Identificaiton and characterization of a novel hemoglobin gene (Tgr-HbIII) from blood clam Tegillarca granosa.

Hemoglobin (Hb) is the major protein component of red blood cells (hemocytes) of the blood clam Tegillarca granosa. Three T. granosa hemoglobin genes have been mentioned in the literature, designated Tgr-HbI, Tgr-HbIIA and Tgr-HbIIB. Previously, our group identified another novel gene, Tgr-HbIII, in the Hb cluster of the chromosome-level genome but the issue of whether this Hb gene expresses functional protein remains unclear. In the current study, phylogenetic analysis revealed that Tgr-HbIII resembles an ancient Hb gene. Sequence alignment and three-dimensional structural modeling results showed that Tgr-HbIII does not bind heme due to the completely different structure at amino acid position 96-100 and replacement of the N residue in known Tgr-Hbs with Q, what causes loss of a single hydrogen bond linking heme with the globin fold. Interface prediction data suggest that Tgr-HbIII forms a homodimer (ΔG = -5.6 kcal/mol) with a similar conformation to the Tgr-HbI homodimer (ΔG = -3.5 kcal/mol). In adult T. granosa, mRNA expression of Tgr-HbIII was lower than that of Tgr-HbIIA and Tgr-HbIIB (up to 100 × ), but comparable to that of Tgr-HbI. Notably, protein expression of Tgr-HbIII was extremely low. Single-cell RNA sequencing analysis of Hb expression showed that all adult hemocytes expressed Tgr-HbI, Tgr-HbIIA and Tgr-HbIIB, while only 43 % (3872 of 8978) expressed Tgr-HbIII. Based on the collective data, we speculate that Tgr-HbIII carried oxygen prior to mutation of N to Q and subsequently evolved into a known functional remnant of Hb with an adequate mRNA/low protein expression profile. The current study provides a foundation for further research on the origin, evolution and function of molluscan Hbs.