N-terminal Amino Acid Sequence Comparison of Hemocyanin Subunits from the Spiders, Argiope amoena, Argiope bruennichii, Araneus ventricosus, Nephila clavata and Dolomedes sulfureus.

Abstract

By two dimensional electrophoresis using acrylamide disc and slab gels, hemocyanin hexamers of four araneid spiders Argiope amoena, A. bruennichii, Araneus ventricosus, Nephila clavata, and a pisaurid spider Dolomedes sulfureus were separated completely into individual subunits. Direct protein sequencing of hemocyanin monomer subunits gave the N-terminal sequences for the first 10-25 amino acid residues, except for subunits with blocked amino terminals. From the sequence comparison between the araneid spider's hemocyanin subunits, it is supposed that the araneid spiders share the subunits with similar N-terminal sequences derived from a common ancestral sequence and that duplications of the ancestral sequence occurred in the lineages of A. amoena and N clavata or in the common ancestor immediately before the divergence of these two spiders and then in the lineage of N clavata. On the contrary, the pisaurid spider has hemocyanin subunits with unique N-terminal sequences which are not found in any hemocyanin until now.

Evolution of Hemocyanin Subunits in Mygalomorph Spiders. Distribution of Hemocyanin Subunits and Higher Classification of the Mygalomorphae.

“…1 and analyzed the N-terminals of these monomer subunits. The Nterminal amino acid sequences of twenty-two monomer subunits were determined, because the subunits AR1-3, AY1-3 and AK1 could not be sequenced for the N-terminals, suggesting blocked amino terminals, which were reported in hemocyanin subunits of a scorpion (Ali et al, 1995) and araneomorph spiders (Takasu and Sugita, 1997). Direct protein sequencing of the hemocyanin monomers presented the sequences for the first 13 -43 amino acid residues.…”

“…The hemocyanin subunits in a disc gel were re-electrophoresed into a slab gel to separate hemocyanin subunits completely (Takasu and Sugita, 1997) and the subunits in the slab gel were transferred to a polyvinylidene difluoride (PVDF) membrane.…”

“…On the other hand, from comparison of N-terminal amino acid sequences of hemocyanin subunits, the orthologous subunits which are available for making evolutionary tree of animals were found in horseshoe crabs (Sugita and Murayama, 1998), scorpions (Sugita et al, 1999) and araneomorph spiders (Takasu and Sugita, 1997), respectively. Furthermore, they could discuss origins and duplications of the hemocyanin subunits during the evolution of animals having these subunits.…”

Hemocyanin Subunits of a Whipscorpion, Typopeltis crucifer, and a Primitive Spider, Heptathela kimurai: Orthologous Hemocyanin Subunits in Arachnids

“…The monomer subunits associate noncovalently in most cases, although a few hemocyanins contain one or more disulfide-linked dimers (Van Holde and Miller, 1995). The heterogeneity of the monomer subunits of spider and scorpion hemocyanins has been demonstrated by means of polyacrylamide gel electrophoresis (Sugita and Sekiguchi, 1975;Takasu and Sugita, 1997;Sugita et al, 1999;Kuwada and Sugita, 2000), but each of these subunits has a mass of about 75KDa and carries one oxygen-binding site, suggesting that all are clearly derived from a common ancestral protein (Van Holde and Miller, 1995).…”

Orthologous Hemocyanin Subunits between Asian and American Horseshoe Crabs and Duplication of Hemocyanin Subunits in American Horseshoe Crab Lineage.

“…As the first step to make species tree of spiders using the hemocyanin subunit sequence data, Takasu and Sugita (1997) analyzed the N-terminal amino acid sequences of hemocyanin monomer subunits from four araneid spiders and reported that duplications of monomer subunit occurred in the lineages of Argiope amoena and Nephila clavata or in the common ancestor immediately before the divergence of these two spiders and then in the lineage of N clavata. Thus, they could show that N-terminal amino acid analysis is available not only to find orthologous subunits but also to study the molecular evolution of hemocyanin subunits.…”