Abstract
ADAMTS9 is a secreted, cell-surface-binding metalloprotease that cleaves the proteoglycans versican and aggrecan. Unlike most precursor proteins, the ADAMTS9 zymogen (pro-ADAMTS9) is resistant to intracellular processing. Instead, pro-ADAMTS9 is processed by furin at the cell surface. Here, we investigated the role of the ADAMTS9 propeptide in regulating its secretion and proteolytic activity. Removal of the propeptide abrogated secretion of the ADAMTS9 catalytic domain, and secretion was inefficiently restored by expression of the propeptide in trans. Substitution of Ala for Asn residues within each of three consensus N-linked glycosylation sites in the propeptide abrogated ADAMTS9 secretion. Thus, the propeptide is an intramolecular chaperone whose glycosylation is critical for secretion of the mature enzyme. In addition to two previously identified furin-processing sites (Arg74 downward arrow and Arg287 downward arrow) the ADAMTS9 propeptide was also furin-processed at Arg209. Substitution of Ala for Arg74, Arg209, and Arg287 resulted in secretion of an unprocessed zymogen. Unexpectedly, versican incubated with cells expressing this pro-ADAMTS9 was processed to a greater extent than when incubated with cells expressing wild-type, furin-processable ADAMTS9. Moreover, cells and medium treated with the proprotein convertase inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone had greater versican-cleaving activity than untreated cells. Following furin processing of pro-ADAMTS9, propeptide fragments maintained a non-covalent association with the catalytic domain. Collectively, these observations suggest that, unlike other metalloproteases, furin processing of the ADAMTS9 propeptide reduces its catalytic activity. Thus, the propeptide is a key functional domain of ADAMTS9, mediating an unusual regulatory mechanism that may have evolved to ensure maximal activity of this protease at the cell surface.
Highlights
The ADAMTS9 protease domain contains an N-terminal propeptide, which, as in most zymogens, is believed to maintain enzyme latency and to be proteolytically excised to permit catalytic activity, a process termed “activation.” The ADAMTS9 propeptide contains a number of predicted furin-processing sites, and as in many metalloproteases, propeptide excision is mediated by proprotein convertases (PCs) such as furin
This suggested that the contiguous propeptide and catalytic domain were essential for cellsurface binding and that furin processing either disrupted a cell-binding site or led to a conformational change that resulted in loss of cell-surface binding
ADAMTS9 Catalytic Domain Lacking the Propeptide Is Not propeptide diminished the levels of each construct within the Secreted from Cells—We previously showed that both full- cell lysate (Fig. 2B) but could not restore their secretion to the length ADAMTS9 and propeptide and catalytic domain (Pro-Cat) (Fig. 1A) were efficiently pro- robust levels seen in conditioned medium upon expression of cessed by furin at the surface of HEK293F cells
Summary
The ADAMTS9 protease domain contains an N-terminal propeptide, which, as in most zymogens, is believed to maintain enzyme latency and to be proteolytically excised to permit catalytic activity, a process termed “activation.” The ADAMTS9 propeptide contains a number of predicted furin-processing sites, and as in many metalloproteases, propeptide excision is mediated by proprotein convertases (PCs) such as furin. The propeptide alone (ADAMTS91–286) was efficiently secreted into the conditioned medium, and underwent furin processing to generate fragments similar to those previously seen upon Pro-Cat expression
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