Primary and tertiary structures of the first domain of Panulirus interruptus hemocyanin and comparison of arthropod hemocyanins

Abstract

The amino acid sequence of the first domain (positions 1 -175) of Panulirus interruptus hemocyanin subunit a has been determined. The sequence of residues 1 -158 (18-kDa fragment obtained by limited proteolysis) was derived from peptides obtained by digestion of this fragment with CNBr and trypsin and by subdigestion of these peptides with other enzymes. The peptides were sequenced automatically or manually. The amino acid sequence has been fitted into the electron-density map at 0.32-nm resolution.The residues of domain 1 are folded into a larger, mainly helical, globular part, containing one disulfide bridge, and a smaller part near the molecular twofold axis. The latter part consists of an c( helix and a fl strand which contains a covalently attached carbohydrate moiety. The sites susceptible to limited proteolytic cleavage of the subunit are discussed.Comparison of the N-terminal sequence with those of other arthropod hemocyanins revealed, besides an Nterminal extension of five residues, the presence of a 21-residue loop (positions 22 -42) in the crustacean sequences. This loop contains helix 1.2, a less defined region in the electron-density map. It is absent in chelicerate sequences. Strong evidence is presented that: (a) the structure of the first 21 residues (including helix 1.1) is the same in all arthropod hemocyanins with known amino acid sequence; (b) a stretch containing about 15 residues (including part of helix 1.3) following the 21-residue loop has a different structure in crustaceans and chelicerates; (c) the rest of domain 1 has the same structure again. It is shown that all conserved residues are in the contact region with the other two domains.Hemocyanins are copper-containing oxygen carriers occurring in the hemolymph of arthropods and molluscs. Arthropod hemocyanins consist of hexamers or multihexamers [I, 21. In the spiny lobster, Panulirus interruptus, hemocyanin molecules are present as hexamers built from three different subunits, called a, b and c, each with a molecular mass of about 75 kDa [3]. These were formerly called 94 K, 90 K and 80 K, respectively, according to their apparent molecular mass on SDS/polyacrylamide gels. The N-terminal sequences of the three subunits have been published [4]. Subunits a and b are very similar, subunit c is quite different [5].Limited tryptic cleavage of subunit a gives rise to three fragments: an N-terminal 18-kDa fragment, a C-terminal 55-kDa fragment and a glycopeptide of 13 amino acid residues located in between [6].Both the primary and the three-dimensional structures are under investigation. The three-dimensional structure has been determined at a resolution of 0.32 nm. It is the only structure of an arthropod hemocyanin in which the polypeptide chain has been traced. The polypeptide chain, which is 657 residues Correspondence to N. M . Soeter, Rudolf Magnus Instituut voor

Panulirus interruptus hemocyanin. The elucidation of the complete amino acid sequence of subunit a

“…The sites of limited proteolysis are indicated. The amino acid sequence of the glycopeptide [lo], the carbohydrate-containing CNBr peptide [ll] and the 18-kDa fragment [12] have already been published. Some additional evidence regarding the region near the first limited-proteolytic cleavage site (Lys-158) came from a peptide designated CBO ( Fig.…”

“…In contrast to the 18-kDa fragment [12], the molecular mass of the 55-kDa fragment (55435 Da) is overestimated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (71 kDa). Therefore, the 55-kDa fragment is to blame for the major part (16 kDa) of the total overestimation of 17 kDa for whole subunit a.…”

Panulirus interruptus hemocyanin

“…The presence of glutamic acid at position 139 (peptide T15a and a tryptic peptide CB4T2 derived from CB4) is a correction of the previous assignment in subunit a [19].…”