Erythropoiesis is a complicated process in which red blood cells are formed from haematopoietic stem cells in the bone marrow. A research team led by Assistant Professor Rajesh Chandramohanadas at Singapore University of Technology and Design (SUTD) is now able to explain this fascinating biological phenomenon. The mystery behind the red blood cells maturation published in the British Journal of Haematology solved through quantitative profiling of protein composition and imaging.
To yield highly deformable biconcave erythrocytes, immature red blood cells also known as reticulocytes undergo dynamic re-arrangements, towards the end of this process. The deformable biconcave erythrocytes (also known as normocytes), which perform gaseous and nutrient exchange throughout the body. Molecular mechanisms underpinning these remarkable morphological and biomechanical transformations remained largely unknown.
Many times, less than 2% of the total red blood cells, reticulocytes form a small proportion of human peripheral blood. Therefore, for large-scale experiments, it is difficult to purify them in sufficient quantities and quality. SUTD researchers to circumvent this problem, purified young reticulocytes from cord blood (which contains ~4% reticulocytes) from normal term pregnancies.
To isolate immature red blood cells a magnetic selection protocol using an antibody against transferrin receptor was used. These cells again fractionated into membrane and soluble samples, to reduce sample complexity and increase overall coverage. For the identification and quantification of more than 1800 proteins, a sophisticated quantitative mass spectrometry technique was employed. This is by far the most comprehensive information on protein composition for human red blood cells.
Proteins such as talin and tubulin are short-listed, which dramatically changed between immature and mature blood cells, research team carefully analyzed in this large dataset. Although such proteins are known to confer stiffness to biological cells, these proteins were confirmed as residues that remained in the reticulocytes from precursor cells.
The researchers then looked at the abundance of other proteins such as a group of proteins called spectrins, which are responsible for maintaining cellular architecture. The arrangements of these proteins were drastically different in reticulocytes as observed through microscopy, but the overall amount of spectrins remained comparable between reticulocytes and normocytes. During maturation, filaments that formed spectrin-based network shrunk by roughly 20% and this accounts for the transition in shape and deformability of reticulocytes.
"This robust dataset on the protein composition of human red blood cells could help promote understanding of pathological conditions that affect blood cell maturation and function. Furthermore, these results will facilitate targeted analysis of interactions between blood cells and infectious agents- such as Plasmodium vivax malaria parasites, which only infect young human reticulocytes," said by Prof Chandramohanadas Principal Investigator.