Inhibition of sonic hedgehog autoprocessing in cultured mammalian cells by sterol deprivation

Abstract

Sonic hedgehog (Shh) is a signaling molecule that is important for defining patterning in the developing vertebrate central nervous system. After translation, Shh autoproteolyzes and covalently attaches cholesterol to the newly formed carboxyl terminus, a modification crucial for normal Shh signaling. Presented here is evidence that acute severe sterol deprivation in cultured Chinese hamster ovary cells expressing mouse Shh (mShh) inhibits autoprocessing of the protein. These conditions allowed the first detailed kinetic analysis of mShh autoprocessing and turnover rates revealing that cells rapidly degrade both precursor and mature mShh regardless of sterol content and sterol deprivation increases the rate of precursor degradation. Inhibition of mShh autoprocessing also allowed the determination of the subcellular localization of mShh precursor which accumulates in a pre-medial Golgi intracellular compartment. Finally, the precursor form of mShh that results from autoprocessing inhibition appears to accumulate as an amide rather than a stable thioester.

Figure 1

Inhibition of mShh autoprocessing by sterol deprivation. (A) Schematic representation of the precursor and mature forms of recombinant mShh protein. FLAG indicates the double copy of the FLAG-epitope tag. The inverted filled triangle indicates the site of autoproteolytic cleavage, and the filled oval indicates the site of concomitant attachment of cholesterol. The fork indicates a site for N-linked glycosylation. (B) Pulse-chase experiment demonstrating that sterol deprivation inhibits mShh processing and mShh remains cell associated. CHO-7 cells, where indicated stably expressing mShh, were grown overnight in medium A. One hour before metabolic labeling, the medium was replaced, as indicated, with sterol-depriving medium (see Materials and Methods). Cells were subjected to a 30-min pulse labeling with ³⁵S-labeled Met and 30-min chase. Recombinant mShh was immunoprecipitated from detergent lysates or media with mouse α-FLAG(M2)⋅agarose and subjected to SDS/PAGE (12% gel).

Figure 2

Pulse-chase experiment showing the time course of mShh autoprocessing and turnover of precursor and mature forms of mShh in normal and sterol-deprived cells. CHO-7 cells, where indicated stably expressing mShh, were grown overnight in medium A. One hour before metabolic labeling, the medium was replaced, as indicated, with sterol-depriving medium. Cells were labeled for 30 min with ³⁵S-labeled Met followed by a variable-length chase (see Materials and Methods). Recombinant mShh was immunoprecipitated from detergent lysates of cell pellets with mouse α-FLAG(M2)⋅agarose and subjected to SDS/PAGE (12% gel).

Figure 3

Glycosidase-sensitivity experiment demonstrating the glycosylation state of mShh precursor in normal and sterol-deprived cells. (A) Effects of sterol deprivation on the subcellular localization of mShh and the steady-state partition between precursor and mature forms of mShh. Treatment of samples with EndoH is indicated with an “E” whereas treatment of samples with PNGase F is indicated with a “P”. (B) Effects of sterol deprivation on the subcellular localization of the transferrin receptor (Tf^R). CHO-7 cells, where indicated stably expressing mShh, were grown for 48 h in medium A. Four hours before harvest, the media was replaced, as indicated, with sterol-depriving medium. At harvest, membrane fractions were prepared, denatured, and incubated in the presence or absence of PNGase F or Endo H (see Materials and Methods). The samples were then subjected to SDS/PAGE (9% for mShh; 6.5% for Tf^R control), transferred to Immobilon-P, and immunoblotted with either mouse α-FLAG(M2) or mouse α-Tf^R primary antibodies and donkey α-mouse⋅horseradish peroxidase secondary antibody. Blots were developed by using a chemiluminescent substrate for horseradish peroxidase.

Figure 4

Resistance of mShh precursor to neutral hydroxylamine. (A) Schematic representation of the chemical reactions carried out during mShh autoprocessing. First, mShh precursor converts to a semistable thioester intermediate by attack of the side chain thiol of Cys-199 on the carboxamide of the immediately amino-terminal Gly. Next, the intermediate form of mShh is converted to the cholesterol ester mature form by the attack of cholesterol on the thioester. Amides and esters are resistant to cleavage by neutral hydroxylamine whereas thioesters are labile to such cleavage. (B) Resistance of mShh precursor to neutral hydoxylamine. CHO-7 cells, where indicated stably expressing mShh, were grown overnight in medium A. One hour before metabolic labeling, the medium was replaced, as indicated, with sterol-depriving medium. Cells were labeled for 30 min with ³⁵S-labeled Met followed by a 15-min chase. Recombinant mShh was immunoprecipitated from detergent lysates with mouse α-FLAG(M2)⋅agarose (see Materials and Methods). Before elution from the beads, immunoprecipitated mShh proteins were treated, as indicated, with either 1 M Tris·HCl (pH 7.4) or 1 M hydroxylamine (pH 7.4) for 30 min at 37°C. The resulting protein mixtures were eluted as described and subjected to SDS/PAGE (12% gel). (C) Lability of the palmitate thioester of G protein α to neutral hydroxylamine. Control immunoprecipitates were prepared from cells treated as described above without radiolabeling. Immediately before hydroxylamine treatment, the samples were treated with in vitro-translated G protein α that had been labeled with tritiated palmitate (24,000 cpm per sample). Before elution from the beads, these samples were then subjected, as indicated, to treatment with either 1 M Tris·HCl (pH 7.4) or 1 M hydroxylamine (pH 7.4) for 30 min at 37°C. The resulting protein mixtures were eluted from the beads, subjected to SDS/PAGE (12% gel), and transferred electrophoretically to an Immobilon P membrane.