The mast cell transcriptome and the evolution of granule proteins and Fc receptors

University dissertation from Uppsala : Acta Universitatis Upsaliensis

Abstract: Protection against disease-causing pathogens, known as immunity, involves numerous cells organs, tissues and their products. To able to understand the biology of immune cells (hematopoietic cells) and their role in an immune system, we have used several different methods, including transcriptome analyses, bioinformatics, production of recombinant proteins and analyses of some of them, focusing on the granule proteases by substrate phage display.Hematopoietic cells express surface receptors interacting with the constant region of immunoglobulins (Igs) known as Fc receptors (FcRs). These receptors play major roles in the immune system, including enhancing phagocytosis, activating antibody dependent cellular cytotoxicity and cell activation. A detailed bioinformatics analysis of FcRs reveals that the poly-Ig receptors (PIGR), FcR-like molecules and common signalling γ chain all appeared very early with the appearance of the bony fishes, and thereby represent the first major evolutionary step in FcR evolution. The FcμR, FcαμR, FcγR and FcεR receptors most likely appeared in reptiles or early mammals, representing the second major step in FcR evolution.Cells of several of the hematopoietic cell lineages contain large numbers of cytoplasmic granules, and serine proteases constitute the major protein content of these granules. In mammals, these proteases are encoded from four different loci: the chymase, the met-ase, the granzyme (A/K) and the mast cell tryptase loci. The granzyme (A/K) locus was the first to appear and came with the cartilaginous fishes. This locus is also the most conserved of the three. The second most conserved locus is the met-ase locus, which is found in bony fishes. The chymase locus appeared relatively late, and we find the first traces in frogs, indicating it appeared in early tetrapods.To study the early events in the diversification of these hematopoietic serine proteases we have analyzed key characteristics of a protease expressed by an NK-like cell in the channel catfish, catfish granzyme–like I. We have used phage display and further validated the results using a panel of recombinant substrates. This protease showed a strict preference for Met at the P1 (cleavage) position, which indicates met-ase specificity. From the screening of potential in vivo substrates, we found an interesting potential target caspase 6, which indicates that caspase-dependent apoptosis mechanisms have been conserved from fishes to mammals.A larger quantitative transcriptome analysis of purified mouse peritoneal mast cells, cultured mast cells (BMMCs), and mast cells isolated from mouse ear and lung tissue identified the major tissue specific transcripts in these mast cells as the granule proteases. Mast cell specific receptors and processing enzymes were expressed at approximately 2 orders of magnitude lower levels. The levels of a few proteases were quite different at various anatomical sites between in vivo and cultured BMMCs. These studies have given us a new insights into mast cells in different tissues, as well as key evolutionary aspects concerning the origins of a number of granule proteases and FcRs.