A faster migrating variant masquerades as NICD when performing in vitro gamma-secretase assays with bacterially expressed Notch substrates

Abstract

Intramembrane proteolysis is a new and rapidly growing field. In vitro assays utilizing recombinant substrates for gamma-secretase, an intramembrane-cleaving enzyme, are critically important in order to characterize the biochemical properties of this unusual enzyme. Several recombinant Notch proteins of varying length are commonly used as in vitro substrates for CHAPSO-solubilized gamma-secretase. Here we report that several recombinant Notch constructs undergo limited or no proteolysis in vitro. Instead, upon incubation with or without gamma-secretase, variants of the intact protein migrate during SDS-PAGE at the location expected for the gamma-secretase specific cleavage products. In addition, we show that addition of aspartyl- and gamma-secretase specific protease inhibitors are able to retard the formation of these variants independent of gamma-secretase, which could lead to the erroneous conclusion that Notch cleavage by solubilized gamma-secretase was achieved in vitro even when no proteolysis occurred. In contrast, substrates produced in mammalian or insect cells are cleaved efficiently in vitro. These observations suggest that in vitro studies reliant on recombinant, bacterially produced Notch TMD should be performed with the inclusion of additional controls able to differentiate between actual cleavage and this potential artifact.

Figure 1. No γ-Secretase cleavage occurs in vitro with the HAN100Flag substrate

(A) Schematic representation of HAN100Flag indicating the γ-secretase cleavage sites within the Notch TMD (S4, S3) and the expected N-terminal (HA-tagged) and C-terminal (Flag-tagged) cleavage products. (B) HAN100Flag was incubated at −20°C or at 37°C (4 hrs) in the presence of either CHAPSO-solubilized Hek293 membranes containing γ-secretase or in extracts enriched for γ-secretase by IP with a PS-1 antibody (9N14 (44)). (C) Cleavage is not completely inhibited by the γ-secretase inhibitors DAPT and DupE (D). (E) HAN100Flag was incubated in cleavage buffer alone (no γ-secretase). Faster protein species, migrating at the expected size for a C-terminal (NICD; anti-Flag) and N-terminal (Nβ; anti-HA) cleavage products are observed. The appearance of the C-terminal (NICD) product was partially blocked when HAN100Flag was incubated in the presence of 10uM DAPT but not an equivalent volume of vehicle (“). Notice that asterisk (B, E) marks a band detectable with the amino terminal tag (anti-HA) and corresponding to the C-terminal cleavage product NICD.

Figure 2. Putative cleavage products are eliminated at low pH

HAN100Flag was incubated in cleavage buffer in the presence of the aspartyl protease inhibitor Pepstatin A at the concentrations indicated. The apparent inhibition of cleavage by Pepstatin A is attributable to the lowering of the cleavage reaction buffer pH by the acetic acid/ethanol solution in which Pepstatin A is dissolved. Appearance of the faster migrating band is inhibited at acidic pH.

Figure 3. Summary of data demonstrating that HAN100Flag preparations contain a faster migrating conformational variant

(A) The MALDI-TOF traces for the “intact” (band B in Figure 1A) and “variant” (band C in Figure 1A) forms of HAN100Flag. The trace shown is representative of multiple independent experiments with several preparations of HAN100Flag. (B) Expected (left hand column) and observed (right hand column) results for cleavage of a bacterial substrate by γ-secretase. ND indicates not determined.

Figure 4. Substrate from non-bacterial sources are cleaved efficiently by γ-secretase

In this figure γ-secretase is produced in Sf9 cells doubly transfected with virus expressing Pen2/PS1 (PEHP) and NCT/Aph (N/A). The identity of the viruses in all experiments is described in the line labeled “source”. (A) N100ppt, purified from baculovirus infected Sf9 cells is clearly cleaved when mixed with γ-secretase-containing membranes. The NICD product is detected by the VLLS epitope arising only after cleavage at S3; DAPT blocks NICD formation, (B) No cleavage product is detected when N100P is incubated in membrane preparations containing inactive γ-secretase (D385N) (C, D) Cleavage of Notch also occurs when Sf9 membranes containing active γ-secretase (WT) mixed with a longer substrate derived from a mammalian cells (DKO ΔICE). This demonstrates that γ-secretase can cleave substrate from an exogenous source provided they are pre-inserted into a membrane environment. (E) HAN100Flag was incubated for 16 hours at −20°C ((Inc; −) or 37°C ((Inc; +) in buffer alone, (F) with Sf9 cell membranes containing reconstituted γ-secretase (WT) or (G) in Sf9 cell membranes containing inactive γ-secretase (D385N). A faster migrating species appears at the expected location for NICD in all preparations containg HAN100Flag, and is unaffected by DAPT.

Figure 5. Bacterially produced Notch can undergo limited cleavage by γ-secretase

The bacterially produced Notch substrate N102FmH was incubated with γ-secretase containing Sf9 membranes (PNAP, cells infected with 4 viruses, see methods) for 16 hrs at −20°C (Inc.; −) or 37°C (Inc.; +). Conformational variants were detected with anti-c-myc antibody 9B11. When substrate was incubated at 37°C in buffer alone (Buffer), the appearance of the conformational variant was retard with 1µM DAPT (arrow in A). Incubation with enzyme from Sf9 cells (wt) showed an increase in the faster migrating species relative to the fractions lacking the enzyme. In (B), N102FmH was incubated in the absence of enzyme (Buffer; −), with human HeLa cell membranes containing γ-secretase (HeLa), or with membranes from Sf9 with active (active) or inactive (D385A) enzyme. Using the monoclonal M2-Flag antibody, conformational variants were observed under all condition. Again, the faster migrating species was increased when incubated with active enzyme from mammalian or insect cells, but not in buffer, inactive (D385A) or inhibited (DAPT; 1µM). (C) Detection of N102FmH with a polyclonal Flag antibody (pAb-Flag) showed increased signal in lanes that contain actual NICD (compared with B and D). (D) The Val 1744 antibody can easily and definitively discriminate between conformational variants and the actual, cleaved NICD product. A slower migrating bend of undetermined identity appears in all Hela extract. The enzyme produced by PNAP is equal in activity to that produced by PEHP/NA cells (not shown).