Protein Structure, Gene Organization, and Evolution

Protein Structure, Gene Organization, and Evolution

J Mol Evol (2006) 62:362 374
DOI: 10.1007/s00239-005-0160-x
The Hemocyanin from a Living Fossil, the Cephalopod
Nautilus pompilius: Protein Structure, Gene Organization, and Evolution
Sandra Bergmann,1 Bernhard Lieb,1 Peter Ruth,2 Jürgen Markl1
1
2
Institute of Zoology, Johannes Gutenberg University, D-55099 Mainz, Germany
Institute of Zoology, Justus Liebig University, D-35390 Giessen, Germany
Received: 29 June 2005 / Accepted: 3 October 2005 [Reviewing Editor: Dr. Axel Meyer]
Abstract. By electron microscopic and immunobiochemical analyses we have confirmed earlier evidence that Nautilus pompilius hemocyanin (NpH) is a
ring-like decamer (Mr = 3.5 million), assembled
from 10 identical copies of an 350-kDa polypeptide.
This subunit in turn is substructured into seven
sequential covalently linked functional units of 50
kDa each (FUs a g). We have cloned and sequenced
the cDNA encoding the complete polypeptide; it
comprises 9198 bp and is subdivided into a 5¢ UTR of
58 bp, a 3¢ UTR of 365 bp, and an open reading
frame for a signal peptide of 21 amino acids plus a
polypeptide of 2903 amino acids (Mr = 335,881).
According to sequence alignments, the seven FUs of
Nautilus hemocyanin directly correspond to the seven
FU types of the previously sequenced hemocyanin
‘‘OdH’’ from the cephalopod Octopus dofleini. Thirteen potential N-glycosylation sites are distributed
among the seven Nautilus hemocyanin FUs; the
structural consequences of putatively attached glycans are discussed on the basis of the published X-ray
structure for an Octopus dofleini and a Rapana
thomasiana FU. Moreover, the complete gene structure of Nautilus hemocyanin was analyzed; it resembles that of Octopus hemocyanin with respect to
linker introns but shows two internal introns that
differ in position from the three internal introns of the
The sequence reported in this paper has been deposited in the
EMBL/GenBank database under accession number AJ619741.
Correspondence to: Prof. Dr. Jürgen Markl; email: markl@
uni-mainz.de
Octopus hemocyanin gene. Multiple sequence alignments allowed calculation of a rather robust phylogenetic tree and a statistically firm molecular clock.
This reveals that the last common ancestor of
Nautilus and Octopus lived 415 ± 24 million years
ago, in close agreement with fossil records from the
early Devonian.
Key words: Hemocyanin — Nautilus pompilius —
Protein structure — cDNA sequence — Gene structure — Cephalopod evolution
Introduction
Molluscan hemocyanins are very large extracellular
copper proteins that serve as oxygen carriers and
possess a blue color in their oxygenated form. Their
basic quaternary structure is a decamer assembled
from 10 copies of a 350- to 400-kDa subunit. The
decamer is a ring-like structure, 35 nm in diameter
and 18 nm in height. An outer protein wall can be
distinguished from an internal collar complex. In
gastropods and bivalves the typical quaternary
structure for hemocyanin is a cylindrical didecamer,
formed by face-to-face assembly of two decamers
(review: van Holde and Miller 1995). In many marine
gastropods tube-like multidecamers are also present,
with decamers added sequentially at one or both ends
of a ‘‘nucleating’’ didecamer (Herskovits and Ham-
363
ilton 1991; Markl et al. 2001). Several gastropod
hemocyanins are heterogeneous in that they consist
of two immunologically very distinct isoforms that
are differentially expressed (Streit et al. 2005). In a
well-studied example, the hemocyanin isoform pair
HtH1 and HtH2 from the abalone Haliotis tuberculata share only 66% sequence identity, and according
to molecular clock calculations the divergence of the
two isoforms occurred by a gene duplication event ca.
320 million years ago (Altenhein et al. 2002).
The subunit of gastropod hemocyanin is an 400kDa polypeptide folded into eight different covalently
linked globular functional units (FUs) of Mr 50
kDa, termed FU-a to FU-h (from the N- to the
C-terminal). Each FU carries an active site that
reversibly binds one dioxygen molecule. This site
contains two copper ions, each complexed by three
highly conserved histidine residues, thereby forming
the CuA and CuB complex. The X-ray structures of
FU-g from Octopus dofleini and FU2-e from Rapana
thomasiana are available (Cuff et al. 1998; Perbandt
et al. 2003). They each show two structural domains
and confirm, for example, that FUs are indeed glycosylated, which has been suggested from sugar
analyses (e.g., Hall et al. 1977; Kurokawa et al. 2002).
The eight functional units in each subunit are connected by short linker regions of the polypeptide
chain (Lang and van Holde 1991; Miller et al. 1998;
Lieb et al. 2000). While the first six FUs build the
protein wall, the internal collar complex is formed by
FU-g and FU-h. Within this collar complex, a more
central ‘‘arc’’ formed by FU-g can be distinguished
from a more peripheral collar formed by FU-h
(Meissner et al. 2000).
In contrast to the scenario described so far, the
hemocyanin molecules from cephalopods and chitons are restricted to single decamers. Chiton
hemocyanin is an asymmetrical decamer with a
peripheral collar complex at one edge that resembles
decamers obtained by partial dissociation of gastropod hemocyanin molecules (Lambert et al. 1994).
Cephalopod hemocyanins, however, are structurally
symmetrical decamers, with a central collar complex. Octopus dofleini hemocyanin (OdH) lacks FUh, and consequently, its polypeptide has a molecular
mass of only 350 kDa (Miller et al. 1990, 1998);
FU-h is also absent at the gene level (Lieb et al.
2001). FU-g forms the central collar, which structurally corresponds to the ‘‘arc’’ of gastropod hemocyanins (Lamy 1993). OdH consists of two
isoforms, which share a sequence identity of 96%;
this corresponds to a comparatively recent gene
divergence, ca. 40 million years ago (Miller et al.
1998; Miller and van Holde 2003). Sepia officinalis
hemocyanin (SoH), on the other hand, is built from
an 400-kDa subunit, and the presence of eight
different FUs has been demonstrated immunobio-
chemically (Lamy 1998). However, the additional
FU results from a duplication of FU-d, and is
probably integrated into the central collar complex.
This raises questions concerning the structure-function consequences of these FU differences, questions
that can possibly be addressed by studying hemocyanins from other cephalopod branches, notably at
the amino acid primary sequence and gene structural level. Within the cephalopods, the complete
cDNA and genomic sequence of Octopus dofleini
hemocyanin is available and has been compared to
the corresponding data from the gastropods Haliotis
tuberculata and Aplysia californica (Miller et al.
1998; Lieb et al. 2000, 2001, 2004; Altenhein et al.
2002). In addition, two partial amino acid sequences
of Sepia officinalis hemocyanin (SoH) have been
published, namely, of SoH-d (Top et al. 1990) and
SoH-g (Declerq et al. 1990); the latter sequence has
also been confirmed at the cDNA level (Beuerlein et
al., unpublished).
All five extant cephalopod orders of the class
Cephalopoda, the Octopoda, Sepioidea, Teuthoidea,
Vampyromorpha, and Nautiloidea, express hemocyanin. As deduced from the few species studied, it
appears that Sepioidea and Teuthoidea (traditionally
combined as the ‘‘Decabrachia’’ and represented in
hemocyanin studies by Sepia officinalis, Sepioteuthis
lessoniana, and Loligo pealei) possess Sepia-type
hemocyanin (Chignell 1997; Lamy et al. 1998; Mouche et al. 1999), whereas Octopoda and Vampyromorpha (traditionally combined as ‘‘Octobrachia’’
and represented by Octopus dofleini, Octopus vulgaris,
Benthoctopus sp., and Vampyroteuthis infernalis)
possess Octopus-type hemocyanin (Miller et al. 1990;
Chignell 1997). In this context we decided to study
Nautilus pompilius hemocyanin, because according to
fossil records this animal represents, within the
Cephalopoda, the most ancient extant branch (the
‘‘Tetrabranchiata’’; Fig. 1) and marks a divergence
point from the four other cephalopod orders (the
‘‘Dibranchiata’’) that existed at least 400 million
years ago (Pojeta 1987). Physicochemical as well as
functional aspects of Nautilus pompilius hemocyanin
have already been studied by Bonaventura et al.
(1981), which provide a firm basis for our present
investigation.
Materials and Methods
Preparation of RNA and RT-PCR
RNA was extracted from the midgut gland of N. pompilius using
the GTC method (Chomczynski and Sacchi 1987; Sambrook et al.
2001). Reverse transcription and subsequent PCR were performed
with Expand Reverse Transcriptase and Expand DNA polymerase,
respectively (Roche Diagnostics, Mannheim, Germany), following
the procedure of the manufacturer’s instruction. The missing 5¢ and
364
Fig. 1. The tetrabranchiate cephalopod Nautilus
pompilius.
3¢ ends were obtained using the 5¢ and 3¢ Race Kit (Invitrogen,
Karlsruhe, Germany), respectively.
Preparation of DNA and PCR
Genomic DNA was extracted from mantle tissue of N. pompilius
according to the protocol of Stratagene DNA Extraction Kit (La
Jolla, CA, USA). PCR reactions were performed using specific
primers derived from the cDNA sequence. PCR fragments up to
3 kb were generated by a standard three-step protocol with the
Invitrogen Taq polymerase (Invitrogen). For larger fragments, the
Expand PCR system was used according to the manufacturer’s
protocol (Roche Diagnostics).
Purification and Sequencing of PCR Fragments
PCR samples were analyzed in standard agarose gels in 1 · TBE
(89 mM Tris/chloride, 89 mM sodium borate, 2 mM sodium
EDTA, pH 7.5) and purified by the gel extraction kit from Qiagen
(Hilden, Germany). Prior to sequencing, PCR fragments were either cloned into TOPO pCR 2.1 and pCR-XL-TOPO (Invitrogen)
or sequenced directly (Genterprise, Mainz, Germany).
Sequence Data Analyses and Phylogenetic Studies
Data obtained from the automated sequencer were computeranalyzed using CHROMAS (http://trishul.sci.gu.edu.au/conochromas.html), TRANSLATE (http://www.expasy.ch), ALIGN
(http://www.expasy.ch), and BLAST Japan (http://blast.genome.
ad.jp). SIGNALP V1.1 was used for prediction of the signal peptide (http://www.expasy.ch). Multiple sequence alignments and
identity matrices were calculated by CLUSTALX 1.83 (Thompson
1997) after manual optimization with the aid of GENEDOC 2.6
(Nicholas and Nicholas 1997). Phylogenetic analyses were performed with the PHYLIP package (Felsenstein 2001) and
MRBAYES 2.01, assuming different matrices (Huelsenbeck and
Ronquist 2001). For Baysian analyses Metropolis-coupled Markov
chain Monte Carlo sampling was performed with four chains that
were run for 150,000 generations. Prior probabilities for all trees
were equal, starting trees were randomly assigned, and tree sam-
pling was done every 10 generations. Posterior probabilities were
estimated on 5000 trees (burn-in = 10,000). For molecular clock
calculation, the software Protdist, using the JTT-matrix of the
PHYLIP package, was applied.
SDS-PAGE and Crossed Immunoelectrophoresis
SDS-PAGE was performed according to the procedure of Laemmli
(1970). Crossed and crossed-line immunoelectrophoreses were
carried out following the methods described by Weeke (1973) and
Kroll (1973). NpH-specific antibodies were raised in rabbits as
described by Markl and Winter (1989).
Limited Proteolysis
Total NpH was dialysed overnight in glycine buffer (pH 9.6).
Subsequently, limited proteolysis was performed at 37C for 4 h
using the following enzymes: bovine pancreatic elastase type IV (E0258), bovine pancreatic trypsin type XIII (T-8642) (Sigma, Deisenhofen, Germany), and Staphylococcus aureus V8 protease type
XVII (45172) (Fluka, Neu-Ulm, Germany). After dissolving the
proteases in 0.1 M NH4HCO3 (pH 8.0) a final concentration of 2%
(w/w) was used. The hemocyanin concentration was between
1 mg/ml and 50 mg/ml. Proteolytic fragments were applied to a
Q-Sepharose anion-exchange column (Pharmacia, Freiburg,
Germany), equilibrated with 0.02 M Tris/HCl, pH 8.0, and eluted
by a linear sodium chloride gradient (0 0.5 M NaCl) in the same
buffer, at a flow rate of 1 ml-min)1. Proteolytic fragments were
isolated by cutting the bands from a native PAGE gel after inverse
staining with Roti-White system (Roth, Karlsruhe, Germany).
Amino Acid Analysis
Proteolytic fragments were separated by SDS-PAGE (Laemmli
1970) and subsequently electrotransferred to ProBlot membranes
(Applied Biosystems, Weiterstadt, Germany) in a vertical blotting
chamber. The transferred bands were detected by Ponceau S
staining. Polypeptides of interest were cut and sequenced by a
365
this corresponds to seven FUs, as also shown by
Miller et al. (1998) for Octopus dofleini hemocyanin.
Electron microscopy of negatively stained native
hemocyanin molecules revealed top views and side
views, similar to the Octopus-type decamers (Fig. 3),
in agreement with earlier results (Bonaventura et al.
1981).
Immunoelectrophoresis
Fig. 2. Molecular mass determination of the NpH subunit by
SDS-PAGE. A 7.5% polyacrylamide gel was used. As standards,
we used commercial molecular mass markers (M: myosin, bgalactosidase, phosphorylase A, bovine serum albumin) and, in
addition, several molluscan hemocyanin subunits known to contain
eight FUs (KLH1/2 and RtH1: 400 kDa), plus two subunit fragments known to contain six and two FUs, respectively (RtH2.1,
300 kDa; RtH2.2, 100 kDa). Note that NpH migrates predominantly as a single band of 350 kDa, confirmed by calibration plot
of molecular mass versus migration distance (not shown); the small
fraction of faster-moving material might result from partial proteolytic cleavage as usually observed in molluscan hemocyanin
preparations (for references, see Gebauer et al. 1999; Söhngen et al.
1997; Idakieva et al. 1993).
commercial service (Dr. Hans Heid, DKFZ, Heidelberg, Germany).
Electron Microscopy and Protein Structure Modeling
Negative staining was performed using the single droplet procedure
(Harris and Horne 1991). Carbon support films were initially glowdischarged for 20 s to render them hydrophilic and adsorptive for
the protein. Hemocyanin samples (0.1 mg/ml) were adsorbed to
the carbon film, then washed twice with droplets of water, and,
finally, negatively stained with 2% (w/v) uranyl acetate. After
drying, the grids were studied in a Zeiss EM 900 transmission
electron microscope at 80 kV. Deep View/SwissPdbViewer (Guex
and Peitsch 1997) was applied to model NpH-g, using the crystal
structure of OdH-g as a template (Cuff et al. 1998).
Results
Biochemical Analysis and Electron Microscopy
Nautilus pompilius hemocyanin (NpH) was purified
from cell-free hemolymph by ultracentrifugation. In
SDS-PAGE, the NpH polypeptide showed a single
band, migrating faster than several gastropod
hemocyanin subunits known to contain eight FUs
(e.g., KLH1/2 from Megathura crenulata and RtH1
from Rapana thomasiana), but slower than a fragment of RtH2 known to contain six FUs (Fig. 2) (for
references, see Söhngen et al. 1997; Gebauer et al.
1999). From such experiments, a molecular mass of
350 kDa was calculated for the NpH polypeptide;
Crossed immunoelectrophoresis using rabbit antibodies developed against purified, at pH 9.6 dissociated NpH yielded a single precipitation peak,
indicating the presence of either a single polypeptide
or almost-identical polypeptides (Fig. 4A). To analyze the subunit organization, we applied the limited
proteolysis technique combined with HPLC separation of the resulting fragments, as described in detail
elsewhere (Gebauer et al. 1994; Söhngen et al. 1997).
The cleavage products representing either single FUs
or larger fragments composed of two or more FUs
were studied by SDS-PAGE, N-terminal sequencing
(Table 1), and crossed as well as crossed-line immunoelectrophoresis (Figs. 4B E). As with other molluscan hemocyanins studied previously, the largest
number of single FUs released proteolytically was
obtained using elastase (Fig. 4B). Within the pattern
of the elastase cleavage products, most of the precipitation peaks could be assigned to individual FUs.
Identification of the biochemically isolated cleavage
products was greatly facilitated by comparing their
N-terminal protein sequence (see legend to Fig. 4) to
the complete polypeptide sequence as predicted from
the cDNA sequence (Fig. 5).
cDNA Sequencing
From the midgut gland, which had been previously
identified as the site of biosynthesis (Ruth et al. 1988,
1999), total RNA was isolated, and cDNA encoding
the complete NpH polypeptide was obtained by
RT-PCR. As a first approach, various hemocyaninspecific sequences were obtained using degenerated
primers derived from known molluscan hemocyanin
sequences and coding for the conserved copper
binding sites. The predicted amino acid sequences
encoded by the resulting cDNA fragments were
analyzed by multiple sequence alignments and
thereby assigned to their respective FUs. Using these
cDNAs, NpH-specific primers were designed and
used to fill the absent sequences between the fragments by RT-PCR. The 5¢ UTR and 3¢ UTR were
obtained by 5¢ and 3¢ RACE. In order to ensure that
only one single cDNA type was present, overlaps of
200 300 bp between the various fragments were
analyzed. The complete cDNA sequence comprises
366
Fig. 3. Transmission electron microscopy of NpH
decamers. Negatively stained NpH decamers visible
in side views (rectangular) and in top views (circles),
suggesting a typical Octopus-type structure (Sepiatype decamers would exhibit a more pronounced
central ring, rather than the five distinct central
masses as seen here: see Fig. 2 and Mouche et al.
1999). Image obtained by W. Gebauer.
Fig. 4. Immunoelectrophoresis (IE) of the NpH subunit and its
seven functional units. The starting points are marked by asterisks.
In the first dimension, the anode was on the left. In the second
dimension, the anode was at the bottom. Rabbit antibodies against
NpH were applied. A Crossed IE of the 350-kDa subunit as obtained by overnight dialysis of native NpH against alkaline buffer
(pH 9.6); the single immunoprecipitate suggests homogeneity at the
subunit level. B Crossed IE of the same material as in A, but after
limited proteolysis by elastase, showing a variety of immunologically distinct fragments; most of them could be assigned to individual FUs as exemplified in C E. C Crossed-line IE of a sample
as in B, with HPLC-purified FU-c in the line to identify its peak in
the whole pattern (by its fusing with the line). D, E Experiments
corresponding to C, but with FU-g and FU-e in the line. Note that,
in spite of many experiments it remained unclear whether ‘‘peak?’’
in B is FU-d, FU-e, or their dimer d-e.
9198 bp (EMBL/GenBank database, accession
number AJ619741). The first potential start codon is
at position 59, yielding a 5¢ UTR (untranslated region) of 58 bp. The first stop codon (TAG) is at
position 2925, followed by a 3¢ UTR of 365 bp. Close
to the end of the latter, a typical polyadenylation
signal (AATAAA) and, 11 bp farther downstream, a
poly(A) tail are present.
The deduced primary structure of the hemocyanin
polypeptide starts with a signal peptide of 21 amino
acids as predicted using the SIGNALP V1.1 software; such a peptide exists also in other previously
sequenced molluscan hemocyanins (Lieb et al. 2001),
all of which are secreted via the endoplasmic reticulum (Sminia 1977; Ruth et al. 1988; Albrecht et al.
2001). With the signal peptide excluded, the deduced
polypeptide comprises 2901 amino acids and is subdivided into seven paralogous regions of 404 421
amino acids, representing the seven functional units
NpH-a to NpH-g; according to sequence alignments,
they correspond to the seven equivalent FUs of
O. dofleini hemocyanin (Table 2, Fig. 5). A radial
phylogenetic tree was constructed using a multiple
sequence alignment of the seven functional units of
N. pompilius hemocyanin in comparison to the
completely sequenced FUs of the gastropods
H. tuberculata and A. californica and the cephalopods O. dofleini and S. officinalis (Fig. 6); to construct this tree, the Baysian inference method was
applied. A relative rate test according to Tajima
(1993) revealed that the intact hemocyanin subunits
possess a relatively low evolutionary rate of about
10)9 mutations per site per year and follow an
approximately linear evolution. This allowed calcu-
367
Table 1. Comparison of the protein sequences derived from translated DNA sequences to N-terminal sequences of proteolytic fragments
of FU-a, -c, -f, and -g from NpH
Protein sequence translated from DNA sequencing
Protein sequence deduced from N-terminal sequencing
NpH-a
NpH-c
NpH-f
NpH-g
TSDPTN
TSDPTN
VKSMNISH
VKSMNISH
EHHEVHHPLN
EHHEVHHELH
DTKQAERERRISGG
NTKQAERERRISGG
lation of a rather robust molecular clock based on
divergence rates, as presented in Table 2. The obtained time-scale confirmed our previous calculations
(see Lieb et al. 2000, 2001; Altenhein et al. 2002) and
yielded the additional information that the Tetrabranchiata-Dibranchiata (=Nautiloidea-Coloidea)
split occurred ca. 415 million years ago (Fig. 7).
Using the X-ray structure of OdH-g (Cuff et al.
1998), the conformation of NpH-g was predicted
from its sequence. This revealed that in NpH-g a
characteristic loop between strand b2 and strand b3
is missing, which in OdH-g carries the single N-glycan side chain; instead, NpH-g has two potential Nglycosylation sites, located at the strand b5 and helix
a12 positions (Fig. 8).
Genomic DNA Sequencing
The gene encoding NpH was sequenced by genomic
PCR using NpH-specific primers derived from the
cDNA sequence. The complete cDNA sequence has
a length of 18.5 kb and is structured into 10 exons
and 9 introns; 6 of the latter are linker introns
separating the seven regions encoding functional
units (Fig. 9). All of the introns are spliced according to the GT/AG rule (Table 3). This resembles the
situation in the previously analyzed molluscan
hemocyanin genes (Lieb et al. 2001, 2004; Altenhein
et al. 2002); indeed, direct comparison among NpH,
OdH, and HtH1 at the protein level reveals a
striking correspondence of the linker intron positions (Fig. 10). One intron of nearly 1000 bp is located within the signal peptide and is in phase 0.
Additionally, two internal introns have been found
in NpH. The first internal intron lies in NpH-a. It
has a length of approximately 5000 bp and is in
phase 1. The second internal intron of 819 bp is
localized within the region encoding FU-g and is in
phase 1. Another specific feature of the NpH gene is
that introns are not present between the signal
peptide and NpH-a, or within the 3¢ UTR.
Discussion
Quaternary Structure and Biochemical Studies
Early electron microscopic work of Bonaventura
et al. (1981) showed that Nautilus hemocyanin is
present in the hemolymph exclusively as decamers
that resemble the structure found for Octopus rather
than Sepia hemocyanin. Our own electron microscopic data confirmed this (see Fig. 3). Moreover, we
have clearly demonstrated, immunochemically as well
as at the DNA level, that in contrast to gastropod
and S. officinalis hemocyanin, the polypeptide of N.
pompilius hemocyanin consists of only seven FUs and
that, as in O. dofleini, the missing component is FU-h.
From biochemical and functional evidence, Bonaventura et al. (1981) suggested the presence of three
different hemocyanin subunits in N. pompilius.
However, we have not been able to confirm this
immunoelectrophoretically or at the cDNA and
genomic DNA levels. Certainly, the existence of two
or more very similar polypeptides cannot be rigorously excluded and could well result from
posttranslational modification, but we present here
strong evidence that a single hemocyanin gene exists
in N. pompilius, in contrast to O. dofleini and
H. tuberculata, which possess two distinct hemocyanin genes (Lieb et al. 2001; Altenhein et al. 2002). The
presence of two genes, however, apparently arose by
two independent gene duplication events in completely different geological eras.
Potential Disulfide Bridges and N-Glycosylation Sites
The primary structure of the N. pompilius hemocyanin polypeptide chain, as predicted from its cDNA
sequence, shows a variety of features and motifs
typical for all the molluscan hemocyanins sequenced
to-date (see Fig. 5). A detailed discussion of these
aspects has been published by Miller et al. (1998).
The most conserved regions surround the seven active sites, each with two copper ions and six histidines. In contrast, the linker peptides between
neighboring FUs are more variable (see Fig. 10).
Among the strictly conserved features are two
disulfide bridges (Cys64 Cys75 and Cys190
Cys257). In the two available X-ray structures of
molluscan hemocyanin FUs, a copper-containing
core domain can be distinguished from a more
peripheral b-sandwich domain (Cuff et al. 1998;
Perbandt et al. 2003). From their positions, the two
disulfide bridges could stabilize the core domain,
and indeed, oxygen-binding capacity is completely
lost after disulfide bond reduction (Topham et al.
368
Fig. 5. Primary structure of the seven functional units of NpH as
predicted from their cDNA sequence. A multiple sequence alignment in comparison to Octopus dofleini hemocyanin is shown
(OdH; Miller et al. 1998). A consensus numbering system was
applied. The position of secondary structure features in OdH-g
(Cuff et al. 1998) is indicated by blocks (see also Fig. 7). Potential
N-glycosylation sites (14 in NpH, 7 in OdH) are highlighted in dark
blue for NpH and in light blue for OdH. Putative disulfide and
thioether bridges are marked. Note that the third disulfide bridge is
absent in FU-b and FU-c. The alignment was performed with
ClustalX and was edited by GeneDoc.
369
Table 2. Matrix of percentage identity between known hemocyanin sequences of cephalopods
ObdH-a NpH-b OdH-b NpH-c OdH-c NpH-d OdH-d OdH-e SoH-e NpH-e OdH-f SoH-f NpH-f OdH-g SoH-g NpH-g
61
44
43
44
44
59
41
40
42
46
42
42
43
46
57
42
44
46
47
47
43
43
44
45
44
42
44
63
43
40
44
46
41
42
46
44
43
41
43
46
44
44
46
46
77
44
41
46
45
45
44
47
44
61
63
44
43
42
44
44
44
43
43
42
44
45
42
42
42
46
43
43
44
44
43
45
44
72
46
46
44
46
45
42
49
48
43
44
46
56
55
43
44
45
48
45
44
46
43
43
43
46
47
45
48
44
45
45
46
44
45
47
45
46
46
46
48
47
49
76
47
45
48
50
47
46
52
50
46
47
49
47
46
52
65
67
NpH-a
OdH-a
NpH-b
OdH-b
NpH-c
OdH-c
NpH-d
OdH-d
OdH-e
SoH-e
NpH-e
OdH-f
SoH-f
NpH-f
OdH-g
SoH-g
Note. The sequences for the Octopus hemocyanin were taken from Miller et al. (1998). Identities between the corresponding FUs from NpH
and OdH are in boldface; from NpH and SoH, in cursive; and from SoH and OdH, underlined.
Fig. 6. Phylogenetic analysis of molluscan
hemocyanin functional units. A radial
phylogenetic tree with hemocyanin
sequences from Nautilus pompilius (NpH),
Octopus dofleini (OdH), Sepia officinalis
(SoH), and Haliotis tuberculata (two
isoforms: HtH1, HtH2) was constructed
assuming the Dayhoff substitution matrix.
The values at the branches represent
Bayesian posterior probabilities (values
below 0.8 are omitted). Note that eight
major branches are obtained that
correspond to the eight different FU types.
Also note that SoH[h]-g (originally termed
functional unit ‘‘h’’ and renamed, for
obvious reasons, in our recent publications
[e.g., Lieb et al. 2000]) is of significantly
greater similarity to OdH-g than to NpH-g.
1999). A third disulfide bridge lies between Cys348
and Cys360, which is at the surface of the b-sandwich domain. However, in all studied molluscan
hemocyanins FU-b lacks this bridge. Interestingly,
in N. pompilius and O. dofleini it is also absent from
FU-c, which might be a specific feature of cephalopod hemocyanins. This is interesting in view of the
evidence that in gastropod di- and multidecamers,
FU-c, FU-b, and FU-a are apparently located at the
contact region between neighboring decamers
(Meissner et al. 2000; Gebauer at al. 2001). One
might argue that the third S S bridge enables FU-c
to stabilize didecamer formation. Otherwise, this
formation is more likely a result of a divalent or
monovalent salt interaction (Miller et al. 1998).
However, whether or not the third disulfide bridge is
involved in this decamer-dimerization process has
still to be determined.
370
Fig. 7. Molecular clock calculated from the distance values of
various molluscan hemocyanin sequences. On the basis of fossil
records (Benton 1993), the splitting point of gastropods and
cephalopods was used for calibration (520 million years ago;
asterisk). Note that the Tetrabranchiata-Dibranchiata split occurred 105 million years later, followed by the separation into
Octobrachia and the Decabrachia after a further 170 million years.
Distance values are given in Table 2.
The sequence of N. pompilius hemocyanin reveals
13 potential N-glycosylation sites with the motifs
‘‘NXT’’ or ‘‘NXS’’; in addition, in NpH-a the sequence ‘‘NPT’’ exists, but according to Bause (1983)
this site should be blocked by the central proline. A
common feature of most molluscan hemocyanins is
the lack of an N-glycosylation site on FU-c, and N.
pompilius hemocyanin FU-c concurs; in O. dofleini,
OdH-c contains a sequence ‘‘NPT.’’ OdH-d and
NpH-d are strikingly similar with respect to their
potential N-glycosylation sites (see Fig. 5). The most
frequent potential N-glycosylation site in molluscan
hemocyanin FUs is localized in the b-sandwich domain, between strand b11 and strand b12 (Lieb et al.
2000); indeed, NpH-a, NpH-b, NpH-d, and NpH-f
also share this feature. Most of the indels in the
alignment comprise only a single amino acid, located,
as deduced from the available X-ray structures, in the
loops between secondary structure elements. However, NpH-g contains a conspicuous gap of four
amino acids between strand b2 and strand b3 which,
in the other previously sequenced molluscan hemocyanins, seem to form a highly conserved loop (see
Fig. 8). Superposition of the X-ray structure of OdHg (Cuff et al. 1998) and the ternary structure of NpHg, as predicted from molecular modeling, reveals a
convincing structural fit except for this loop. Moreover, in OdH-g, these amino acids encompass a glycosylation site actually occupied by an N-glycan
which apparently fills, though from an opposite
direction, the same space as would a glycan attached
between strand b11 and strand b12 (see Discussion in
Lieb et al. 2000). However, in NpH-g this alternative
region between strand b11 and strand b12 is also
devoid of such a site. Instead, NpH-g exhibits two
N-glycan attachment sites on two other faces of the
molecule (see Fig. 8). Consequently, the putative
glycosylation pattern in NpH-g differs drastically
Fig. 8. Projection of the two potential N-glycosylation sites of
NpH-g into the X-ray structure of OdH-g. Note that OdH-g contains a single N-glycan attached between strand b2 and strand b3
(arrow). As deduced from comparative modeling (DeepView/
SwissPdbViewer [Guex and Peitsch 1997]) of the NpH-g sequence
with the ternary structure of OdH-g (Cuff et al. 1998) as template,
the exposed b2/b3 loop is missing in NpH-g. Instead, N-glycans in
NpH-g would attach at two other faces of the functional unit (flash
symbols).
from that in OdH-g. Another feature of N. pompilius
hemocyanin is attachment sites for N-glycans within
linker peptides, namely, the linkers NpH-b/NpH-c as
well as NpH-d/NpH-e; in all other available molluscan hemocyanin sequences the linkers are devoid of
such a site. The few detailed sugar analyses of
molluscan hemocyanins already available have revealed a rich collection of different N-glycans, indicating that most, if not all, of the potential sites are
indeed glycosylated (Hall et al. 1977; Kurokawa et al.
2002; Stoeva et al. 2002); for OdH-g and RtH2-e, this
has been directly confirmed by X-ray crystallography
(Cuff et al. 1998; Perbandt et al. 2003). Although
hemocyanin glycosylation patterns in molluscs are
now being slowly revealed, there is no meaningful
functional concept of their biological significance and
variability.
Gene Structure
The gene structure of N. pompilius hemocyanin (see
Fig. 9) fits in principle the previously published sit-
371
Fig. 9. Gene structures of NpH and OdHG. Large boxes, exons
encoding functional units (FU-a to FU-g); small boxes, exons
encoding the signal peptide (S) and the 3́ untranslated region
(UTR); white rods, phase 1 linker introns (between two functional
units or signal peptide and FU-a); black rods, internal introns
(within functional units); gray rods, introns within the signal peptide and close to the FU-g/3́ UTR border, whereas the latter is
probably a result of intron sliding.
Table 3. Position, length, and phase of the introns in the NpH encoding gene
Domain
Type
Position
NpH-s1/s2
NpH-a1/a2
NpH-a/b
NpH-b/c
NpH-c/d
NpH-d/e
NpH-e/f
NpH-f/g
NpH-g1/g2
‘‘Internal’’
‘‘Internal’’
Linker
Linker
Linker
Linker
Linker
Linker
‘‘Internal’’
CTCTCTGCAG
ACTTCTGACC
TCTTCTGACC
GTTCCAGCAC
GCGGCTCTTG
CCTGCACCAG
CCAGCTGCAG
CCAGGAAGAG
CCTGGAAGAG
GT...CAG
GT...TAG
GT...CAG
GT...CAG
GT...TAG
GT...CAG
GT...TAG
GT...CAG
GT...CAG
CTCCTTGTCT
CCACCAATAT
CACCCATGCG
ATGTAAAAAG
GTACCTATGG
GATCCAAAAA
AACATCACGA
ATACCAAACA
GAGCCCAGCA
Size (bp)
Phase
1000
5000
487
277
487
600
367
300
819
0
1
1
1
1
1
1
1
1
Note. The splicing of the introns follows exclusively the GT/AG rule. The intron of NpH-s1/NpH-s2 is marked with quotation marks
because the origin of this intron is still unclear with respect to the intron-late/intron-early hypothesis.
uation for H. tuberculata and O. dofleini hemocyanin
(Lieb et al. 2001). The different FUs, serially arranged within the polypeptide, are represented in the
gene by a series of exons separated by phase 1 linker
introns. These introns are apparently very ancient
and can be used as examples for an ‘‘intron-early’’
scenario (Lieb et al. 2001). In N. pompilius, intron
size ranges from 200 to 500 bp, which resembles the
situation in O. dofleini and on average is significantly
smaller than in H. tuberculata. In addition to linker
introns, internal introns have been observed (i.e.,
introns located within a region encoding a specific
FU), namely, three in Octopus and six in Haliotis;
they vary in position and phase and therefore have
been discussed as examples of an ‘‘intron-late’’ scenario (Lieb et al. 2001). In the N. pompilius hemocyanin gene two internal introns are present. The
first internal intron within the functional units lies 10
bp downstream of the start of exon NpH-a. It
comprises approximately 5000 bp and is in phase 1.
Another phase 1 internal intron of 819 bp is found
in the exon of NpH-g, localized only 11 bp upstream
of the stop codon (see Fig. 9). Both introns are absent in the hemocyanins of O. dofleini as well as of
H. tuberculata. However, an intron is not present
between the signal peptide and NpH-a, or within the
3¢ UTR of the N. pompilius hemocyanin gene,
whereas in the other two mollusks an apparently
very ancient phase 1 intron exists ahead of FU-a and
a few base pairs downstream from the stop codon
(Lieb et al. 2001). Assuming that the internal intron
within the exon of FU-a corresponds to the intron
between the signal peptide and FU-a in H. tuberculata and O. dofleini, an intron displacement could
have been occurred. Similar observations have been
made in Artemia hemoglobin (Jellie et al. 1996). This
supposition is supported by the fact that the position
of the intron in NpH-a conforms to the linker intron
between the signal peptide and FU-a of H. tuberculata and O. dofleini. One possibility for the location of the internal intron in NpH-g is that it is
indeed the missing intron of the 3¢ UTR and has
been displaced by intron sliding. Alternatively, the
internal intron in N. pompilius stems from a former
linker intron between FU-g and FU-h that has been
conserved after the loss of FU-h in the cephalopod
lineage. Interestingly, according to the protein sequence alignment, the internal intron of N. pompilius
lies at exactly the same position as the FU-g/FU-h
linker intron in both isoforms of H. tuberculata
(Altenhein et al. 2002). Of course, due to the fact
that this internal intron is missing in the Octopus
hemocyanin gene, it cannot be excluded that the
ancestors of N. pompilius gained this internal intron
372
Fig. 10. Sequence surrounding the linker introns
in H. tuberculata, O. dofleini, and N. pompilius. The
conserved residues are marked in gray. The
beginning of every FU is variable until the first
b-sheet and therefore gaps are introduced with
undefined position. Secondary structure elements as
deduced from OdH-g (Cuff et al. 1998) are indicated
along the top of the sequence. All of the linker
introns are inserted in a clearly defined region
between conserved residues of two FUs.
much more recently. But in each case, the overall
exon-intron pattern of N. pompilius hemocyanin
represents the most ancient situation so far
encountered. Furthermore, investigation of the 18S
rDNA sequence and karyotype analysis does indeed
suggest a most ancient situation in Nautilus (Bonnaud et al. 2004).
Phylogeny
The seven FUs of N. pompilius hemocyanin share
protein sequence identities of only 41 52% (see Table 2), whereas they are 55 67% identical to orthologous FUs from O. dofleini or S. officinalis. Sequence
identity of corresponding FUs from the latter two
hemocyanins is significantly higher (ca. 75%). This
fits the current view of phylogenetic relationships
within the Cephalopoda (see below). Using the Baysian inference method we constructed a radial phylogenetic tree based on the present N. pompilius
hemocyanin sequences and other completely sequenced FUs of molluscan hemocyanins (see Fig. 6).
Molluscan hemocyanin is phylogenetically related to
tyrosinase (e.g., van Gelder et al. 1997) and catechol
oxidase (Klabunde et al. 1998), which is also indicated by the very similar active site in these proteins
and the weak phenol oxidase activity of molluscan
hemocyanin (Salvato et al. 1998). However, at the
level of the primary structure this relationship is so
remote that to use a tyrosinase as outgroup is inefficient; therefore, we choose an unrooted representation of the phylogenetic tree. The tree reveals eight
stable branches representing the eight functional
units, thereby emphasizing their very early divergence
from a single ancestral FU by three subsequent gene
duplication and fusion events (Lieb et al. 2001; Altenhein et al. 2002). In this tree, as in former phylogenetic trees of molluscan hemocyanin, no pedigree
of the different functional units emanates from the
analyses. Likewise, the branching order of the FUs
are not strongly supported by the posterior probabilities. From this it follows that the duplication
events must have happened within a short period of
time (Lieb et al. 2001).
Within the major branches of the phylogenetic
tree, we found a very stable separation into gastropod
and cephalopod hemocyanin, which has been previously used to calibrate a molecular clock based on
fossil records of the gastropod-cephalopod split in the
late Cambrian (Benton 1993; Lieb et al. 2000;
Altenhein et al. 2002). According to other fossils,
separation of the branches leading to the extant
Nautiloidea (Nautilus) and Coleoidea (Octopus,
Sepia), respectively, might have already happened
470 million years ago, close to the Cambrian-Ordiv-
373
ician transition, but definitely it had occurred by the
early Devonian, 400 million years ago. In this
context, it is remarkable that the present molecular
clock indicates a Nautiloidea-Coleoidea split 415 ±
24 million years ago (see Fig. 7). Moreover, the
phylogenetic tree in Fig. 6 suggests that both the
Cephalopoda and the Coleoidea are monophyletic.
Although these clades are well supported by a variety
of independent data, they are still debated (Bonnaud
1997), and to confirm the Coleoidea hypothesis still
requires sequence data from the Vampyromorpha
and Theutoidea.
Within the Cephalopoda, the basal position of the
branch leading to N. pompilius is without doubt (see
Fig. 6). Even so, a clear structural correspondence
between NpH-a-b-c-d-e-f-g and OdH-a-b-c-d-e-f-g
can be emphasized (see Fig. 5), and this is also
reflected by the similar quaternary structure of Nautilus and Octopus hemocyanin (see Fig. 3 and Chignell et al. 1997). Therefore, the situation in Sepia and
Loligo hemocyanin, with an additional FU yielding a
more compact collar, and the evidence that this FU
apparently originated from one of the central ‘‘standard’’ FUs (Chignell et al. 1997; Lamy et al. 1998),
clearly appears as a synapomorphic character and
supports the concept that the Decabrachia (i.e., Sepioidea and Teuthoidea) are monophyletic. The position of the Vampyromorpha is still uncertain
because they combine morphological characters of
both Decabrachia and Octobrachia (Jeletzky 1966;
von Boletzky 1992). With only seven FUs, their
hemocyanin is clearly Octopus-like (Mouche et al.
1999), and also mitochondrial DNA sequences indicate this (Bonnaud et al. 1997). The present results
conclusively demonstrate that from its exceptional
size, complexity, and variability, hemocyanin is a
very suitable molecule from which to unravel deep
phylogenetic branching orders within the Cephalopoda. Completion of the Sepia hemocyanin sequence
(currently in progress) and sequencing of hemocyanin
from a vampyromorph and Loligo will hopefully allow a more detailed phylogenetic analysis in the nearfuture.
Acknowledgments.
We thank Prof. Dr. J. Robin Harris (Institute
of Zoology, University of Mainz) for critically reading the manuscript and correcting the language and our colleague Wolfgang
Gebauer for providing the electron micrographs. This work was
financially supported by DFG grants to J.M. (Ma843) and by the
biosyn company (Fellbach, Germany).
References
Albrecht U, Keller H, Gebauer W, Markl J (2001) Rhogocytes
(pore cells) as the site of hemocyanin biosynthesis in the marine
gastropod Haliotis tuberculata. Cell Tissue Res 304:455 462
Altenhein B, Markl J, Lieb B (2002) Gene structure and hemocyanin isoform HtH2 from the mollusc Haliotis tuberculata indicate early and late intron hot spots. Gene 301:53 60
Bause E (1983) Structural requirements of N-glycosylation of
proteins. Studies with proline peptides as conformational
probes. Biochem J 209:331 336
Benton MJ (1993) In: The fossil record 2. Chapman and Hall,
London, pp 125 270
Bonaventura C, Bonaventura J, Miller KI, van Holde KE (1981)
Hemocyanin of the chambered Nautilus: structure-function
relationships. Arch Biochem Biophys 211:589 598
Bonnaud L, Boucher-Rodini R, Monnerot M (1997) Phylogeny of
cephalopods inferred from mitochondrial DNA sequences. Mol
Phylogenet Evol 7:44 54
Bonnaud L, Ozouf-Costaz C, Boucher-Rodini R (2004) A molecular and karyological approach to the taxonomy of Nautilus.
CR Biol 327:133 138
Chignell D, van Holde KE, Miller KI (1997) The hemocyanin of
the squid Sepioteuthis lessoniana: Structural comparison with
other cephalopod hemocyanins. Comp Biochem Physiol
118:895 902
Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform
extraction. Anal Biochem 162:156 159
Cuff ME, Miller KI, van Holde KE, Hendrickson WA (1998)
Crystal structure of a functional unit from Octopus hemocyanin. J Mol Biol 278:855 870
Declerq L, Witters R, Preaux G (1990) Partial sequence determination of Sepia officinalis hemocyanin via cDNA. In: Preaux G,
Lontie R (eds) Invertebrate dioxygen carriers. Leuven University Press, Louvain, Belgium, pp 131 134
Felsenstein J (2001) PHYLIP (phylogeny inference package), version 3.6a2. Distributed by the author. Department of Genetics,
University of Washington, Seattle
Gebauer W, Harris JR, Heid H, Süling M, Hillenbrand R, Söhngen
S, Wegener-Strake A, Markl J (1994) Quaternary structure,
subunits and domain patterns of two discrete forms of keyhole
limpet hemocyanin: KLH1 and KLH2. Zoology 98:51 68
Gebauer W, Stoeva S, Voelter W, Danese E, Salvato B, Beltramini
M, Markl J (1999) Hemocyanin subunit organization of the
gastropod Rapana thomasiana. Arch Biochem Biophys
372:128 134
Guex N, Peitsch MC (1997) SWISS-MODEL and the Swiss-PdbViewer: An environment for comparative protein modeling.
Electrophoresis 18:2714 2723
Hall RL, Wood EJ, Kamberling JP, Gerwig GJ, Vliegenthart FG
(1977) 3-O-Methyl sugars as constituents of glycoproteins.
Identification of 3-O-methylgalactose and 3-O-methylmannose
in pulmonate gastropod haemocyanins. Biochem J
165:173 176
Harris JR, Horne RW (1991) Negative staining. In: Harris JR (ed)
Electron microscopy in biology. IRL Press, Oxford, UK, pp
203 228
Herskovits TT, Hamilton MG (1991) Higher order assemblies of
molluscan hemocyanins. Comp Biochem Physiol B 99:19 34
Huelsenbeck JP, Ronquist F (2001) MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17:754 755
Idakieva K, Severov S, Svendsen I, Genov N, Stoeva S, Beltramini
M, Tognon G, Di Muro P, Salvato B (1993) Structural properties of Rapana thomasiana Grosse hemocyanin: isolation,
characterization and N-terminal amino acid sequence of two
different dissociation products. Comp Biochem Physiol
106B:53 59
Jeletzky JA (1966) Comparative morphology, phylogeny, and
classification of fossil Coleoidea. The University of Kansas
paleontological contributions. Mollusca 7:1 162
Jellie AM, Tate WP, Trotman CN (1996) Evolutionary history of
introns in a multidomain globin gene. J Mol Evol 42:641 647
374
Klabunde T, Eicken C, Sacchettini JC, Krebs B (1998) Crystal
structure of a plant catechol oxidase containing a discopper
center. Nat Struct Biol 5:1084 1090
Kroll J (1973) Crossed-line immunoelectrophoresis. Scand J
Immunol 2:79 81
Kurokawa T, Wuhrer M, Lochnit G, Geyer H, Markl J, Geyer R
(2002) Hemocyanin from the keyhole limpet Megathura crenulata
(KLH) carries a novel type of N-glycans with Gal(beta1-6)Manmotifs. Eur J Biochem 269:5459 5473
Laemmli UK (1970) Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature 227:680 685
Lang WH, van Holde KE (1991) Cloning and sequencing of
Octopus dofleini hemocyanin cDNA: derived sequences of
functional units Ode and Odf. Proc Natl Acad Sci USA
88:244 248
Lambert O, Boisset N, Penczek P, Lamy J, Taveau JC, Frank J,
Lamy JN (1994) Quaternary structure of Octopus vulgaris
hemocyanin. Three-dimensional reconstruction from frozenhydrated specimens and intramolecular location of functional
units Ove and Ovb. J Mol Biol 238:75 87
Lamy J, Gielens C, Lambert O, Taveau JC, Motta G, Loncke P,
De Geest N, Preaux G, Lamy J (1993) Further approaches to
the quaternary structure of octopus hemocyanin: A model
based on immunoelectron microscopy and image processing.
Arch Biochem Biophys 305:17 29
Lamy J, You V, Taveau JC, Boisset N, Lamy JN (1998) Intramolecular localization of the functional units of Sepia officinalis
hemocyanin by immunoelectron microscopy. J Mol Biol
284:1051 1074
Lieb B, Altenhein B, Markl J (2000) The sequence of a gastropod
hemocyanin (HtH1 from Haliotis tuberculata). J Biol Chem
275:5675 5681
Lieb B, Altenhein B, Markl J, Vincent A, van Olden E , van Holde
KE , Miller KI (2001) Structures of two molluscan hemocyanin
genes: Significance for gene evolution. Proc Natl Acad Sci USA
98:4546 4551
Lieb B, Boisguerin V, Gebauer W, Markl J (2004) cDNA sequence,
protein structure, and evolution of the single hemocyanin from
Aplysia californica, an opisthobranch gastropod. J Mol Evol
59:1 10
Linn JF, Black P, Derksen K, Rubben H, Thuroff JW (2000)
Keyhole limpet haemocyanin in experimental bladder cancer:
Literature review and own results. Eur Urol 37:34 40
Markl J, Winter S (1989) Subunit-specific monoclonal antibodies
to tarantula hemocyanin, and a common epitope shared with
calliphorin. J Comp Physiol 159B:139 151
Markl J, Lieb B, Gebauer W, Altenhein B, Meissner U, Harris JR
(2001) Marine tumor vaccine carriers: structure of the molluscan hemocyanins KLH and HtH. J Cancer Res Clin Oncol
127:R3 R9
Meissner U, Dube P, Harris JR, Stark H, Markl J (2000) Structure
of a molluscan hemocyanin didecamer (HtH1 from Haliotis
tuberculata) at 12 Å resolution by cryoelectron microscopy. J
Mol Biol 298:21 34
Miller KI, van Holde KE (2003) XIIIth Conference on Dioxygen
Binding Proteins. Mainz, Germany
Miller KI, Schabtach E, van Holde KE (1990) Arrangement of
subunits and domains within the Octopus dofleini hemocyanin
molecule. Proc Natl Acad Sci USA 87:1496 1500
Miller KI, Cuff ME, Lang WF, Varga-Weisz P, Field KG, van
Holde KE (1998) Sequence of the Octopus dofleini hemocyanin
subunit: structural and evolutionary implications. J Mol Biol
278:827 842
Mouche F, Boisset N, Lamy J, Zal F, Lamy JN (1999) Structural
comparison of cephalopod hemocyanins: Phylogenetic significance. J Struct Biol 127:199 212
Nicholas KB, Nicholas HB Jr (1997) GeneDoc: Analysis and
visualization of genetic variation. Distributed by the authors
Perbandt M, Guthohrlein EW, Rypniewski W, Idakieva K, Stoeva
S, Voelter W, Genov N, Betzel C (2003) The structure of a
functional unit from the wall of a gastropod hemocyanin offers
a possible mechanism for cooperativity. Biochemistry
42:6341 6346
Pojeta J Jr (1980) Molluscan phylogeny. Tul Stud Geol Paleontol
16:55 80
Ruth P, Schipp R, Klüssendorf B (1988) Cytomorphology and
copper content of the basal cells in the midgut-gland of Nautilus
(Cephalopoda, Tetrabranchiata). A contribution to the localization of hemocyanin synthesis. Zoomorphology 108:1
11
Ruth P, Schimmelpfennig R, Schipp R (1999) Comparative
immunohistochemical and immunocytochemical investigations
on the location of haemocyanin synthesis in dibranchiate and
tetrabranchiate Cephalopods (Sepia and Nautilus). In: Olóriz F,
Rodriguez-Tovar FJ (eds) Advancing research on living and
fossil cephalopods. Plenum Press, New York, pp 189 202
Salvato B, Santamaria M, Beltramini M, Alzuet G, Casella L
(1998) The enzymatic properties of Octopus vulgaris hemocyanin: o-diphenol oxidase activity. Biochemistry 37:14065
14077
Sambrook J, Russel DW (2001) Molecular cloning: a laboratory
manual. Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, NY
Sminia T (1977) Haemocyanin-producing cells in gastropod molluscs. In: Bannister JV (ed) Structure and function of haemocyanin. Springer, Berlin, pp 279 288
Söhngen SM, Stahlmann A, Harris JR, Muller SA, Engel A, Markl
J (1997) Mass determination, subunit organization and control
of oligomerization states of keyhole limpet hemocyanin (KLH).
Eur J Biochem 248:602 614
Stoeva S, Idakieva K, Betzel C, Genov N, Voelter W (2002) Amino
acid sequence and glycosylation of functional unit RtH2-e from
Rapana thomasiana (gastropod) hemocyanin. Arch Biochem
Biophys 399:149 158
Streit K, Jackson D, Degnan BM, Lieb B (2005) Developmental
expression of two Haliotis asinina hemocyanin isoforms. Differentiation 73:341 349
Tajima F (1993) Simple methods for testing molecular clock
hypothesis. Genetics 135:599 607
Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins
DG (1997) The ClustalX Windows interface: flexible strategies
for multiple sequence alignment aided by quality analysis tools.
Nucleic Acids Res 24:4876 4882
Top A, Gielens C, Witters R, van Beumen J, Preaux G (1990)
Partial amino acid sequence and location of the carbohydrate
chain in functional unit of Sepia officinalis. In: Preaux G, Lontie
R (eds) Invertebrate dioxygen carriers. Leuven University Press,
Louvain, Belgium, pp 119 124
Topham R, Tesh S, Westcott A, Cole G, Mercatante D, Kaufman
G, Bonaventura C (1999) Disulfide bond reduction: A powerful,
chemical probe for the study of structure-function relationships
in the hemocyanins. Arch Biochem Biophys 369:261 266
van Gelder CWG, Flurkey WH, Wichers HJ (1997) Sequence and
structural features of plant and fungal tyrosinases. Phytochemistry 45:1309 1323
van Holde KE, Miller KI (1995) Hemocyanins. Adv Protein Chem
47:1 81
von Boletzky S (1992) Evolutionary aspects of development, life
style, and reproductive mode in incirrate octopods (Mollusca,
Cephalopoda). Rev Suisse Zool 99:755 770
Weeke B (1973) Crossed immunoelectrophoresis. Scand J Immunol
2:47 56