Expression and Characterization of Membrane-Type 4 Matrix Metalloproteinase (MT4-MMP) and its Different Forms in Melanoma

Abstract

Background/Aims: Membrane-type matrix metalloproteinases (MT-MMPs) are expressed on the cell surface and hydrolyze extracellular matrix components and signaling molecules by which they influence cancer cell migration and metastasis. Two of the six known MT-MMPs are anchored to the plasma membrane via a GPI anchor, one of which is MT4-MMP. Only little is known about MT4-MMP expression, synthesis, regulation and degradation. Methods: We analyzed several human cancer cell lines as well as tissue homogenates using Western blotting and quantitative PCR for the expression of MT4-MMP. Organelles of SK-Mel-28 cells were separated using continuous Iodixanol gradients. Glycosylation of the SK-Mel-28 protein was studied via glucosidases and site directed mutagenesis of the MT4-MMP cDNA prior to transfection. Results: We found the MT4-MMP highly expressed in human melanoma cell lines as well as skin and melanoma tissue samples. Three forms of MT4-MMP with molecular masses of 45 kDa, 58 kDa and 69 kDa were detected. Further, we demonstrate that the 58 kDa form is the mature protein in the cell membrane, while the 69 kDa form is its precursor found in intracellular compartments. The 69 kDa forms are processed by furin cleavage in the Golgi apparatus. Moreover, we identified Asn³¹⁸ as the single N-glycosylation site of MT4-MMP. Conclusion: We demonstrate the novel expression of MT4-MMP in melanocytic tissues and propose a precursor/product-relationship of the different forms of MT4-MMP in melanoma cells.