Abstract
Processing of polypeptide precursors by proprotein convertases (PCs) such as furin typically occurs within the trans-Golgi network. Here, we show in a variety of cell types that the propeptide of ADAMTS9 is not excised intracellularly. Pulse-chase analysis in HEK293F cells indicated that the intact zymogen was secreted to the cell surface and was subsequently processed there before release into the medium. The processing occurred via a furin-dependent mechanism as shown using PC inhibitors, lack of processing in furin-deficient cells, and rescue by furin in these cells. Moreover, down-regulation of furin by small interference RNA reduced ADAMTS9 processing in HEK293F cells. PC5A could also process pro-ADAMTS9, but similarly to furin, processed forms were absent intracellularly. Cell-surface, furin-dependent processing of pro-ADAMTS9 creates a precedent for extracellular maturation of endogenously produced secreted proproteins. It also indicates the existence of a variety of mechanisms for processing of ADAMTS proteases.
Highlights
Zinc metalloendopeptidases, like most proteases, are synthesized as zymogens, and the N-terminal propeptide is usually excised
The Proteolytically Processed ADAMTS9 Propeptide Is Present in the Conditioned Medium but Not in the Cell Lysate—The ADAMTS9 propeptide (Fig. 1, A and B) has five consensus furin-recognition sequences (Arg-Xaa-Xaa-Arg)
We examined ADAMTS9 propeptide processing using various approaches, none of which provided any evidence for intracellular processing
Summary
Like most proteases, are synthesized as zymogens, and the N-terminal propeptide is usually excised. Furin Processing of Pro-ADAMTS9 signal peptide, propeptide, and catalytic domain (Pro-Cat) with C-terminal Myc and His tags were described previously [30]. The results were identical, i.e. antiRP4 detected the intact Pro-Cat zymogen (55 kDa) only in the cell lysate, and 37- and 20 –22-kDa fragments were found only in the conditioned medium (Fig. 2D).
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