The Regulated Cell Surface Zymogen Activation of the Proprotein Convertase PC5A Directs the Processing of Its Secretory Substrates

Gaétan Mayer,Josée Hamelin,Marie-Claude Asselin,Antonella Pasquato,Edwidge Marcinkiewicz,Meiyi Tang,Siamak Tabibzadeh,Nabil G Seidah

doi:10.1074/jbc.m708763200

Gaétan Mayer, Josée Hamelin + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m708763200

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Abstract

The proprotein convertases are synthesized as zymogens that acquire activity upon autocatalytic removal of their NH(2)-terminal prosegment. Based on the convertase furin, to fold properly and gain activity, the convertases PC5A, PACE4, and PC7 are presumed to undergo two sequential prosegment cleavages in the endoplasmic reticulum and then in the trans-Golgi network. However, biochemical and immunocytochemical experiments revealed that mouse PC5A is complexed to its prosegment at the plasma membrane. This labeling is lost upon treatment with heparin and is increased by overexpressing members of the syndecan family and CD44, suggesting attachment of secreted PC5A-prosegment complex to heparan sulfate proteoglycans. Following stimulation of Y1 cells with adrenocorticotropic hormone or 8-bromo-cyclic AMP, the cell surface labeling of the prosegment of PC5A is greatly diminished, whereas the signal for mature PC5A is increased. Moreover, after stimulation, the protease activity of PC5A is enhanced, as evidenced by the cleavage of the PC5A substrates Lefty, ADAMTS-4, endothelial lipase, and PCSK9. Our data suggest a novel mechanism for PC5A activation and substrate cleavage at the cell surface, through a regulated removal of its prosegment. A similar mechanism may also apply to the convertase PACE4, thereby extending our knowledge of the molecular details of the zymogen activation and functions of these heparan sulfate proteoglycan-bound convertases.

Highlights

heparan sulfate proteoglycans (HSPGs) Are Required for the Cell Surface Anchoring of PC5A and PACE4—Our previous observations have shown that the secreted PC5A and PACE4 can be anchored to the cell surface through their cysteine-rich domain (CRD) [17]
To determine how this binding occurs, COS-1 cells expressing FL-PC5A were treated with suramin, a highly sulfated compound or with heparinase III, which cleaves at the 1,4 linkages between hexosamine and glucuronic acid residues in heparan sulfate, thereby removing cell surface heparan sulfate chains
Immunolabelings of pgsD-677 cells expressing FL-PC5A, FL-PACE4, or the CRDs alone revealed no signal at the plasma membrane, demonstrating that PC5A and PACE4 are anchored at the cell surface through binding of their CRD to HSPGs (Fig. 1, I–L)

Summary

Introduction

This demonstrates that the prosegment is not produced in the R116A mutant, as expected, membrane (Fig. S3), indicating that at the cell surface, some since cleavage at Arg1162 is the primary processing site. The data ciate the possible enzyme-substrate relationship between PC5 revealed that ACTH treatment significantly increased the and Lefty, we first compared the distribution of their mRNAs in endogenous PC-like activity found in the medium of Y1 cells, as mouse tissues using in situ hybridization in adult whole animal compared with control untreated cells (Fig. 5F).

Results

Conclusion