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Characterization of the Cancer-Associated Meprin Βeta Variants G45R and G89R
Antonin Gellrich1, Franka Scharfenberg1, Florian Peters2
1Biochemical Institute, Kiel University, Kiel, Germany.
Abstract:
Meprin β is a metalloprotease associated with neurodegeneration, inflammation, extracellular matrix homeostasis, transendothelial cell migration, and cancer. In this study, we investigated two melanoma-associated variants of meprin β, both exhibiting a single amino acid exchange, namely, meprin β G45R and G89R. Based on the structural data of meprin β and with regard to the position of the amino acid exchanges, we hypothesized an increase in proteolytic activity in the case of the G45R variant due to the induction of a potential new activation site and a decrease in proteolytic activity from the G89R variant due to structural instability. Indeed, the G89R variant showed, overall, a reduced expression level compared to wild-type meprin β, accompanied by decreased activity and lower cell surface expression but strong accumulation in the endoplasmic reticulum. This was further supported by the analysis of the shedding of the interleukin-6 receptor (IL-6R) by meprin β and its variants. In transfected HEK cells, the G89R variant was found to generate less soluble IL-6R, whereas the expression of meprin β G45R resulted in increased shedding of the IL-6R compared to wild-type meprin β and the G89R variant. A similar tendency of the induced shedding capacity of G45R was seen for the well-described meprin β substrate CD99. Furthermore, employing an assay for cell migration in a collagen IV matrix, we observed that the transfection of wild-type meprin β and the G45R variant resulted in increased migration of HeLa cells, while the G89R variant led to diminished mobility.
Insights
Meprin β G45R variant enhances proteolytic activity and cell migration, while G89R variant shows reduced expression and activity. These findings highlight differential impacts of meprin β variants in biological processes.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Meprin β is a metalloprotease implicated in various physiological and pathological processes, including neurodegeneration, inflammation, and cancer.
- Melanoma-associated variants of meprin β, such as G45R and G89R, involve single amino acid substitutions that may alter its function.
- Understanding the functional consequences of these variants is crucial for elucidating their roles in disease.
Purpose of the Study:
- To investigate the functional impact of two melanoma-associated meprin β variants, G45R and G89R, on proteolytic activity, protein expression, and cell migration.
- To compare the activity and cellular localization of these variants with wild-type meprin β.
- To assess the effect of meprin β variants on the shedding of specific substrates like IL-6 receptor and CD99.
Main Methods:
- Site-directed mutagenesis was used to generate meprin β G45R and G89R variants.
- HEK cells were transfected to express wild-type and variant forms of meprin β.
- Proteolytic activity was assessed by measuring the shedding of IL-6 receptor and CD99.
- Cell surface expression and intracellular localization were analyzed.
- Cell migration assays were performed using HeLa cells in a collagen IV matrix.
Main Results:
- The G89R variant exhibited reduced expression, decreased proteolytic activity, and lower cell surface presence, accumulating in the endoplasmic reticulum.
- The G45R variant demonstrated increased proteolytic activity, leading to enhanced shedding of IL-6 receptor and CD99 compared to wild-type meprin β.
- Both wild-type meprin β and the G45R variant promoted HeLa cell migration, whereas the G89R variant diminished it.
Conclusions:
- Meprin β variants G45R and G89R display distinct functional profiles, with G45R enhancing activity and G89R impairing it.
- The differential activities of these variants influence substrate shedding and cell migration, suggesting specific roles in melanoma and other associated conditions.
- Structural alterations in meprin β can lead to significant functional divergence, impacting cellular processes and disease pathology.
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