Tài liệu Báo cáo khoa học: Complete subunit sequences, structure and evolution of the 6 · 6-mer

Complete subunit sequences, structure and evolution

of the 6 · 6-mer hemocyanin from the common house centipede,

Scutigera coleoptrata

Kristina Kusche*, Anne Hembach, Silke Hagner-Holler, Wolfgang Gebauer and Thorsten Burmester

Institute of Zoology, Molecular Animal Physiology, University of Mainz, Germany

Hemocyanins are large oligomeric copper-containing pro￾teins that serve for the transport of oxygen in many arth￾ropod species. While studied in detail in the Chelicerata and

Crustacea, hemocyanins had long been considered unnec￾essary in the Myriapoda. Here we report the complete

molecular structure of the hemocyanin from the common

house centipede Scutigera coleoptrata (Myriapoda: Chilo￾poda), as deduced from 2D-gel electrophoresis, MALDI￾TOF mass spectrometry, protein and cDNA sequencing,

and homology modeling. This is the first myriapod hemo￾cyanin to be fully sequenced, and allows the investigation of

hemocyanin structure–function relationship and evolution.

S. coleoptrata hemocyanin is a 6 · 6-mer composed of

four distinct subunit types that occur in an approximate

2 : 2 : 1 : 1 ratio and are 49.5–55.5% identical. The cDNA

of a fifth, highly diverged, putative hemocyanin was

identified that is not included in the native 6 · 6-mer

hemocyanin. Phylogenetic analyses show that myriapod

hemocyanins are monophyletic, but at least three distinct

subunit types evolved before the separation of the Chilo￾poda and Diplopoda more than 420 million years ago. In

contrast to the situation in the Crustacea and Chelicerata,

the substitution rates among the myriapod hemocyanin

subunits are highly variable. Phylogenetic analyses do not

support a common clade of Myriapoda and Hexapoda,

whereas there is evidence in favor of monophyletic

Mandibulata.

Keywords: hemocyanin; subunit diversity; structure; Arthro￾poda; evolution.

Oxygen transport in the body fluid of many arthropod and

molluscan species is mediated by large copper-containing

proteins that are referred to as hemocyanins [1–3]. Mollu￾scan hemocyanins, however, are not significantly related to

the arthropod proteins and are most likely of divergent

evolutionary origin [3–5]. Arthropod hemocyanins typically

form hexamers or oligo-hexamers (up to 8 · 6)of subunits

with about 620–660 amino acids [1,2,6]. Oxygen binding

is mediated by a pair of copper atoms that are coordinated

by six histidine residues per monomer.

In the past 30 years, hemocyanins have been studied

thoroughly in the Chelicerata and Crustacea in terms of

function, structure, sequence and evolution [1,2,6]. By

contrast, little is known about hemocyanins in Onycho￾phora, Myriapoda and Hexapoda. Oxygen supply in these

taxa is mediated via trachea [7]. Therefore, the presence of

respiratory proteins had long been considered unnecessary,

with the exception of some insect species that live under

hypoxic conditions and possess extracellular hemoglobins

[8, but also see 9]. A putative hemocyanin, however, has

been identified in the embryonic hemolymph of the

grasshopper Schistocerca americana [10], although this

respiratory protein is absent in adult animals and hemocy￾anins were lost later in hexapod evolution [6]. Hemocyanins

also occur in the Onychophora, suggesting an ancient

evolutionary origin of this type of respiratory protein [11].

Mangum et al. [12] were the first who unambiguously

demonstrated the occurrence of hemocyanins in the Myr￾iapoda. The centipede Scutigera coleoptrata (Chilopoda)

possesses a 36-mer (6 · 6)hemocyanin that closely resem￾bles other arthropod hemocyanins [12,13]. This protein is

composed of four electrophoretically and immunologically

distinct subunits (termed a : b : c : d)in the range of 74–

80 kDa, which occur in a ratio of approximately a/b/c/d ¼

2 : 2 : 1 : 1 [14]. Structurally similar 6 · 6-mer hemocya￾nins also occur in at least in one family of the Diplopoda, i.e.

the Spirostreptidae, suggesting that despite the well-devel￾oped tracheal system hemocyanins are widespread among

the Myriapoda [15,16].

Myriapod, crustacean, and chelicerate hemocyanins

strikingly differ in subunit composition and quaternary

structure [1]. In previous analyses, the complete subunit

sequences of one crustacean [17–19] and two chelicerate

hemocyanins [20,21] have been determined. Based on

sequence comparison and immunological studies, a remark￾ably different pattern of hemocyanin subunit evolution has

been observed in these two arthropod subphyla [4,6,22], but

little was known about the Myriapoda. Here we report the

complete cDNA-cloning and biochemical analyses of the

Correspondence to T. Burmester, Institute of Zoology,

University of Mainz, D-55099 Mainz, Germany.

Fax: + 49 6131 3924652, Tel.: + 49 6131 3924477,

E-mail: [email protected]

Abbreviation: Hc, hemocyanin.

*Present address: Institute of Animal Physiology, University of

Mu¨nster, Germany.

(Received 21 March 2003, revised 7 May 2003,

accepted 12 May 2003)

Eur. J. Biochem. 270, 2860–2868 (2003) FEBS 2003 doi:10.1046/j.1432-1033.2003.03664.x