDSVQGAGEQTIDQDAVSYCGA.KDQK..YPDK~GYPFDRPIQ~TPSQFKTP~FQEIIIQYEGHKH
............................
YAQDAVNGHGENAECVDAVSYCGA.KDQK..YPDKKPMGFPFDRVIEGLTLEEFLTPSMSCTDVRIKYTDIK
.........
HDEDTVAGLSENAVCSDAVSYCGA.RDDR..YPDKKAMGFLTDIKIKFHG...................._
.......
..FD...NKG ...... ..KD.LAP.FESF..VLDNNLLASLWIAPLLMHYSR.FLTCISRIFSFTT.R~GSLTNSIFL~~I~FQKIKF
..............
YQ......AVP ...... .KE.MEP.FKSI..VPDSKPFGYPFDRPVHPEYFKQPNMHV
VK .. .TNLLLPNLDMNIMKERKTC.AGAS..VSTRCRSGFPFDRKIDMTHFFT~FTD~IFRKDLSLS~IKD~MSD~KDDLTYLDSD~~
W
.
VR .. .TGMLLPTLDMTMMKDRCAC.RWSS..CISTMPLGYPFDRPID~SFFTS~FAD~IYRKDLGMSNTSKTTSE~..KDDLTYLDSD~
...........
LEKDQVD.QPAGPRR..CASFCGILDSKFPDKRPMGFPFD~PPPRLQDAE~SVADY~LS~TVQDITITFLTTASRSRHDGPI
.......................
YSQDSVE.QPKTPNDACSTAYSFCGLKDKLYPDRRTMGYPFDRRLPN~TELVGAFG~TDLRI~NDRVID~
.........................................................
LAFDSA..FTIKTKIVAQNGTELPASILPEATVIRIPPSKQDA
..........................................................
LKVNNAASYQLMrEIKAVPGTLLDPHILPDPSIIFEPGTKER
LSIKRPSKLTASPGPIPESLKYLAPDGKYTDWIVNVRAQKLIVSGT
....................................................................................................
491
subunit e, GB number X16650; Anda
= Androctonus austrulis hemocyanin subunit 6, SP number P80476; BombA = Bombyx mori storage
protein 2, GB numbers M24370, 504829; MsexA = Munduca sextu arylphorin subunit alpha, GB numbers M28396, 505092, 505093; TniM =
Trichoplusiu ni basic juvenile hormone sensitive hemolymph protein 1, GB number LO3280; BombM = Bombyx mori sex-specific storage
protein 1, GB numbers X12978, 503722; PapPO = Pucifastucus leniusculus prophenoloxidase,
GB number X83494; DrpPO = Drosophila
melunoguster prophenoloxidase,
GB number D45835; Octoe = Octopus dojeini hemocyanin domain e, GB number M57288; Hpomd = Helix
pomutiu hemocyanin domain d, SP number P1203 1; NeuTy = Neurosporu crussu tyrosinase, GB numbers M3327 1, 505052; HumTy = Human
tyrosinase, GB number M743 14.
272
Durstewitz
and Terwilliger
these hexamerins is that of storage proteins during insect
metamorphosis
(Telfer and Kunkel 1991). Hexamerins
include the arylphorins, proteins rich in aromatic amino
acids, and the methionine-rich
storage proteins.
Although structurally similar to arthropodan hemocyanins
(Markl and Winter 1989), hexamerins contain no copper.
Indices of structural features of the arthropodan
proteins aligned in figure 2 were predicted by the PEPTIDESTRUCTURE
program (GCG Sequence analysis
software package, Devereux,
Haeberli,
and Smithies
1984, data not shown). As could be expected for a globular protein occurring
freely dissolved in the hemolymph, Cmag6 shows no extensive hydrophilic
or hydrophobic domains. Indices for all three structural features, hydrophilicity,
surface probability based on amino
acid side-chain
solvent accessibilities,
and regional
backbone
flexibility
are strikingly
similar among all
crustacean hemocyanins
as well as among the other arthropodan
subgroups. These subgroups include chelicerate hemocyanins
(Euryd, Eurye, Anda6, and LimII),
methionine-rich
storage proteins (BombM and TniM),
arylphorins
(BombA and MsexA), and prophenoloxidases (PapPO and DrpPO). Some motifs appear to be
conserved in all arthropodan hemocyanin-type
proteins.
None of the three indicators suggests any structural homology between the arthropodan
proteins mentioned
above on the one hand and the molluscan hemocyanins
and tyrosinases
on the other. The high degree of sequence similarity
among arthropodan
hemocyanins
(30%-70%
sequence identity) suggests a common tertiary structure. X-ray crystallography
of hemocyanin
from Panulirus and Limulus (Volbeda and Ho1 1989b;
Hazes et al. 1993) has shown that arthropodan hemocyanins consist of three domains. Domain 1 (residues
1-174 in C. magister subunit 6) is quite variable and
mainly o-helical in structure. Domain 2 (residues 175399 in C. magister) contains the oxygen-binding
CuA
and CUB sites and is the most conserved part of the
protein. CuA and CUB each consist of an antiparallel
helix pair containing
three Cu-binding
histidine residues. In C. magister subunit 6, the CuA helix pair extends from residue 186 to residue 200 (helix 2.1) and
from residue 215 to residue 239 (helix 2.2). The Cubinding histidines are located at positions 192 and 196
(helix 2.1) as well as 224 (helix 2.2). The CUB helix
pair extends from residue 341 to residue 353 (helix 2.5)
and from residue 378 to residue 396 (helix 2.6). Its
Cu-binding
histidines are located at positions 343 and
347 (helix 2.5) as well as 383 (helix 2.6). Domain 3 is
rich in P-sheets and forms a P-barrel structure (Hazes
and Ho1 1992).
Phylogenetic
analysis using parsimony
was performed with the PAUP program (Swofford 1991). Sequence comparison (fig. 2) revealed varying degrees of
sequence similarity among taxa. Amino acid sequence
identity between Cmag6 and CmagX was 85%. Among
any two crustacean hemocyanins
it was approximately
60%, with chemical similarity of over 80%. Sequence
identity among chelicerate
hemocyanins
ranged from
53% to 65%; among molluscan
hemocyanins
it was
42%.
131
BombA
MsexA
Tni M
BombM
,A
155
63%
‘35
LimII
Euryd
210
PapPO
DrpPO
247
98%
lmJ7.
1
205
321
FIG. 3.-Single
aligned in figure 2.
5,045 substitutions.
stitutions (indicated
are indicated below
114
Hpomd
NeuTy
HurnTy
most-parsimonious
unrooted tree
Gaps are treated as missing data.
Branch lengths are proportional to
above branches). Bootstrap values
branches.
of the proteins
Total tree size:
number of sub(500 replicates)
The single most-parsimonious
phylogenetic
tree
consistent with the data set (total size = 5,045 substitutions) is shown in figure 3. It was obtained through a
heuristic search algorithm treating gaps as missing data.
Various search options (simple and random addition,
branch and tree swapping) gave the same result. The
resulting tree represents a molecular phylogeny of hemocyanin-class
proteins, not a phylogeny
of the involved species.
Sequence alignment of the functionally
important
CuA and CUB sites (fig. 4) illustrates several points: (1)
The histidine ligands are conserved
in those proteins
that bind Cu, i.e., the arthropodan
and molluscan hemocyanins, the tyrosinases, and the prophenoloxidases.
In the non-Cu-binding
insect hexamerins these residues
are not conserved, although the overall sequence similarity of the hexamerins
to crustacean hemocyanins
is
high. (2) The CUB site is the only region that exhibits
significant homologies
in all taxa surveyed, including
the molluscan hemocyanins
and tyrosinases.
This suggests a common origin for at least part of the molecule.
(3) The CuA site is either of the arthropodan
or the
molluscan
type. Sequence
homology
between these
types is marginal at best. All arthropod proteins in this
study form a monophyletic
group relative to molluscan
hemocyanins
and tyrosinases.
Phylogeny
l
181
P
13
RKGE
scssw
Qagx
P
13
RKGE
s.
PFW
Pintc
LIW HIM E P P
12
RKGE
s .
FPW
Pinta
PWHKDF
II
P
12
RKGE
.LFPW
P
12
RKGE
DVGINAH
cl
DIGINSH
P
13
RKGE
P
13
RKGE
DVGTNAH
II
DIGINAH
P
13
RKGE
Penvl
Euryd
Eurye
Anda
YFT E
I II
D I "1"1"1"
MsexA
IY F(YIE
Tni_M
/Y F(TIE DAD
D IjLINIS
i
Y YIY
Y YIY
P
13
RKGE
‘1’1”1”
H L P
13
YIF~Y~M
H;
13
RRGE
r-l
RRGE.IY
13
RRGE.IM
LI NTYHYYLHUSY
P
P
!d-ld-
BombM
PapPO
DrpPO
DVDLNTYMY
P
DFGINSHEW
P
DICVNSHHW
P
d
Octoe
S PIBIG
RRGE.IM
u
RKGE.LF
13
'8
11
I
l--l-J
RRGE.LF
16
IRIG M P T/PIP
S
::[r::
[z$J:
NeuTy
HumTy
residues
3 42
deleted
l
.
TAE
TAB
Pintc
.
.
Pinta
.TAE
Cmag6
CmagX
.
.
Penvl
LimII
TAB
residues
deleted
r
KHK
II
I
EHK
cl
TAB
KHT
EH
I
WGlIi
EY
Euryd
EY
Eurye
FIDNIFQ
VMl4ANIT
AndaB
YQRS
E VFAR
MsexA
YQRSYEINARHV
Tni_M
MMH....LMKRL
RV
IL G]A
A P MIPI
18
/RI.
. . .
.ID PIAlP
Octoe
KE..
L H Y A A YID
Hpomd
HA..
LDYTA
NeuTy
HumTy
273
PH
Hpomd
BombA
Gene Family
.
CmagC
LimII
of Hemocyanin
PlIlF
KY
YIQ
FIDNIFQ
LJ
.
L Y N R I V E Y I \
Y L
H RiS N VlDlR
EH
1
E F
L W V IIW
LEVSA
NG..
.ALEIYM..NG..
Ll
VQGSA
residues
deleted
FIG. 4.-Sequence
conservation
in the copper-binding
sites of hemocyanin-type
proteins. Top, CuA site; bottom, CUB site. Numbering of
residues is according to Cmag6. Residues conserved in more than half of the taxa or in one complete group of taxa are boxed. Asterisk indicates
conserved histidine, presumably acting as copper ligand.
Discussion
The phylum Arthropoda is composed of three major taxa: The Chelicerata, the Insecta, and the Crustacea.
Traditionally,
the latter two have been considered to be
more closely related and were grouped together as Mandibulata (Remane, Starch, and Welsch 1980, p. 227).
This relationship is supported by 18s r-RNA (Turbeville
et al. 1991; Garey et al. 1996) and mitochondrial
12s
rRNA sequence comparisons (Ballard et al. 1992). Three
minor taxa, the Myriapoda, the Onychophora,
and the
Tardigrada, are placed at the base of the arthropod lineage by these studies, a placement that also is supported
274
Durstewitz and Terwilliger
f
of cu
t
gene duplications
gene
duplications
and fusions
addition
of
domains 1 and
ancestral
lbinuclear
I
t
I
molluscan
Cu protein1
I
3
I
ancestral
arthropod
binuclear
> gene duplication
Cu
protein
and fusion
uniquely
molluscan
CuA peptide
ICu binding
FIG. 5.-Possible
Durstewitz 1996).
helix
pair]
evolutionary relationships between respiratory proteins (based on Volbeda and Ho1 1989~; van Holde and Miller 1995;
by their greater morphological
similarity to the annelids.
However, this phylogeny is by no means certain, and the
problem is compounded
by the question of whether the
arthropods form a monophyletic
group at all or arose
from annelid-like
ancestors in several independent
lineages.
The most parsimonious
phylogenetic tree of the 19
taxa aligned in figure 2 indicates four monophyletic
groups within the arthropods: the crustacean hemocyanins, the insect hexamerins,
the chelicerate hemocyanins, and the prophenoloxidases
(fig. 3). These arthropodan proteins are clearly monophyletic
with respect to
the molluscan hemocyanins
and tyrosinases. These conclusions are supported by very robust nodes in the phylogenetic tree as indicated by bootstrap values well over
80%. They are also in agreement with the comparison
of predicted structural parameters (data not shown) that
suggests a significant degree of structural conservation
among the arthropod proteins, but not between the arthropodan and the molluscan
groups. However, parsimony analysis fails to resolve the relative arrangement
of crustacean and chelicerate hemocyanins,
hexamerins,
and prophenoloxidases
within the arthropod lineage, as
indicated by low bootstrap values (58% and 63%) for
the two major arthropodan branches in figure 3. These
data suggest that (1) the common ancestor of all arthropodan hemocyanins,
hexamerins, and prophenoloxidases
was a Cu-binding
arthropodan hemocyanin-type
protein
and (2) the insect hexamerins lost their Cu-binding
capabilities after the insects diverged from the crustaceans,
presumably due to the development
of the tracheal system that made respiratory proteins obsolete.
Aspan et al. (1995) recently discovered certain sequence similarities between arthropodan prophenoloxi-
dases and arthropodan hemocyanins.
The prophenoloxidases represent nonhemocyanin
proteins that bind copper and occur in both insects (DrpPO) and crustaceans
(PapPO). Although identical in function to tyrosinases
(NeuTy and HumTy), their sequences show only a slight
resemblance to them. Instead, prophenoloxidases
appear
to be most closely related to the hexamer-type family of
arthropodan proteins and feature a typical arthropodan
CuA site. Tyrosinases from most nonarthropod phyla of
the animal kingdom as well as from plants, fungi, and
procaryotes contain a CuA site of the mollusc type (van
Holde and Miller 1995). It is therefore reasonable to
assume that after the arthropods and molluscs diverged,
an ancestral
arthropod-type
binuclear
Cu protein
evolved into four classes of proteins: crustacean hemocyanins, chelicerate hemocyanins,
arthropodan prophenoloxidases, and-through
loss of Cu-insect
hexamerins. Prophenoloxidases,
with two functional
copper
sites, are structurally
more similar to arthropodan hemocyanins
than are the hexamerins.
Detection of prophenoloxidase
in the tracheal cuticle of insects (which
are thought not to have respiratory proteins) has suggested the fascinating possibility of a respiratory function for prophenoloxidases
in that taxon (Kawabata et
al. 1995). The length of the branches leading to both the
insect and crustacean prophenoloxidases
illustrates the
long independent
evolutionary
history of these proteins
and suggests that they diverged from the other lineages
early in arthropodan evolution.
The multitude of different subunit types found in
crustacean and chelicerate hemocyanins
is probably the
result of gene duplications
that occurred independently
after these taxa diverged (Neuteboom et al. 1990). This
Phylogeny
split occurred about 600 MYA, during the early Cambrian, after the arthropods and molluscs diverged.
The absence of a true outgroup (hemocyanin occurs
only in arthropods
and molluscs)
makes speculation
about the relationship of arthropodan and molluscan hemocyanins difficult. The CUB sites are homologous and
not the result of convergence
(van Holde and Miller
1995). This means that all arthropodan
hemocyanins,
hexamerins, and prophenoloxidases
share a common ancestor with the molluscan hemocyanins
and tyrosinases.
The tyrosinases in particular appear to be phylogenetitally very old, because they are found in animals, plants,
fungi, and even procaryotes, and the degree of sequence
similarity between human and procaryotic
(Streptomyces) tyrosinase, for example, is remarkable. Since there
apparently
are no procaryotic
hemocyanins,
it seems
reasonable to assume that molluscan hemocyanins
arose
from tyrosinase-like
ancestors.
A speculative
model of hemocyanin
evolution is
given in figure 5. Our analysis supports the view that
both arthropodan
and molluscan
hemocyanins
arose
from a common ancestral Cu protein (Durstewitz 1996).
Whether this ancestor was mono- or binuclear cannot be
decided from our data. van Holde and Miller (1995)
assume a common origin for the arthropodan and molluscan CUB sites and consider the arthropodan CuA site
a result of gene duplication and fusion in that lineage.
This notion is supported by the fact that in arthropods,
the CuA site is very similar in sequence and structure
(HXXXH for the first two Cu ligands) to the CUB site
(Volbeda and Ho1 1989a), while in molluscs it is not.
The ancestral arthropodan hemocyanin
would, then, be
a binuclear Cu-binding
protein, corresponding
roughly
to domain 2 of today’s arthropodan
hemocyanin.
Domains 1 and 3 would have been added later, following
an evolutionary
trend to provide sites for allosteric regulation and multisubunit
cooperativity.
In this scenario,
the CuA site of molluscan hemocyanins
and tyrosinases
is of separate origin from the CuA site of arthropods,
and the molluscan hemocyanins
are the fusion product
of this uniquely molluscan CuA peptide and a CUB site
shared with the arthropods. The weak tyrosinase activity
of molluscan hemocyanins
(Nakahara, Suzuki, and Kino
1983; Salvato et al. 1983; Ma&l and Decker 1992) is
further evidence for a common origin of tyrosinases and
molluscan hemocyanins.
The huge multidomain
hemocyanins of modern-day molluscs would have arisen from
this monomeric binuclear Cu-protein through a series of
gene duplication
and fusion events. A recent report of
residual o-diphenol
oxidase activity in crustacean hemocyanins, particularly in the dissociated subunit, under
nonphysiological
conditions
(Zlateva et al. 1996) will
provide new opportunities
to explore the evolutionary
relationships
between the molluscan hemocyanins
and
nonarthropodan
tyrosinases on the one hand and the arthropodan hemocyanins
and prophenoloxidases
(tyrosinases) on the other hand.
Two approaches were used in this study to investigate hemocyanin
evolution. The results of both parsimony analysis of a protein sequence alignment and comparison of conserved
structural features are consistent
of Hemocyanin
Gene Family
275
with a monophyletic
origin of arthropodan and molluscan hemocyanins.
Homology between the two, however,
is limited to a small portion of the molecule, and both
classes of proteins, the cylindrical molluscan hemocyanins and the arthropodan
hexamer-type
proteins, diverged during the early stages of life on this planet (and
have evolved quite differently ever since). Prophenoloxidases and hexamerins
are proteins homologous
to arthropodan hemocyanin that have left oxygen binding to
hemocyanin
and taken on a variety of other functions
within the animal.
Acknowledgments
We thank Robert Hanner for his valuable ideas on
phylogenetic
reconstruction.
This study was supported
by NSF grants DCB 89-08362 and IBN 92-17530 to
N.B.T.
LITERATURE
CITED
ASPAN, A., T-S. HUANG, L. CERENIUS,and K. S~DERH;~LL.
1995. cDNA cloning of prophenoloxidase from the freshwater crayfish Pacifastacus leniusculus and its activation.
Proc. Natl. Acad. Sci. USA 92:939-943.
BAK, H. J., and J. J. BEINTEMA. 1987. Panulirus interruptus
hemocyanin: the elucidation of the complete amino acid
sequence of subunit a. Eur. J. Biochem. 169:333-348.
BALLARD,J. W. O., G. J. OLSEN, D. l? FAITH, W. A. ODGERS,
D. M. ROWELL,and I? W. ATKINSON.1992. Evidence from
12s ribosomal RNA sequences that Onychophorans are
modified arthropods. Science 258: 1345-1348.
BEINTEMA,J. J., W. T. STAM, B. HAZES, and M. I? SMIDT.
1994. Evolution of arthropod hemocyanins and insect storage proteins (hexamerins). Mol. Biol. Evol. 11:493-503.
DEVEREUX,J., I? HAEBERLI,and 0. SMITHIES. 1984. A comprehensive set of sequence analysis programs for the VAX.
Nucleic Acids Res. 12:387-395.
DREXEL,R., S. SIEGMUND,H. J. SCHNEIDER,B. LINZEN, C.
GIELENS,G. PREAUX,J. KELLERMANN,
and E LO~TSPEICH.
1987. Complete amino acid sequence of a functional unit
from a molluscan hemocyanin (Helix pomatia). Biochem.
HS 368:617-635.
DURSTEWITZ,G. 1996. Molecular studies of hemocyanin expression in the Dungeness crab. Dissertation, University of
Oregon, Eugene, Oreg.
DURSTEWITZ,G., and N. B. TERWILLIGER.1997. Developmental changes in hemocyanin expression in the Dungeness
crab, Cancer magister. J. Biol. Chem. (in press).
GAREY, J. R., M. KROTEC, D. R. NELSON, and J. BROOKS.
1996. Molecular analysis supports a tardigrade-arthropod
association. Invertebr. Biol. 115:79-88.
HAZES, B., and W. G. J. HOL. 1992. Comparison of the hemocyanin B-barrel with other greek key B-barrels: possible
importance of the “P-zipper” in protein structure and folding. Proteins 12:278-298.
HAZES, B., K. A. MAGNUS,C. BONAVENTURA,
J. BONAVENTURA, Z. DAUTER, K. H. KALK, and W. G. J. HOL. 1993.
Crystal structure of deoxygcnated Limulus polyphemus subunit II hemocyanin at 2.18A resolution: clues for a mechanism for allosteric regulation. Protein Sci. 2:597-619.
KAWABATA,T., Y. YUSAHARA,M. OCHIAI,S. MATSUURA,and
A. MASAAKI. 1995. Molecular cloning of insect pro-phenol
oxidase: a copper-containing protein homologous to arthropod hemocyanin. Proc. Natl. Acad. Sci. USA 92:77747778.
276
Durstewitz and Terwilliger
and D. F. DOOLITTLE. 1982. A simple method for
displaying the hydropathic character of a protein. J. Mol.
Biol. 157: 105-132.
LARSON,B. A., N. B. TERWILLIGER,and R. C. TERWILLIGER.
1981. Subunit heterogeneity of Cancer magister hemocyanin. Biochim. Biophys. Acta 667:294-302.
LERCH, K., M. HUBER, H.-J. SCHNEIDER,R. DREXEL,and B.
LINZEN. 1986. Different origins of metal binding sites in
binuclear copper proteins tyrosinase and hemocyanin. J. Inorg. Biochem. 26:213-217.
LIN, H. C., S. LEI, and G. WILCOX. 1985. An improved DNA
sequencing strategy. Anal. Biochem. 147: 114-l 19.
LINZEN,B., N. M. SOETER,A. E RIGGS et al. (17 co-authors).
1985. The structure of arthropod hemocyanins. Science
2295 19-524.
MARKL, J., and H. DECKER. 1992. Molecular structure of the
arthropod hemocyanins. Pp. 325-376 in C. I? MANGUM,ed.
Advances in comparative and environmental physiology.
Vol. 13. Springer Verlag, Berlin, Heidelberg, New York.
MARKL,J., and S. WINTER. 1989. Subunit-specific monoclonal
antibodies to tarantula hemocyanin, and a common epitope
shared with calliphorin. J. Comp. Physiol. B 159:139-151.
MUNN, E. A., and G. D. GREVILLE.1969. The soluble proteins
of developing Calliphora erythrocephala, particularly calliphorin, and similar proteins in other insects. J. Insect
Physiol. 15: 1935-1950.
NAKAHARA,A., S. SUZUKI,and J. KINO. 1983. Tyrosinase activity of squid hemocyanin. Pp. 319-322 in E. J. WOOD,
ed. Structure and function of invertebrate respiratory proteins. Life Chemistry Reports Supplement 1. Harwood,
London.
NEUTEBOOM,B., I? A. JEKEL,R. M. W. HOFSTRA,S. J. SIERDSEMA,and J. J. BEINTEMA. 1990. Structure, function and
evolution of crustacean hemocyanins. Pp. 85-88 in G.
PREAUXand R. LONTIE,eds. Invertebrate dioxygen carriers.
Leuven University Press, Leuven, Belgium.
OCHIAI, E. I. 1983. Copper and the biological evolution. Biosystems 16:81-86.
REMANE, A., V. STORCH, and U. WELSCH. 1980.
Systematische Zoologie. Gustav Fischer Verlag, Stuttgart
and New York.
SALVATO,B., G. JORI, A. PIAZZESE,E GHIRETTI,M. BELTRAMINI, and K. LERCH. 1983. Enzymic activities of type-3
copper pair in Octopus vulgaris hemocyanin. Pp. 3 13-3 17
in E. J. WOOD, ed. Structure and function of invertebrate
respiratory proteins. Life Chemistry Reports Supplement 1.
Harwood, London.
KYTE, J.,
SAMBROOK,J., E. E FRITSCH,and T. MANIATIS. 1989. Molecular cloning. 2nd edition. Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y.
SWOFFORD,D. L. 1991. PAUP: phylogenetic analysis using
parsimony. Version 3.1. Illinois Natural History Survey,
Champaign, Ill.
TELFER, W. H., and J. G. KUNKEL. 1991. The function and
evolution of insect storage hexamers. Annu. Rev. Entomol.
36:205-228.
TELFER,W. H., and H. C. MASSEY. 1987. A storage hexamer
from Hyalophoru that binds riboflavin and resembles the
apoprotein of hemocyanin. Pp. 305-314 in J. H. LAW, ed.
UCLA Symposium on Molecular and Cellular Biology,
New Series. Vol. 49. Liss, New York.
TERWILLIGER,N. B., and A. C. BROWN. 1993. Ontogeny of
hemocyanin function in the Dungeness crab Cancer magister: the interactive effects of developmental stage and divalent cations on hemocyanin oxygenation properties. J.
Exp. Biol. 183: 1-13.
TERWILLIGER,N. B., and G. DURSTEWITZ.1996. Molecular
studies of the sequential expression of a respiratory protein
during crustacean development. Pp. 353-368 in J. D. FERRARISand S. R. PALUMBI,eds. Molecular zoology: advances, strategies and protocols. Wiley-Liss, New York.
TERWILLIGER,N. B., and R. C. TERWILLIGER.1982. Changes
in the subunit structure of Cancer magister hemocyanin
during larval development. J. Exp. Zool. 221: 181-191.
TURBEVILLE,J. M., D. M. PFEIFER, K. G. FIELD, and R. A.
RAFF. 1991. The phylogenetic status of arthropods, as inferred from 18s rRNA sequences. Mol. Biol. Evol. 8:669686.
VAN HOLDE, K. E., and K. I. MILLER. 1995. Hemocyanins.
Adv. Protein Chem. 47: 1-81.
VOLBEDA,A., and W. G. J. HOL. 1989~. Pseudo 2-fold symmetry in the copper-binding domain of arthropodan hemocyanins. J. Mol. Biol. 206:531-546.
-.
19896. Crystal structure of hexameric hemocyanin
from Punulirus interruptus refined at 3.2A resolution. J.
Mol. Biol. 209:249-279.
VOLL, W., and R. VOIT. 1990. Characterization of the gene
encoding the hemocyanin subunit e from the tarantula Eurypelma californicum. Proc. Natl. Acad. Sci. USA 87:53125316.
ZLATEVA,T., l? DI MURO, B. SALVATO,and M. BELTRAMINI.
1996. The o-diphenol oxidase activity of arthropod hemocyanin. FEBS Lett. 384:25 l-254.
CLAUDIA KAPPEN, reviewing
Accepted
November
11, 1996
editor