Abstract
Matriptase plays important roles in epithelial integrity and function, which depend on its sorting to the basolateral surface of cells, where matriptase zymogen is converted to an active enzyme in order to act on its substrates. After activation, matriptase undergoes HAI-1-mediated inhibition, internalization, transcytosis, and secretion from the apical surface into the lumen. Matriptase is a mosaic protein with several distinct protein domains and motifs, which are a reflection of matriptase's complex cellular itinerary, life cycle, and the tight control of its enzymatic activity. While the molecular determinants for various matriptase regulatory events have been identified, the motif(s) required for translocation of human matriptase to the basolateral plasma membrane is unknown. The motif previously identified in rat matriptase is not conserved between the rodent and the primate. We, here, revisit the question for human matriptase through the use of a fusion protein containing a green fluorescent protein linked to the matriptase N-terminal fragment ending at Gly-149. A conserved seven amino acid motif EEGEVFL, which is similar to the monoleucine C-terminal to an acidic cluster motif involved in the basolateral targeting for some growth factors, has been shown to be required for matriptase translocation to the basolateral plasma membrane of polarized MDCK cells. Furthermore, time-lapse video microscopy showed that the motif appears to be required for entry into the correct transport vesicles, by which matriptase can undergo rapid trafficking and translocate to the plasma membrane. Our study reveals that the EEGEVFL motif is necessary, but may not be sufficient, for matriptase basolateral membrane targeting and serves as the basis for further research on its pathophysiological roles.
Highlights
The type 2 transmembrane serine protease (TTSP) matriptase was initially identified as the major secreted gelatinolytic activity present in conditioned medium from breast cancer cells in an effort to identify and characterize cancer cell-derived extracellular matrix-degrading proteases involved in cancer invasion and metastasis [1,2]
Matriptase is synthesized as a type 2 transmembrane protein, and the transmembrane domain is positioned between a short N-terminal cytoplasmic domain (54 amino acids) and a C-terminal extracellular protein domain known as a SEA domain [4,5]
Analysis of the subcellular localization and trafficking of the matriptase N-terminal fragment fused with EGFP in HaCaT and polarized Madin-Darby Canine Kidney (MDCK) cells, allowed us to identify a protein motif within the cytoplasmic domain that appears to be required for matriptase basolateral targeting
Summary
The type 2 transmembrane serine protease (TTSP) matriptase was initially identified as the major secreted gelatinolytic activity present in conditioned medium from breast cancer cells in an effort to identify and characterize cancer cell-derived extracellular matrix-degrading proteases involved in cancer invasion and metastasis [1,2]. Only activated matriptase in complex with HAI-1 is secreted from the apical plasma membrane of polarized Caco-2, and not the zymogen form of the enzyme [7] It remains unclear if matriptase can be secreted from the basolateral plasma membrane of cells in vivo. The presence of activated matriptase in complex with HAI-1 without measurable matriptase zymogen in human body fluids is consistent with the pattern of matriptase secretion observed in vitro in polarized Caco-2 cells [9] These in vivo and in vitro studies illustrate several milestones throughout the matriptase lifespan: 1) synthesis as zymogen, 2) targeting to the basolateral plasma membrane, 3) conversion to an active enzyme and action on its substrates in the basolateral milieu, 4) enzymatic inhibition through the formation of a very stable complex with HAI-1 on the basolateral plasma membrane, 5) internalization of the activated matriptase-HAI-1 complex from the basolateral plasma membrane, 6) transcytosis to the apical face of the cell and 7) shedding from the apical plasma membrane into the lumen of the secretory glands as the activated matriptase-HAI-1 complex, which has been detected in body fluids
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