REPOSITORY > RESULTS

Abstract

Processing of extracellular membrane proteins also known as ectodomain shedding is a biochemical process that has recently started getting increasing attention. Initially, it was believed that only metalloproteases were able to act as sheddases, but nowadays there are more and more proteases, including cysteine proteases and gingipains, which are recognized as shedases.
Among others, cysteine cathepsins, especially cathepsins L and S, can act as sheddases under special circumsatances, i.e. when released into extracellular millieu. In the tumor microenvironment, both cathepsins are secreted by tumor cells, as well as by immune and other cells that have infiltrated into the tumour stroma. We observed that cathepsin L affects the activation of the complement system on the surface of tumor cells. After treatment with the enzyme, complement activation on the surface of breast cancer tumour cells was reduced. Our aim was therefore to determine which pathway of complement activation is the most affected by the cathepsins and at what step of this pathway it has the biggest effect. Furthermore, we wanted to identify the physiological substrates of cathepsin L that are responsible for reduction of complement activation. We found out that shedding of cathepsin L and S predominantly affects the classical pathway of complement activation. Moreover, the main target molecule in this cascade is C1q. This is the most upstream molecule of the classical pathway of activation, which is responsible for recognition of non-self cells. Further experiments have shown that the binding partner of C1q is the adhesion molecule CD44 and that its shedding is a step in which the complement activation is reduced. We proposed that after shedding of CD44, the cleaved fragment exposes some cryptic sites that bind C1q with an affinity, thereby preventing further activation of the complement although C1q can also bind to CD44 on intact cells. We further suggest that this is a new way how tumour cells evade the immune system.
In addition to cathepsins, we also worked with cysteine proteases of bacterial origin - gingipains secreted by the pathogenic bacteria Porphyromonas gingivalis, the causative agent of periodontitis. It was assumed that gingipains were also shedases and that the proteolysis of surface proteins dysregulated the activation of the complement system. However, it turned out that gingipains mostly cleave adhesion proteins, while we could not identify any complement regulators as gingipain substrates. It should be noted that gingapin shedding is only the first step in the process of degradation of extracellular membrane proteins. The extent of shedding or degradation depends on the concentration of gingipains, which is associated with the progression of the disease. In the initial phase of the periodontitis, the concentration of gingipains and P. gingivalis bacteria is low in the oral cavity, which is why the processing of extracellular substrates is limited and shedding can be observed. However, when the disease develops and the concentration of gingipains is higher, the unregulated degradation of membrane proteins prevails. In this way, gingipains dysregulate the immune system and regulatory and signaling pathways, and trigger anoikis, programmed cell death of host epithelial cells, resulting from the loss of contacts with adjacent cells. In particular, the latter is probably of great significance, since most of the identified gingipain substrates in our experiments were adhesion molecules. At the same time, one of the main symptoms and the consequence of periodontitis is the destruction of soft gum tissue and alveolar bone in later stages, which can largely be due to anoikis.