Introduction. Siglec (sialic acid binding immunoglobulin-like(Ig-like) lectins) is I-type lectin receptors that binds to sialic acid and mostly expressed on immune cells. Siglec plays a crucial role in discriminating Self from Non-Self, also controlling and inhibiting inappropriate activation of the immune cells. Human neutrophils are the most abundant leukocytes that circulate in the bloodstream. Neutrophils are essential immune cells that initiate inflammatory responses against pathogens and microbes. Human neutrophils express two inhibitory (Siglec 5 and 9) and one activation (Siglec 14) Siglec receptor. Molecular characterization these Siglec receptors has been done on transfected and mammalian cell lines, rather than on the primary human neutrophils.
Aims. The aim of this work is to undertake molecular characterization of Siglecs on the human neutrophils by western blot and sucrose density centrifugation.
Methods and Materials. Neutrophil prep conducted applying Percoll Density gradient (55%,71%. And 80%) (GE Healthcare). The cells were lysed in with nonionic detergent (50mM Tris- HCL buffer, pH7.4, 1% Tritone 100X, 150 mM NaCl, 1 mM EDTA including a protease inhibitor) and with non-detergent lysis buffer (50mM Tris-HCL buffer, pH7.4, 150 mM NaCl, 1 mM EDTA with protease inhibitor). To isolate membrane proteins from the cell materials used sucrose density gradient, centrifuged by ultracentrifuge (100.000g). Siglecs determined on western blot, using anti-Siglec 5/14 (AI5) and anti-Siglec 9 (KALI) antibodies. To reduce glycosylation used PNGase.
Result and Discussion. Flow cytometry detected siglec 5/14 and 9 on human neutrophils. After examining Siglecs expression level on the human neutrophil, it has been concluded that Siglec 5/14 expression is high level in the human; in contrast, Siglec 9 expressed at a lower level. Sucrose density gradient allowed to differentiate transmembrane proteins from the rest cell material. The Siglec 9 concentration increased using immunoprecipitation (IP) method and it allowed to continue work at molecular level. Molecular weight of the Siglecs were 100-110 kDa, which comes against to the GeneCard data (50-45 kDa). It was due to glycosylation sites on the siglecs, they were around 8. Each glycosylation site theoretically adds 5 kDa of glycan, which adds 40-45 more kDa to the siglecs. It was proved by adding glycosylation reductase, PNGase. Siglecs reduced in molecular weight and they were at the same level as predicted by the gene/amino acid sequence.
Conclusion. To study siglecs on the human neutrophils required a great effort, simply because of siglec 9 expressed in low level and Siglec 5 could not easily be differentiated from siglec 14. The human neutrophils one on the most important and at the same time the most dangerous immune cells in our body in inappropriate activation. While targeting pathogenic microorganisms, it might react to the host cells, which lead to the autoimmune chronic inflammation and no cure yet available. Siglecs already being therapeutically targeted to suppress allergic response and yet not fully studied. It demonstrated for the first time a study of siglecs on the molecular level, and it gives an opportunity to explore siglecs with different manipulations and detection on the western blot.
- .Parham P. The Immune System. 4th ed. New York: Garland Science; 2014. 532 p.
- . Crocker PR, Varki A. Siglecs, sialic acids and innate immunity. Trends Immunol [Internet]. 2001;22(6):337-42.
- .Crocker PR, Varki A. Siglecs in the immune system. Immunology. 2001;103(2):137-45.
- .Crocker PR, Redelinghuys P. Siglecs as positive and negative regulators of the immune system. Biochem Soc Trans [Internet]. 2008;36(Pt 6):1467-71.
- .Macauley MS, Crocker PR, Paulson JC. Siglec-mediated regulation of immune cell function in disease. Nat Rev Immunol [Internet]. 2014;14(10):653-66.