Characterization and Expression of the Lucina pectinata Oxygen and Sulfide Binding Hemoglobin Genes

Abstract

The clam Lucina pectinata lives in sulfide-rich muds and houses intracellular symbiotic bacteria that need to be supplied with hydrogen sulfide and oxygen. This clam possesses three hemoglobins: hemoglobin I (HbI), a sulfide-reactive protein, and hemoglobin II (HbII) and III (HbIII), which are oxygen-reactive. We characterized the complete gene sequence and promoter regions for the oxygen reactive hemoglobins and the partial structure and promoters of the HbI gene from Lucina pectinata. We show that HbI has two mRNA variants, where the 5’end had either a sequence of 96 bp (long variant) or 37 bp (short variant). The gene structure of the oxygen reactive Hbs is defined by having 4-exons/3-introns with conservation of intron location at B12.2 and G7.0 and the presence of pre-coding introns, while the partial gene structure of HbI has the same intron conservation but appears to have a 5-exon/ 4-intron structure. A search for putative transcription factor binding sites (TFBSs) was done with the promoters for HbII, HbIII, HbI short and HbI long. The HbII, HbIII and HbI long promoters showed similar predicted TFBSs. We also characterized MITE-like elements in the HbI and HbII gene promoters and intronic regions that are similar to sequences found in other mollusk genomes. The gene expression levels of the clam Hbs, from sulfide-rich and sulfide-poor environments showed a significant decrease of expression in the symbiont-containing tissue for those clams in a sulfide-poor environment, suggesting that the sulfide concentration may be involved in the regulation of these proteins. Gene expression evaluation of the two HbI mRNA variants indicated that the longer variant is expressed at higher levels than the shorter variant in both environments.

SAXS structure of homodimeric oxyHemoglobin III from bivalve Lucina pectinata

“…There was no statistical difference in hemoglobin presence within tissues, supporting the formation of HbII‐HbII, HbIII‐HbIII, and HbII‐HbIII dimers. [ 30 ] However, in the clam's internal environment, the H 2 S concentration has been correlated with Hb's expression levels. [ 30 ] Therefore, the hydrogen sulfide concentration is a factor regulating the expression of HbII and HbIII and may also control the formation of both dimeric and tetrameric species.…”

“…[ 30 ] However, in the clam's internal environment, the H 2 S concentration has been correlated with Hb's expression levels. [ 30 ] Therefore, the hydrogen sulfide concentration is a factor regulating the expression of HbII and HbIII and may also control the formation of both dimeric and tetrameric species. Another potential factor regulating the protein oligomerization is the CRP present in the clam.…”

“…Another potential factor regulating the protein oligomerization is the CRP present in the clam. [ 3,4,29,30 ] However, this is an open question, and additional studies are necessary to reveal the role of the CRP in this pathway.…”

De Novo Assembly of Lucina pectinata Genome using Ion Torrent Reads

“…As mentioned before, only a few proteins have been characterized for L. pectinata. In terms of gene sequence, the gene structure for HbI from L. pectinata is partially characterized, while the genes coding for HbII and HbIII are fully characterized having a 4exon/3intron gene structure [30]. We wanted to find the complete HbI gene sequence in both of our draft genome assemblies.…”

“…Of all of these, three matches had an alignment of more than 500 bp with identities above 98%, covering part of the first incomplete intron an exon 2, the end of intron 2, exon 3-intron 3, and intron 3 and most of exon 4. Most of the sequences obtained with the Blastn results using the HbI incomplete gene sequence as query, align with the regions of HbI where repetitive regions are found [30]. Similar searches were performed with the sequences of the HbII and HbIII genes.…”