Recombinant functional multidomain hemoglobin from the gastropod Biomphalaria glabrata

Area of Science:

• Biochemistry
• Molecular Biology
• Parasitology

Background:

• Biomphalaria glabrata is the intermediate host for Schistosoma mansoni, a human parasite.
• The extracellular hemoglobin (Hb) of B. glabrata is a large multimer (1.44 MDa) composed of 240 kDa subunits.
• Previous work established the amino acid sequences for two B. glabrata Hb subunits (BgHb1, BgHb2) and a partial sequence for a third (BgHb3).

Purpose of the Study:

• To investigate the structure-function relationships of B. glabrata extracellular hemoglobin (BgHb).
• To produce functional BgHb subunits and dimers using recombinant expression systems.
• To characterize the oxygen-binding properties and aggregation behavior of recombinant BgHb.

Main Methods:

• Recombinant expression of BgHb subunits and fragments in Escherichia coli and insect cells.
• Purification and characterization of recombinant BgHb products.
• Oxygen-binding measurements (P50, cooperativity, Bohr effect) of recombinant BgHb.
• Electron microscopy to observe aggregation of recombinant BgHb.

Main Results:

• Functional BgHb1 and BgHb2 subunits and BgHb2 fragments were successfully expressed recombinantly.
• Recombinant BgHb1 expressed as a disulfide-bridged dimer (480 kDa).
• Oxygen-binding studies revealed a P(50) of approximately 7 mmHg with no significant cooperativity or Bohr effect.
• The covalently linked dimer of BgHb1, but not the monomer, formed aggregates similar to native BgHb molecules under electron microscopy.

Conclusions:

• Recombinant expression systems can produce functional B. glabrata hemoglobin subunits and dimers.
• The dimeric form of BgHb1 is crucial for forming higher-order aggregates resembling native structures.
• These findings contribute to understanding the molecular basis of oxygen transport and multimerization in invertebrate hemoglobins.