Towards the development of an enzyme replacement therapy for the metabolic disorder propionic acidemia

Abstract

Propionic acidemia (PA) is a life-threatening disease caused by the deficiency of a mitochondrial biotin-dependent enzyme known as propionyl coenzyme-A carboxylase (PCC). This enzyme is responsible for degrading the metabolic intermediate, propionyl coenzyme-A (PP-CoA), derived from multiple metabolic pathways. Currently, except for drastic surgical and dietary intervention that can only provide partial symptomatic relief, no other form of therapeutic option is available for this genetic disorder. Here, we examine a novel approach in protein delivery by specifically targeting and localizing our protein candidate of interest into the mitochondrial matrix of the cells. In order to test this concept of delivery, we have utilized cell penetrating peptides (CPPs) and mitochondria targeting sequences (MTS) to form specific fusion PCC protein, capable of translocating and localizing across cell membranes. In vitro delivery of our candidate fusion proteins, evaluated by confocal images and enzymatic activity assay, indicated effectiveness of this strategy. Therefore, it holds immense potential in creating a new paradigm in site-specific protein delivery and enzyme replacement therapeutic for PA.

Keywords: CPPs, cell penetrating peptides; CoA, coenzyme-A; ERT, enzyme replacement therapy; Enzyme replacement therapy; His-tag, six histidines tag; LAD, lipoamine dehydrogenase; MPP, mitochondrial processing peptidase; MTS, mitochondria targeting sequences; Mitochondrial targeting sequences; PA, propionic acidemia; PCC, propionyl coenzyme-A carboxylase; PCCA, PCCα subunit; PCCB, PCCβ subunit; PP-CoA, propionyl coenzyme-A; Propionic acidemia; Propionyl coenzyme-A carboxylase; Protein transduction domains; UPLC-MS/MS, ultra performance liquid chromatography tandem mass spectrometry.

Supplementary Fig. 1

Reducing SDS-PAGE of fusion PCC subunits showing expression level before and after induction in BL21 competent cells. Induced cells were allowed to grow overnight at 18 °C before collection. Lysates of the cells were run for SDS-PAGE. UI: un-induced. I: induced.

Supplementary Fig. 2

Fusion PCC-(TAT) control subunits co-localization. HeLa cells were treated with CF488A green fluorescent labeled PCCA and PCCB fusion variants (0.1 mg/ml final concentration) for 30 min, washed and incubated with MitoView 633 red fluorescent dye for 30 min before fixation with 4% paraformaldehyde. The cells were analyzed for transduction and co-localization by confocal microscopy. Original magnification is 60 ×. (A) PCCB-TAT, (B–D) TAT-PCCA.

Fig. 1

Structural design and construction of 3′ end PCC fusion subunits variants. (a) 3′ end fusion subunits variants were designed to be amplified from original PCC subunits clone. MTS-TAT fusion subunits could be amplified from MTS fusion subunits. (b) A schematic representation of TAT, MTS, and TAT-MTS fusion 5′ end subunits constructs. Control variants are subunits lacking the MTS or TAT domains. (c) PCR amplification results of pccA and pccB variants by using KOD DNA polymerase. (d) Digestion of cloned 3′ end fusion pccA and pccB subunits in pET28a vector by restriction enzymes. pccA-TAT, pccA-MTS and pccA-MTS-TAT clones were digested by EcoRI-HF and HindIII-HF restriction enzymes. pccB-TAT, pccB-MTS and pccB-MTS-TAT clones were digested by NotI-HF and BamHI-HF restriction enzymes. All digestions were carried out in 37 °C overnight and digestion results visualized by GelRed staining on 10% agarose gel.

Fig. 2

Representative of fusion PCC subunits purification and western blot. (a) Reducing SDS-PAGE of a representative PCC subunit lysate is compared with purified one after using nickel affinity column purification method. Gels were stained with InstantBlue™ (Expedeon Ltd., Cambridge, UK). (b) Western blot representative of N-terminal PCCA and C-terminal PCCB original and fusion variants using anti-PCCA chicken polyclonal and anti-PCCB mouse polyclonal as primary antibodies respectively.

Fig. 3

CD spectra of original PCC subunits and fusion variants. The protein concentrations were 0.1 mg/ml and the path length of the cuvette was 10 mm. (a) PCCA variants in PBS, (b) PCCA variants in TAE, (c) PCCB variants in PBS and (d) PCCB variants in TAE. Buffers were adapted to pH 7. Determination were performed at the beginning of the run at 10 °C, in triplicate for each protein.

Fig. 4

Fusion PCC subunits co-localization. HeLa cells were treated with CF488A green fluorescent labeled PCCA and PCCB fusion variants (0.1 mg/ml final concentration) for 30 min, washed and incubated with MitoView 633 red fluorescent dye for 30 min before fixation with 4% paraformaldehyde. The cells were analyzed for transduction and co-localization by confocal microscopy. Original magnification is 60 ×. Localization of TAT-MTS-PCCA and PCCB-MTS-TAT subunits (tests) and PCCB-MTS, TAT-PCCA and original PCC subunits (controls) are shown in this figure. Merged photos indicate merging photos of red and green fluorescent filters.

Fig. 5

Western blot of the fusion and original PCC subunits after delivery to defective lymphocyte cells. Extracted mitochondria and cytosol fractions were analyzed on 12% SDS-PAGE and probed with PCCA and PCCB antibodies. (a) PCCA defective cells treated with TAT-PCCA and TAT-MTS-PCCA are compared with normal lymphocytes and un-treated cells. (b) PCCB defective cells treated with PCCB, PCCB-TAT and PCCB-MTS-TAT are compared with normal lymphocytes and un-treated cells. The purity of the sub-cellular fractions was confirmed using the mitochondrial marker E1α (43 kDa). ImageJ densitometry analysis (National Institutes of Health (NIH) ImageJ 1.47 software) results are presented below each image. Intensity ratio of PCCA and PCCB bands relative to the E1α bands in A and B respectively presented as AUC ratio of corresponding intensity peak (AUC ratio mean ± SD). Anti-chicken HRP conjugated IgG (bovine) and anti-mouse HRP conjugated IgG (goat) (Santa Cruz Biotechnology, CA, USA) were used as secondary antibodies for probing PCCA and PCCB antibodies respectively. PCCA def: PCCA defective lymphocytes, PCCB def: PCCB defective lymphocytes, normal lymph: normal lymphocytes.

Fig. 6

Schematic representation of the fusion proteins activity. The in vitro enzymatic activity assay of delivered fusion and original subunits was performed as described in Materials and methods. PCCA or PCCB defective lymphocytes were treated with (a) TAT-MTS-PCCA, TAT-PCCA, PCCA, and (b) PCCB-MTS-TAT, PCCB-TAT, PCCB at final concentration of 0.1 mg/ml for different time periods (5 min–24 h). PCC activity was analyzed in isolated mitochondria lysate immediately after the incubation time using the UPLC-MS/MS analysis . A set of control was performed by using un-treated PCCA defective lymphocytes (PCCA def), PCCB defective lymphocytes (PCCB def) and normal lymphocyte cells (normal). Activity assays were conducted at least three times and the values presented are the mean values ± SD. The enzymatic activity values are presented as [MM-CoA] μmol/l/min.