Autoproteolysis and feedback in a protease cascade directing Drosophila dorsal-ventral cell fate

Abstract

Three serine protease zymogens, Gastrulation defective (GD), Snake (Snk) and Easter (Ea), and a nerve growth factor-like growth factor ligand precursor, Spaetzle, are required for specification of dorsal- ventral cell fate during Drosophila embryogenesis. The proteases have been proposed to function in a sequential activation cascade within the extracellular compartment called the perivitelline space. We examined biochemical interactions between these four proteins using a heterologous co-expression system. The results indicate that the three proteases do function in a sequential activation cascade, that GD becomes active and initiates the cascade and that interaction between GD and Snk is sufficient for GD to cleave itself autoproteolytically. The proteolytically active form of Ea cleaves GD at a different position, revealing biochemical feedback in the pathway. Both GD and Snk bind to heparin-Sepharose, providing a link between the pipe-defined ventral prepattern and the protease cascade. Our results suggest a model of the cascade in which initiation is by relief from inhibition, and spatial regulation of activity is due to interaction with sulfated proteoglycans.

Fig. 1. Proteolytic processing of Ea by active Snk. An immunoblot, using anti-Ea specific antiserum, of conditioned cell culture medium from co-expression of Ea proenzyme (50 kDa) and increasing amounts of SnkΔn, a constitutively active form of Snk. Lanes (from left to right) correspond to: Ea partially digested in vitro with trypsin; Ea alone; and Ea with 1/100, 1/50, 1/20, one-fifth and an equal volume of SnkΔn (v/v ratio of high titer stocks). The 35 kDa polypeptide corresponds to the size previously reported for the active Ea catalytic chain.

Fig. 2. Proteolytic processing of Spz 8.19 by active forms of Ea. An immunoblot, using anti-Spz C-terminal specific antiserum, of conditioned cell culture medium from co-expression of Spz 8.19 (40 kDa). While the inactive Ea zymogen had no effect upon the 40 kDa polypeptide, a constitutively active Ea catalytic chain, EaΔn, or Ea in combination with one of two different active forms of Snk, SnkΔn and XaSnk, led to generation of a 12 kDa C-terminal Spz fragment.

Fig. 3. Co-expression of GD, Snk, Ea and Spz leads to activation of a protease cascade. Immunoblot of Spz as in Figure 2. Expression of either Spz 8.19 alone or in combination with Ea zymogen and Snk zymogen led to no processing of Spz. However, co-expression of GD plus the other three proteins led to highly efficient processing of the Spz precursor to generate a 12 kDa fragment. Mutation of the active site serine of GD eliminated processing and GD does not bypass Snk to activate Ea.

Fig. 4. Co-expression of GD and Snk results in GD autoproteolysis to generate lower molecular weight polypeptides. Western blot using anti-GD specific antisera. A GD-specific polypeptide of 72 kDa was generated when GD was expressed alone. However, when GD was expressed with either wild-type Snk zymogen or a proteolytically inactive form of Snk, two polypeptides of 44 and 48 kDa were generated. Appearance of the lower molecular weight polypeptides requires integrity of the active site serine of GD.

Fig. 5. Active Snk catalytic chain does not cleave GD. The 72 kDa GD polypeptide was not cleaved by SnkΔn, a constitutively active form of Snk, or by the zymogen form of Ea.

Fig. 6. The active Ea protease specifically processes GD to generate 46 and 50 kDa polypeptide fragments. Western blotting with anti-GD antisera. (A) GD co-expressed with EaΔn but not that co-expressed with proteolytically inactive EaΔn(S to A) led to the generation of two lower molecular weight GD fragments. The 46 kDa band was preferentially cleaved and the 50 kDa band partially suppressed when an active form of Snk was co-expressed with Ea. (B) Ea-generated proteolytic fragments differ in size from those generated by Snk-induced GD autoproteolysis.

Fig. 7. Binding of GD and Snk to heparin–Sepharose. Western blotting with anti-GD antiserum (A) or monoclonal anti-Snk antibody (mAb53) (B) of column chromatography on a 0.5 ml bed volume heparin–Sepharose column. (A) GD binding and elution. (B) Snk binding. Lanes in both panels are as follows: ST, starting material; FT, flowthrough; WA, wash; and EL, eluate. For details see Materials and methods.

Fig. 8. Biochemical model of the dorsal–ventral protease cascade. Based upon our co-expression results, we propose a model in which GD, Snk, Ea and Spz sequentially activate each other. GD, SNK and EA indicate active forms of the proteases. SPZ indicates the ligand form of SPZ. GD, which can become active in the absence of a specific activator, must be kept inactivated via an inhibitory factor. Activation of the cascade would therefore require relief from inhibition by inactivation of this inhibitory factor. Upon complexing with and activating Snk, GD undergoes auto proteolysis to generate cleaved and proteolytically inactive forms represented as GD. An active form of Ea feeds back upon either the proform or the active form of GD to process it and down-regulate the signal.