Secretion of functional α1-antitrypsin is cell type dependent: Implications for intramuscular delivery for gene therapy

Abstract

Heterologous expression of proteins is used widely for the biosynthesis of biologics, many of which are secreted from cells. In addition, gene therapy and messenger RNA (mRNA) vaccines frequently direct the expression of secretory proteins to nonnative host cells. Consequently, it is crucial to understand the maturation and trafficking of proteins in a range of host cells including muscle cells, a popular therapeutic target due to the ease of accessibility by intramuscular injection. Here, we analyzed the production efficiency for α1-antitrypsin (AAT) in Chinese hamster ovary cells, commonly used for biotherapeutic production, and myoblasts (embryonic progenitor cells of muscle cells) and compared it to the production in the major natural cells, liver hepatocytes. AAT is a target protein for gene therapy to address pathologies associated with insufficiencies in native AAT activity or production. AAT secretion and maturation were most efficient in hepatocytes. Myoblasts were the poorest of the cell types tested; however, secretion of active AAT was significantly augmented in myoblasts by treatment with the proteostasis regulator suberoylanilide hydroxamic acid, a histone deacetylase inhibitor. These findings were extended and validated in myotubes (mature muscle cells) where AAT was transduced using an adeno-associated viral capsid transduction method used in gene therapy clinical trials. Overall, our study sheds light on a possible mechanism to enhance the efficacy of gene therapy approaches for AAT and, moreover, may have implications for the production of proteins from mRNA vaccines, which rely on the expression of viral glycoproteins in nonnative host cells upon intramuscular injection.

Keywords: endoplasmic reticulum; protein quality control; proteostasis; proteostasis regulator; serpin.

Fig. 1.

Huh-7 cells efficiently secrete active AAT M1V. (A) Huh-7 cells were pulsed with media containing [³⁵S]-Met/Cys for 30 min and chased with nonradioactive media for the indicated times. Cell lysates (lanes 1 to 8), media (lanes 9 to 16), and detergent insoluble (lanes 17 to 24) fractions were collected. AAT was isolated by IP with anti-AAT antibodies. Samples were resolved by SDS-PAGE and visualized by autoradiography. No significant amounts of AAT were observed in the detergent-insoluble fraction (lanes 17 to 24); therefore, it was not plotted. (B) Band intensities on the SDS-PAGE gels were quantified and plotted over time. The points are the means of three independent biological replicates, and the error bars are the SDs. Secreted fraction of AAT was calculated as media [AAT/total AAT (cell lysate + media + detergent insoluble)] × 100. (C) Cell lysates (lanes 1 to 3) and medium (lanes 4 to 6) immunoprecipitated AAT were treated with glycosidases EndoH (lanes 2 and 5) and PNGaseF (lanes 3 and 6). AAT with complex (AAT^Comp) or high mannose (AAT^HM) carbohydrates are designated along with deglycosylated AAT (AAT^DG). (D) The activity of AAT in the media was determined by treatment with HNE and quantified by dividing the complex amount (HNE-AAT, lane 2) by total AAT (lane 2 reactive AAT plus unreactive AAT) and multiplying by 100. Inhibition of elastase by AAT results in the formation of the higher-molecular-weight AAT-elastase covalent conjugate. The points are the means of three independent biological replicates, and the error bars are the SDs. Molecular weight markers (kDa) are shown on the right of all gels.

Fig. 2.

Secretion of overexpressed AAT-M in CHO, Huh-7, and C2C12 myoblast. AAT-M was exogenously expressed in Huh-7 (A and B), CHO (C and D), and C2C12 myoblast (E and F) cells for 20 h; pulsed with media containing [³⁵S]-Met/Cys for 30 min; chased with nonradioactive media for the indicated times; and processed and quantified as described in Fig. 1. (G) Medium samples were collected after 20 h of transfection, and AAT was isolated by IP and treated with HNE before resolving by SDS-PAGE and immunoblotting to visualize the active HNE-AAT complex. (H) The points are the means of three independent biological replicates, and the error bars are the SDs.

Fig. 3.

Trafficking of overexpressed AAT-Z in CHO, Huh-7, and C2C12 cells. AAT-Z was exogenously expressed in Huh-7 (A and B), CHO (C and D), and C2C12 myoblasts (E and F) for 20 h; pulsed with media containing [³⁵S]-Met/Cys for 30 min; chased with nonradioactive media for the indicated times; and processed and quantified as described in Fig. 1. (G) Medium samples were collected after 20 h of transfection, and AAT-Z was isolated by IP and treated with HNE before resolving by SDS-PAGE and immunoblotting to visualize the active AAT-elastase complex.

Fig. 4.

Treatment with SAHA increases the maturation efficiency of active AAT-M in C2C12 myoblasts. (A) C2C12 myoblasts were transfected with an AAT-M plasmid and grown for 24 h. The medium was replaced and the cells were grown for an additional 24 h in the presence of DMSO alone or one of nine small molecules dissolved in DMSO as indicated. Small molecule compounds include verapamil, dantrolene, and diltiazem, which increase the Ca²⁺ concentration in the ER (56); Tg, which nonspecifically induces UPR by decreasing the Ca²⁺ concentration in the ER (57); compounds 5 (comp5) and 147 (comp147), which up-regulate the ATF6 arm of the UPR (60); SAHA, an HDAC inhibitor (17); salubrinal, an inhibitor of eIF2α phosphorylation (61); and Bix, a small molecule that is reported to induce BiP expression (63, 64). Immunoblots of AAT-M secreted into the media following treatment in the absence (−) or presence (+) of the AAT target protease HNE are shown. (B) Amounts of AAT-M that accumulated in the media were quantified and compared to DMSO control levels that were set at 100%. The mean secretion efficiency (bars) relative to DMSO alone from four biological replicates with the SD given by the error bars. (C) HNE-AAT complex formation was quantified as described in Fig. 1 from at least four biological replicates with the SD given by the error bars. (D) C2C12 cell lysates were immunoblotted for HDAC7, SEL1L, BiP, and GAPDH (loading control) to determine their cellular expression levels after treatment with the various proteostasis regulators for 24 h. (E) Levels of HDAC7, SEL1L, and BiP were quantified from four biological replicates, indicated by color, with error bars representing the SDs.

Fig. 5.

AAV transduction of myotubes supports efficient secretion of active AAT that is increased by SAHA treatment. (A) Timeline of myotube differentiation and transduction of AAT. Cells were plated and differentiated in DMEM containing 2% horse serum (HS) for 7 d. On day 7, cells were transduced with AAV2 containing the AAT plasmid, treated with or without SAHA for 2 d, and harvested on day 9. (B) Immunoblot of AAT expression and activity produced by differentiated myotubes ± SAHA. AAT from the lysate (lanes 1 and 2), medium fractions (lanes 3 and 5), and detergent insoluble pellet (lanes 7 and 8) were probed using an αAAT antibody for cells treated with or without SAHA. A fraction of the media from each sample was tested for activity in its ability to form a complex when incubated with HNE (lanes 4 and 6). (C) Quantification of AAT levels for secreted and total protein from B. Increase in the secreted protein was determined by dividing the amount of AAT in lane 5 by lane 3. The fold increase in total protein was determined by adding AAT in all fractions for each condition (lanes 1, 3, and 7 for − SAHA and lanes 2, 5, and 8 for + SAHA). The total amount for + SAHA was divided by the amount calculated for − SAHA. (D) Percent activity of AAT produced by myotubes ± SAHA from B. Activity of AAT was measured in lane 4 (− SAHA) and lane 6 (+ SAHA). The amount of the complex (HNE-AAT) was divided by the total amount of protein in each lane (HNE-AAT + noncomplexed AAT) and multiplied by 100 to obtain a percentage. Error bars represent the SD of four biological replicates (indicated by color). **** represents P ≤ 0.0001, ** represents P ≤ 0.01, and * represents P ≤ 0.05 for both C and D. (E) Commercial purified AAT (pAAT) (lanes 1 to 3), AAT from the media of untreated cells (lanes 4 to 6), and + SAHA (lanes 7 to 9) were immunoblotted and probed using an αAAT to determine glycan sensitivity. All treatments were compared to undigested protein (−; lanes 1, 4, and 7). Samples were treated with EndoH (E; lanes 2, 5, and 8), a glycosidase unable to digest complex/hybrid glycans, and a lack of digestion suggests protein trafficking to the Golgi. All three samples were also treated with PNGaseF (P; lanes 3, 6, and 9), an enzyme that completely digests glycans regardless of type.