THE GIANT EXTRACELLULAR HEMOGLOBIN FROM THE POLYCHAETE NEANTHES DIVERSICOLOR - THE CDNA-DERIVED AMINO-ACID-SEQUENCE OF LINKER CHAIN L2 AND THE EXON INTRON BOUNDARY CONSERVED IN LINKER GENES/

The 4000 kDa extracellular hemoglobin from the polychaete Neanthes div ersicolor consists of three types of subunits; three 15 kDa monomers ( chains MI, M2 and M3), a 45 kDa disulfide-bonded trimer of chains T1, T2 and T3, and two 50-55 kDa disulfide-bonded homodimeric linkers (cha ins L1 and L2). The latter linker subunits are essential for the assem bly of the other heme-containing subunits, monomers and a trimer. The cDNA encoding the linker chain L2 was amplified by polymerase chain re action (PCR), and the cDNA-derived amino acid sequence of 235 residues has been determined. The sequence showed 22-75% identity with other l inker chains. All of the linker sequences examined so far have a highl y conserved cysteine-rich segment at positions 89-130: -Cys-Xaa(6)-Cys -Asp-Gly-Xaa(2)-Asp-Cys-Xaa(4)-Cys, and the moth corresponds exactly t o the cysteine-rich repeats of the ligand-binding domains of vertebrat e low-density lipoprotein (LDL) receptors (Suzuki, T. and Riggs, A.F. (1993) J. Biol. Chem. 268, 13548-13555). A 287 bp intron interrupts th e coding sequence of Neanthes L2 gene just at the N-terminal boundary of this motif, and the position of the splice junction was exactly con served in Neanthes and Lumbricus linker genes. This suggests that the intron has been conserved for at least 450 million years in annelid li nker genes. The evolutionary origin of the remaining parts of linker c hains is unclear, but it is noteworthy that the topology of the two in trachain disulfide bridges in the C-terminal segment of linker chains is homologous with that of the carbohydrate-recognition domain of anim al C-type lectin.