Glycocapture-based cell surface proteome analysis of human epidermal melanocytes and melanoma cells

Cell surface proteins collectively constitute the surfaceome, a special subset of the plasma membrane proteome. The surfaceome plays crucial roles in determining cellular phenotype and identity, and its qualitative and quantitative composition undergoes significant alterations during healthy and pathological differentiation. Currently, there are no data available concerning the complement of cell surface proteins of healthy and pathological human pigment cells. Therefore, this project is aimed at discovering the epidermal melanocyte and cutaneous melanoma-specific surfaceome.

Three human pigment cell cultures were used in our experiments: primary epidermal melanocytes (from skin samples), WM35 cell line (from in situ melanoma), and A2058 cell line (from metastatic melanoma). Cell surface proteins were selectively labelled by aminooxy-biotin and then isolated by streptavidin-conjugated beads. Isolated proteins were trypsin-digested, and peptides were analysed by high-throughput shotgun mass spectrometry. Identified proteins were then evaluated by bioinformatics tools. For validation, we performed RT-qPCRs, western blots and immunocytochemistry reactions.

The enrichment of the surfaceome was 70,1%, 63,1% and 56,9% in melanocytes, WM35 and A2058 cells, respectively. Based on GO annotations, the proteins constituting the surfaceome were further classified into functional subcategories (transporters, enzymes, receptors, adhesion, miscellaneous proteins). The combined list consisted of 470 different proteins, 112 of which were detected only in melanocytes, whereas 186 proteins were specific to the melanoma cells and 95 proteins were identified in all three cell types. Based on the proteomic analysis we found several proteins worth of further examination. Matrix metalloproteinase inducer protein, basigin (CD147) was one of them and based on mass spectrometry results it was only present in metastatic melanoma cells. However, qPCRs demonstrated basigin mRNA expression in all examined cell cultures, while western blots proved basigin protein expression only in melanoma cells. Surprisingly, immunocytochemistry reactions followed by confocal microscopic evaluation revealed plasma membrane associated basigin expression in all three cell cultures, although intensity of fluorescent signals seemed to increase with malignancy.

Cell surface proteins are main targets of biomedical research due to their potential to become possible biomarkers and therapeutic alternatives. The differences found in the surfaceome of healthy vs malignant pigment cells can lead to the discovery of highly selective biomarkers of disease onset and progression. The protein expression analysis verified our proteomic findings as basigin may become a potent biomarker in melanoma diagnostics.

This work was supported by the Young Researcher Excellence Programme (grant number: FK-134304) of the National Research, Development and Innovation Office, Hungary.