Abstract
The matriptase-prostasin proteolytic cascade is essential for epidermal tight junction formation and terminal epidermal differentiation. This proteolytic pathway may also be operative in a variety of other epithelia, as both matriptase and prostasin are involved in tight junction formation in epithelial monolayers. However, in polarized epithelial cells matriptase is mainly located on the basolateral plasma membrane whereas prostasin is mainly located on the apical plasma membrane. To determine how matriptase and prostasin interact, we mapped the subcellular itinerary of matriptase and prostasin in polarized colonic epithelial cells. We show that zymogen matriptase is activated on the basolateral plasma membrane where it is able to cleave relevant substrates. After activation, matriptase forms a complex with the cognate matriptase inhibitor, hepatocyte growth factor activator inhibitor (HAI)-1 and is efficiently endocytosed. The majority of prostasin is located on the apical plasma membrane albeit a minor fraction of prostasin is present on the basolateral plasma membrane. Basolateral prostasin is endocytosed and transcytosed to the apical plasma membrane where a long retention time causes an accumulation of prostasin. Furthermore, we show that prostasin on the basolateral membrane is activated before it is transcytosed. This study shows that matriptase and prostasin co-localize for a brief period of time at the basolateral plasma membrane after which prostasin is transported to the apical membrane as an active protease. This study suggests a possible explanation for how matriptase or other basolateral serine proteases activate prostasin on its way to its apical destination.
Highlights
The matriptase-prostasin proteolytic cascade is essential for epidermal tight junction formation and terminal epidermal differentiation
Matriptase is at steadystate concentrated and localized mainly at the basolateral plasma membrane together with hepatocyte growth factor activator inhibitor (HAI)-1 [15, 24] while prostasin is mainly concentrated and localized to the apical plasma membrane [17, 18]
Matriptase is mainly located on the basolateral plasma membrane where it is activated, inhibited, endocytosed in complex with its inhibitor HAI-1 and is accumulated in intracellular structures
Summary
Cell Culture—Caco-2 cells were grown in minimal essential medium supplemented with 2 mM L-glutamine, 10% fetal bovine serum, 1ϫ nonessential amino acids, 100 units/ml penicillin, and 100 g/ml streptomycin (Invitrogen) at 37 °C in an atmosphere of 5% CO2. Biotinylation, Internalization, and Biotin-removal—Caco-2 cells grown on transwell filters were washed three times with ice-cold PBSϩ (PBS supplemented with 0.7 mM CaCl2 and 0.25 mM MgCl2) on both apical and basolateral side. Monomeric Avidin/Streptavidin Precipitation of Biotinylated Proteins—Lysates from biotin-labeled cells were centrifuged at 20,000 ϫ g for 20 min to pellet the insoluble material. Biotinylated proteins were eluted from the monomeric avidin agarose with 4 mM biotin (Pierce) in PBS for 30 min followed by addition of SDS sample buffer. Caco-2 cells were biotin-labeled and biotinylated proteins were pulled down with monomeric avidin agarose. The biotin-eluate was separated from the avidin-agarose and incubated with 60 l protease inhibitor-coupled Sepharose in 50 mM TrisHCl, pH 8.75 at 37 °C for 30 min. Gelatin Zymography—Biotinylated monomeric avidin agarose-purified proteins were separated on a 7% SDS-polyacrylamide gel containing 0.1% gelatin. The cells were mounted with Prolong Gold mounting medium (Invitrogen) and subjected to laser scanning confocal microscopy using the Leica TCS SP2 system and Zeiss LS700 system
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