Linker chains of the gigantic hemoglobin of the earthworm Lumbricus terrestris: Primary structures of linkers L2, L3, and L4 and analysis of the connectivity of the disulfide bonds in linker L1

Abstract

The extracellular hemoglobin (Hb) of the earthworm, Lumbricus terrestris, has four major kinds of globin chains: a, b, c, and d, present in equimolar proportions, and additional non-heme, non-globin scaffolding chains called linkers that are required for the calcium-dependent assembly of the full-sized molecule. The amino acid sequences of all four of the globin chains and one of the linkers (L1) have previously been determined. The amino acid sequences via cDNA of each of the three remaining linkers, L2, L3, and L4, have been determined so that the sequences of all constituent polypeptides of the hemoglobin are now known. Each linker has a highly conserved cysteine-rich segment of approximately 40 residues that is homologous with the seven ligand-binding repeats of the human low-density lipoprotein receptor (LDLR). Analysis of linker L1 shows that the connectivity of the three disulfide bonds is exactly the same as in the LDLR ligand-binding repeats. The presence of a calcium-binding site comprising one glutamyl and three aspartyl residues in both the LDLR repeats and in the linkers supports the suggestion that calcium is required for the folding and disulfide connectivity of the linkers as in the LDLR repeats. Linker L2 is markedly heterogeneous and contains unusual glycine-rich sequences near the NH2-terminus and a polar zipper-like sequence with imperfect repeats of Asp-Asp-His at the carboxyl terminus. Similar Asp-Asp-His repeats have been found in a protein homologous to superoxide dismutase in the hemolymph of certain mussels. These repeats may function as metal-binding sites.

Partial characterization of giant extracellular hemoglobin of Glossoscolex paulistus: A MALDI-TOF-MS study

“…Twelve such complexes of heme containing chains are linked together by 36 heme-deficient linker chains to provide the total mass of about 3600 kDa. Recently, the sequences of amino acid residues for the four linker chains (L 1 , L 2 , L 3 and L 4 ) of HbLt have been completely determined [7]. The 1/12 subunit of the whole protein is associated to (abc) 3 d 3 L 3 , where L corresponds to the linker chains.…”

Ferric species equilibrium of the giant extracellular hemoglobin of Glossoscolex paulistus in alkaline medium: HALS hemichrome as a precursor of pentacoordinate species

“…HbLt consists of about 180 polypeptide chains in an arrangement of two heme-containing subunits (monomer M and disulfide-bonded trimer T) and nonglobin linker subunits ("Linker" chains) with molecular weights in the range 24-32 kDa assembled as a "bracelet model" of quaternary structure. In a recent work, the amino acid sequences of the four kinds of "Linker" chains (L1, L2, L3 and L4) have been determined [17]. HbGp is also constituted by a large number of subunits containing heme groups with molecular weights in the range of 16-19 kDa forming a monomer of 16 kDa and a trimer of 52 kDa and non-heme structures with molecular weights in the range 24-32 kDa [18], conferring to the whole protein a minimum molecular weight of 3100 kDa and a double layered hexagonal oligomeric structure [19,20].…”

Effect of heavy metal exposure on blood haemoglobin concentration and methemoglobin percentage in Lumbricus terrestris

“…Earthworm haemoglobin is a large extracellular haemoprotein (Kao et al 2006) organized into a dodecameric assemblage (Strand et al 2004). The overall molecular structure comes from noncovalent complex of globin subassemblies with linker subunits.…”

“…Remarkably, earthworm Hb is more than one order of magnitude larger in molecular mass than the tetramers usually found in vertebrates. It is a gigantic extracellular hemoprotein (Kao et al 2006) organized into a dodecameric assemblage (Strand et al 2004). It has a very high affinity for oxygen (faster oxygenation) and can become 95% saturated at 19-22 mmHg partial pressure of O 2 at room temperature (Weber 1978).…”