ABSTRACT
The erythrocruorin of Lumbricus terrestris (LtEc) is a relatively large macromolecular assembly that consists of at least four different hemoglobin subunits (A, B, C, and D) and four linker subunits (L1, L2, L3, and L4). The complexity and stability of this large structure make LtEc an attractive hemoglobin-based oxygen carrier that could potentially be used as a substitute for donated red blood cells. However, the sequences of the LtEc subunit sequences must be determined before a scalable recombinant expression platform can be developed. The goal of this study was to sequence the L. terrestris genome to identify the complete sequences of the LtEc subunit genes. Our results revealed multiple homologous genes for each subunit (e.g., two homologous A globin genes; A1 and A2), with the exception of the L4 linker. Some of the homologous genes encoded identical peptide sequences (C1 and C2, L1a and L1b), while cDNA and mass spectrometry experiments revealed that some of the homologs are not expressed (e.g., A2). In contrast, multiple sequences for the B, D, L2, and L4 subunits were detected in LtEc samples. These observations reveal novel degeneracy in LtEc and other annelids, along with some new revisions to its previously published peptide sequences.
