Monitoring the secretion and activity of alpha-1 antitrypsin in various mammalian cell types

Abstract

Overexpression of recombinant protein in mammalian cells is widely used for producing biologics, as protein maturation and post-translational modifications are similar to human cells. Some therapeutics, such as mRNA vaccines, target nonnative cells that may contain inefficient secretory machinery. For example, gene replacement therapies for alpha-1 antitrypsin (AAT), a glycoprotein normally produced in hepatocytes, is often targeted to muscle cells due to ease of delivery. In this chapter, we define methods for expressing AAT in representative cell types such as Huh-7, hepatocytes, Chinese hamster ovarian cells (CHO), a common host to produce biologics, and C2C12, a muscle progenitor cell line. Methods for metabolically labeling AAT to monitor secretion in these cell lines are described along with the use of proteostasis activators to increase the amount of AAT secreted in both C2C12 myoblasts and differentiated myotubes. Assays to assess the activity and glycan composition of overexpressed AAT are also presented. The usage of the proteostasis activator SAHA provided a 40% improvement in expression of active AAT in muscle-like cells and may be an advantageous adjuvant for recombinant production of proteins delivered by mRNA vaccines.

1. Introduction

Serpins comprise a superfamily of serine protease inhibitors whose main function is to carefully regulate the activity of proteases [1]. Alpha-1 antitrypsin (AAT) is the most abundant protease inhibitor found in human plasma and targets soluble proteases such as human neutrophil elastase (HNE), cathepsin D, and proteinase 3 [2]. Healthy humans have AAT serum levels of 1-2 g/L and produce approximately 34 mg/(kg*day) with more than 80% of this AAT secreted by liver hepatocytes [3]. Production of AAT begins in the endoplasmic reticulum (ER) where the proteins must fold and receive modifications before being trafficked for secretion [4]. The maturation of AAT requires special care as the protein must fold in a non-sequential manner to a achieve a metastable state required for its activity [2, 5]. Failure to fold correctly, or mutations within the protein sequence, can result in ER retention and degradation of AAT, its inactivity, and lower serum levels. Clinical progression of this trafficking and functional defect underlies the onset of inflammation of lung tissue and emphysema, through unregulated HNE activity [6, 7]. One possible approach to alleviate the symptoms associated with AAT deficiency is to deliver cDNA for the correct variant of AAT through gene replacement therapies.

One route proposed for delivery of AAT cDNA is the use of recombinant adeno-associated virus (rAAV) as a vehicle [8]. This method benefits from ease of production and administration to patients as the vehicle is injected intramuscularly. One drawback, however, is that skeletal muscle, and other possible target cells, may not be optimized for efficient secretion of soluble protein such as AAT [7]. As a result, recipients of these therapies often need to receive multiple injections to maintain therapeutic levels. With this in mind, understanding how recombinant AAT is secreted in representative cell lines and improving the amounts of active protein can provide valuable information for improving future therapies.

Here we report methods for expressing recombinant AAT in three cell lines: Chinese hamster ovarian cells (CHO), a common cell line used in the production of biopharmaceuticals, Huh-7, a human hepatocyte-derived cell line where AAT is expressed endogenously, and C2C12, a mouse myoblast and myotube cell line used to mimic the secretion in skeletal muscle [9, 10]. Additionally, methods are described to metabolically label overexpressed AAT in order to monitor the secretion and to ensure the protein is active through an activity assay. An endoglycosidase assay is also used to determine the composition of appended N-linked glycans as this can provide information on protein maturation within the cell. Finally, protocols for the use of proteostasis activators, such as the histone deacetylase suberoylanilide hydroxamic acid (SAHA), to improve the amount of protein secreted by differentiated C2C12 myotubes are provided. Such assays are useful for monitoring the secretion of active AAT in the hope of improving current therapeutic strategies and patient quality of life of those suffering from AAT deficiency.

2. Materials

2.1. Construction of AAT plasmid for mammalian protein expression

1. Amplify endogenous AAT from Huh-7 genomic DNA. Briefly, extract RNA and synthesize a cDNA library with the d(T)₂₃VN primer (T=thymine, V=adenine or guanine or cytosine and N = can be any nucleotide) set that binds and extends from the poly-A tail of the mRNA transcript. Such primers can be synthesized or bought from New England Biolabs. Incubate 1 μg of extracted RNA with a reverse transcriptase and treat at 42 °C for 1 hr. PCR amplify the AAT gene from the cDNA library with user designed primers. Two example primers are provided below:

   • Fwd: 5’-ATGCCGTCTTCTGTCTCGTGG-3’
   • Rev: 5’-TTATTTTTGGGTGGGATTCACCACTTTTC-3’
     Endogenous AAT from Huh-7 cells has the M1V (Arg101, Val213, Glu376) sequence. Ligate the AAT insert into a mammalian expression vector, such as pcDNA 3.1, with a C-terminal myc tag for immunoblots and immunoprecipitations.

2. Kit to extract RNA from mammalian cells such as PureLink RNA Kit ThermoFisher,

3. Kit to reverse transcribe RNA into cDNA library such as ProtoScript II First Strand cDNA Synthesis Kit from New England Biolabs.

4. Mammalian expression vector

2.2. Cell culture maintenance and transfection

1. 10-cm cell culture treated plates flat bottom plates

2. 3-cm cell culture treated plates flat bottom plates

3. 0.25% trypsin-EDTA (1X)

4. Phosphate-buffered saline (PBS): 137 mM NaCl, 2.7 mM KCl, 1.8 mM KH₂PO₄, 10 mM Na₂HPO₄, pH 7.5

5. See Table 1 for optimal list of cells, media and fetal bovine serum (FBS) (see Note 1)

6. Penicillin-Streptomycin (1000 U/mL) (PenStrep)

7. Low serum media containing 5% or less of FBS, such as Opti-MEM

8. Lipid based transfection reagent, such as Lipofectamine 2000

9. GeneXPlus (ATCC, ACS-4004) (see Note 2)

10. Polyethyleneimine (PEI) “Max” (MW 40,000)

Table 1.

Cell type information and culture media (see Note 1)

Cell Line	Species, Cell Type	Supplier, Product Number	Culture Media	Supplier, Product Number	FBS to complete media (10%)	Supplier, Product Number
Chinese hamster ovarian (CHO)	Hamster, epithelial-like cells	ATCC, CCL-61	MEM Alpha	Gibco, 32561-037	Certified (US origin)	Gibco, 16000044
C2C12	Mouse, myoblasts	ATCC, CRL-1772	DMEM high glucose	Gibco, 11965-092
Huh-7	Human, hepatocytes	CLS, 300156	DMEM low glucose	Gibco, 11885-084	Qualified (NZ origin)	Gibco, A3160901

2.3. C2C12 myotube differentiation and transduction

1. 3-cm cell culture treated plates

2. DMEM high glucose media

3. Certified fetal bovine serum (FBS) (US origin) (Gibco, 16000044) (see Note 1)

4. Heat inactivated horse serum

5. Pre-prepared AAV capsid containing AAT cDNA for overexpression in C2C12 cells [11-14]

6. Low serum media containing 5% or less of FBS, such as Opti-MEM

2.4. Monitoring secretion of AAT from CHO, Huh-7 and C2C12 Cells

1. Phosphate-buffered saline (PBS): 137 mM NaCl, 2.7 mM KCl, 1.8 mM KH₂PO₄, 10mM Na₂HPO₄ pH 7.5

2. DMEM ((−)L-Met/L-Cys) media

3. Sodium pyruvate (100 mM)

4. L-glutamine (100 mM)

5. Cell lifter

6. [³⁵S]-methionine/cysteine ([³⁵S]-Met/Cys) at 11 mCi/mL such as that provided by Promega

7. Huh-7, C2C12, CHO media from section 2.2 for chase

8. Cell lysis buffer: 20 mM MES, 100 mM NaCl, 30 mM Tris-HCl, 0.5% Triton-X 100 (v/v) pH 7.5

9. Broad-spectrum mammalian protease inhibitor cocktail, such as Halt Protease Inhibitor from ThermoFisher

10. N-ethylmaleimide (NEM)

11. Protein-A-Sepharose^® 4B

12. Polyclonal rabbit anti-human alpha-1 antitrypsin antibody

13. Anti-myc monoclonal antibody such as 9B11 offered by Cell Signaling Technology

14. Gel loading buffer: 30 mM Tris-HCl, 9% SDS (w/v), 15% (v/v) glycerol, 0.05% bromophenol blue (w/v), pH 6.8

15. Dithiothreitol (DTT)

16. 9% SDS-PAGE gel

17. PVDF membrane such as that provided Millipore (IPFL00010) (see Note 3)

18. Chromatography paper

19. 1 L salicylate solution: 20 g sodium salicylate, 300 mL methanol, 700 mL Milli-Q water

20. Phosphor screen exposure cassette 8” x 10” (Molecular Dynamics, 184093943970)

2.5. Assessing activity of endogenous or overexpressed AAT

1. Recombinantly purified full length AAT (commercial sources are available from Athens Research Technology)

2. Recombinantly produced human neutrophil elastase (HNE) (commercial sources are available from Athens Research Technology)

3. Trichloroacetic acid (TCA)

4. Acetone

5. Gel loading buffer: 30 mM Tris-HCl, 9% SDS (w/v), 15% glycerol (v/v), 0.05% bromophenol blue (w/v), pH 6.8

6. Dithiothreitol (DTT)

7. 9% SDS-PAGE gel

8. PVDF membrane such as that provided Millipore (IPFL00010) (see Note 3)

9. Phosphate-buffered saline with tween-20 (PBST): 137 mM NaCl, 2.7mM KCl, 1.8 mM KH₂PO₄, 10 mM Na₂HPO₄, 0.1% tween-20, pH 7.5

10. Milk solution: 5% non-fat dry milk powder, 2% bovine serum albumin (w/v%) in PBST

11. Polyclonal rabbit anti-human AAT antibody

12. Anti-rabbit-antibody secondary conjugated to IRDye-680. Commonly purchased from Li-COR

2.6. Measuring effectiveness of proteostasis activators on AAT secretion in cells

1. Low serum media containing 5% or less of FBS, such as Opti-MEM

2. See Table 2 for a list of common proteostasis activators and supplier information

3. Recombinantly produced human neutrophil elastase (HNE) (commercial sources are available from Athens Research Technology)

4. Trichloroacetic acid (TCA)

5. Acetone

6. Phosphate-buffered saline (PBS): 137 mM NaCl, 2.7 mM KCl, 1.8 mM KH₂PO₄, 10 mM Na₂HPO₄, pH 7.5

7. Broad-spectrum mammalian protease inhibitor cocktail, such as Halt Protease Inhibitor from ThermoFisher

8. N-ethylmaleimide (NEM)

9. Cell lysis buffer (20 mM MES, 100 mM NaCl, 30 mM Tris-HCl, 0.5% triton-X 100, pH 7.5)

10. Trichloroacetic acid (TCA)

11. Acetone

12. Gel loading buffer: 30 mM Tris-HCl, 9% SDS (w/v), 15% (v/v) glycerol, 0.05% bromophenol blue (w/v), pH 6.8

13. Dithiothreitol (DTT)

14. 9% SDS-PAGE gel

15. PVDF membrane such as that provided Millipore (IPFL00010) (see Note 3)

16. Phosphate-buffered saline tween-20 (PBST): 137 mM NaCl, 2.7mM KCl, 1.8 mM KH₂PO₄, 10 mM g Na₂HPO₄, 0.1% tween-20, pH 7.5

17. Milk solution: 5% non-fat dry milk powder, 2% bovine serum albumin (w/v) in PBST

18. Anti-BiP antibody rabbit polyclonal

19. Anti-HDAC7 rabbit polyclonal antibody

20. Anti-Sel1L rabbit polyclonal antibody

21. Anti-GAPDH mouse monoclonal antibody

22. Anti-rabbit-antibody secondary conjugated to IRDye-680. Commonly purchased from Li-COR

Table 2.

Use of common proteostasis activators and cellular effect

Compound Name (Abreviaiton)	Working Concentration (μM)	Cellular Effect	Supplier, Product Number	Reference
Verapamil	3	Increase intracellular calcium concentration in the ER	Cayman, 14288
Dantrolene	25		Sigma-Aldrich, D9175	[17]
Diltiazem	10		TOCRIS, 0685
Thapsigargin (Tg)	3	Induces unfolded protein response (UPR) by decreasing ER calcium concentration	Thermo, T7458	[18]
Compound 5 (Comp5)	10	Upregulate ATF6 arm of the UPR pathway	Life Chemicals, HS:3822000000	[19]
Compound 147 (Comp147)	10		Chembridge, 6538059
Suberoylanilide hydroxamic acid (SAHA)	5	Histone deacetylase (HDAC) inhibitor	Sigma, SML0061	[20]
Salubrinal	20	Inhibitor of eIF2α phosphorylation	Sigma, SML1073	[21]
BiP induce X (Bix)	10	Reported to induce BiP expression	Sigma, SML0951	[22, 23]

2.7. Endoglycosidase assay

1. Recombinantly purified full length AAT (commercial sources are available such as from Athens Research Technology)

2. PNGaseF glycosidase to remove most N-linked glycans. A kit is available from New England Biolabs

3. EndoH glycosidase to cleave chitobiose core of high mannose N-linked glycans. A kit is available from New England Biolabs

4. Trichloroacetic acid (TCA)

5. Acetone

6. Gel loading buffer: 30 mM Tris-HCl, 9% SDS (w/v), 15% (v/v) glycerol, 0.05% bromophenol blue (w/v), pH 6.8

7. Dithiothreitol (DTT)

8. 9% SDS-PAGE gel

9. PVDF membrane such as that provided Millipore (IPFL00010) (see Note 3)

10. Milk solution: 5% non-fat dry milk powder, 2% bovine serum albumin (w/v) in PBST)

11. Polyclonal rabbit anti-human alpha-1 antitrypsin antibody

12. Anti-rabbit-antibody secondary conjugated to IRDye-680. Commonly purchased from Li-COR

2.8. Special equipment

1. Cell counter

2. Refrigerated centrifuge

3. Bio Safety cabinet (BSC)

4. Water bath

5. Cell culture incubator

6. Microscope

7. Heat Block

8. A laser scanner for biomolecular imaging, such as a Typhoon FLA 9500

9. An infrared imaging system, such as the Li-Cor Odyssey

10. A gel drier

11. Radiation survey meter to monitor [³⁵S] contamination in lab space, such as those proved by Radiation & Safety & Control Services

3. Methods

3.1. Cell culture maintenance and transfection

3.1.1. Cell thawing and passaging

1. Place cells in a 37 °C water bath for 3 min or until the cells have fully thawed and spin at 250xg for 5 min to pellet cells (see Note 4). In a biosafety cabinet (BSC), remove supernatant and resuspend the cells in 1 mL of the appropriate media (see Table 1).

2. Add resuspended cells to a 10-cm plate containing 5 mL of the correct culture media and place in a 37 °C, 5% CO₂. See Table 1 for culture media for CHO, C2C12, and Huh-7 cells (see Note 5).

3. Ensure cells do not overgrow and split every 2 to 4 days or when plate is 90% confluent (see Note 6).

4. Passage cells in a BSC by first removing the old media, washing with 3 mL of PBS and adding 2 mL of 0.25% trypsin-EDTA. Place the cells back into the 37 °C incubator for 5 min or until cells have dissociated from the plate.

5. Once dissociated, collect cells and pellet at 250xg for 5 min before removing the supernatant and resuspending with culture media. A 1:4 split, or plating 2 X 10⁶ cells, is generally sufficient for maintenance and expansion of cell lines (see Note 7).

3.1.2. Transfection of AAT cDNA into cells

1. One day prior to transfection count cells and plate 7.5 X 10⁵ cells into a 3-cm plate and place into 37 °C, 5% CO₂ incubator for 16-18 hr.

2. The next day, warm low serum media (no more than 5% FBS) in a 37 °C water bath at least 20 min prior to transfection (see Note 8).

3. In a BSC prepare the transfection mixture. For CHO, C2C12 and Huh-7 cells, dilute 2.4 μg of cDNA into 40 μL of prewarmed Opti-MEM.

4. In a separate tube, add 4.8 μL of the transfection reagent and dilute to 40 μL. Incubated for 5 min at room temperature (RT). The most optimum transfection reagent varies with cell type (see Note 2):

   1. CHO: Polyethyleneimine (PEI)
   2. Huh-7: GeneXPlus
   3. C2C12: Lipid based transfection reagent, such as Lipofectamine 2000

5. After incubating, add the 40 μL of transfection mixture to the tube containing the DNA. Incubate the mixture for 20 min at RT (see Note 9).

6. Once complexed, add the 80 μL of the mixture drop wise to the transfection plates prepared the previous day (see Note 10).

7. Place the plates into a 37 °C, 5% CO₂ incubator for 2 hr.

8. Remove the transfection media and replaced with low serum media (no more than 5% FBS) such as Opti-MEM. Allow the cells to express the protein for an additional 16-18 hr prior to downstream analysis (see Note 11).

3.2. C2C12 myotube differentiation and transduction

1. A week prior to transduction plate 2.5 x 10⁵ C2C12 cells into a 3-cm plate containing C2C12 culture media and grow cells in a 37 °C, 5% CO₂ incubator for 24 hr.

2. On day 1, ensure cells have adhered to the plate before removing the media and replacing with 1.5 mL of DMEM media (no FBS) containing 2% HS and place back into 37 °C, 5% CO₂ for 3 days. Cells should appear globular and triangular in shape (Fig. 1).

3. On day 4, remove old media and replace with 1.5 mL of DMEM media containing 2% HS. Place plates back into the incubator for an additional 3 days to complete the differentiation.

4. On day 7, cells should appear elongated (Fig. 1) indicating they are ready for transduction with pre-prepared AAV (see Note 12).

5. The preparation of the AAV capsid containing the AAT plasmid has been reported elsewhere [11-16]. Usage of the AAV capsid provides an additional method to deliver the AAT cDNA to difficult to transfect cells lines, such as C2C12 cells. Prior to transduction, remove the media and replace with 1.5 mL of serum-free DMEM media containing no FBS (see Note 13).

6. Calculate the multiplicity of infection (MOI) using a mock plate to approximate the number of cells in the experimental plates.

7. Transduce the myotubes using a MOI of 2.5 x 10⁵ viral partials per cell (see Note 14).

8. After adding the AAV to the cell plate incubate the cells for an additional 2 hr at 37 °C, 5% CO₂ prior to removing FBS-free DMEM media and replacing with 1.5 mL of low serum media containing 5% or less FBS media (see Note 11).

9. Culture cells for an additional 2 days prior to downstream analysis.

Figure 1. Timeline to differentiate C2C12 myoblasts into myotubes for AAV transduction.

Cells were plated and grown 1 day prior to initiating differentiation. On day 1 media was replaced with DMEM containing 2% horse serum and incubated for 3 days. On day 4 the media was refreshed with additional DMEM+2% HS until day 7 where they were transduced with rAAV containing the AAT cDNA. Cells were incubated for an additional 2 days in the presence or absence of the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) prior to analysis of lysate and media. Representative images of C2C12 differentiated cells in the absence of SAHA provided above for each time point. Figure created using BioRender.

3.3. Monitoring secretion of AAT from CHO, Huh-7 and C2C12 cells

3.3.1. Radioactive labeling of newly synthesized AAT

1. Prior to analysis of secretion, transfect the desired cell type with AAT cDNA (see 3.1.2).

2. The next day remove the media and wash the plate 2 times with 1 mL of sterile PBS.

3. Once washed, add 1 mL completed pulse media to each plate and place into 37 °C, 5% CO₂ incubator for 30 min (see Note 15). Proper radiation personal protection equipment should be used, and rules and regulations followed: The pulse media for each plate contains:

   1. 1 mL of DMEM (-L-Met/L-Cys)
   2. 100 μL of L-glutamine (100 mM)
   3. 100 μL of sodium pyruvate (100 mM)
   4. 6.25 μL of [³⁵S]-Met/Cys at 11 mCi/mL

4. Once pulsed, wash the cells with 1 mL PBS and add 1 mL of appropriate culture media to each plate and incubated for the desired time to chase newly synthesized protein (see Note 16).

5. At each chase time point, collect the media from the plate before washing with 1 mL of ice-cold PBS. Cells should remain on ice.

6. Add 500 μL of cold lysis buffer completed with protease inhibitor cocktail and 20 mM NEM to each plate and scrape the cells off using a cell lifter before collecting the mixture into a clean 1.5 mL tube.

7. Shake the cells vigorously for 10 min at 4°C before spinning them at 20,000xg for 10 min at 4 °C.

8. Remove and save the supernatant for further analysis. Solubilize the insoluble pellet with 20 μL of 1% SDS in 100 mM Tris-HCl, pH 8 and boil the tube for 10 min at 95 °C and vortex for 5 min at RT.

9. Quench the SDS in the insoluble fraction by adding 980 μL of lysis buffer.

10. Incubate the media collected at step 5, soluble lysate, and insoluble fraction overnight with coated beads to immunoprecipitate (preparation described below) and isolate radiolabeled AAT.

3.3.2. Isolation of radioactively labeled AAT by immunoprecipitation

1. Prior to immunoprecipitating AAT from fractions, prepare the resin.

2. For each sample prepare the following (see Note 17):

   1. 10 μL of Protein-A resin
   2. 1 μL of antibody needed for precipitation (see Note 18)

      1. Endogenous AAT – anti-AAT rabbit polyclonal
      2. Transfected AAT – anti-myc mouse monoclonal
   3. 90 μL of PBS

3. Mix gently for at least 1 hr at 4 °C.

4. After mixing add 100 μL of antibody:resin mixture each tube contain the media, lysate, or insoluble fractions.

5. Incubate samples overnight using an end-over-end shaker at 4 °C.

6. The next morning, spin the samples in a 4 °C centrifuge at 250xg for 5 min.

7. Remove the supernatant and wash resin with 500 μL of lysis buffer with no protease inhibitor added.

8. Repeat the washing process twice.

9. After the third wash resuspend the beads with 30 μL of gel loading buffer containing 100 mM of DTT.

10. Vortex the sample before boiling at 95 °C for 10 min and shake vigorously at RT for 10 min. Spin samples down at 250xg for 5 min using a RT centrifuge.

11. Resolve samples by running them on a 9% SDS-PAGE gel at constant 20 mA/gel and max voltage until the dye front has run off the bottom of the gel.

12. Remove the gel and place it in 5 mL of Milli-Q water or any water with a resistivity 18.2 megaohms at 25 °C with total organic carbon ≤ 5 parts per billion. Shake gently for 10 min.

13. After wash, remove water and replace with 5 mL of salicylate solution for 5 min. Repeat wash with salicylate solution once more.

14. After washing, place gel on chromatography paper and dry using a gel drier for 2 hr. Once dry incubate with a phosphor screen for at least 2 days (see Note 19).

15. Image using a laser scanner for biomolecular imaging, such as a Typhoon FLA 9500 and quantify the bands using software that quantifies pixel intensity, such as ImageJ (see Note 20).

16. The percent secreted is calculated by dividing the amount of AAT in the media by the total amount of protein at each chase points (amount in lysate, media, and insoluble fraction) (see Note 21).

3.4. Assessing activity of endogenous or overexpressed AAT (Fig. 2)

Figure 2. Activity assay to monitor functionality of AAT.

AAT (protein data bank accession number (PBD): 1ATU [24] green and red) targets the protease HNE (PDB: 3Q76 [25], gray) (1). HNE docking on the AAT reactive center loop (RCL) leads to the formation of a covalent bond between the HNE active site and the AAT RCL. Subsequent protease cleavage of this AAT loop results in RCL insertion into the central serpin beta-sheet, distorting the elastase active site and kinetically trapping the covalently bound protease (PDB: 2D26 [26]) (2). Misfolded or inactive AAT variants behave as HNE substrates resulting in a 358 amino acid proteolytic fragment (cAAT). Both the $\text{HNE:AAT}$ complex (4) and cleaved AAT can (5) can be visualized by Western blot. The $\text{HNE:AAT}$ complex migrates at ~80 kDa while unbound AAT is at ~50 kDa. Nonfunctional AAT will migrate faster as the detectable cAAT is lower in molecular weight due to dissociation of the 36 C-terminal amino acids. Figure created using BioRender.

1. Prior to analysis of AAT activity, transfect or transduce cells with AAT cDNA (see 3.1.2 or 3.2).

2. After transfection/transduction collect the media from each plate and spin at 20,000xg for 5 min to remove any cellular debris.

3. Aliquot 400 μL of spun media into two separate tubes: a control and a sample tube. The control tube has no protease (HNE) added to it and serves to visualize the migration of AAT secreted from the cells. The sample tube has HNE added (see below) to measure the activity of secreted AAT and the migration of the complex on a gel.

4. Additionally, create two positive control tubes with 400 μL of unspent DMEM media and spike in 1 μL of purified AAT (3.06 mg/mL) into each tube and label one as unconjugated (to have no HNE added to visualize migration of purified AAT on a gel) and one as sample (to have HNE added to it to visualize the migration of the $\text{HNE:AAT}$ complex on a gel).

5. Add 1 μL of HNE (1.27 mg/mL) to the sample tubes and incubate both the control and sample tubes at 37 °C for 30 min with gentle mixing.

6. Precipitate the samples using 10% (v/v) TCA, vortex and spin at 20,000xg for 10 min at 4 °C

7. Remove the supernatant and wash the pellets with 1 mL of ice-cold acetone. Remove the acetone by spinning samples at 20,000xg for 10 min at 4 °C and aspirating the supernatant.

8. Dry pellets at RT for at least an hr.

9. Add 30 μL of gel loading buffer containing 100 mM of DTT and heat at 95 °C for 10 min before shaking at RT for 10 min (see Note 22).

10. Resolve samples by running them on a 9% SDS-PAGE gel at a constant 20 mA/gel and max voltage until the dye front has run off the gel.

11. Set up a Western blot to transfer resolved protein to a PVDF membrane. After transferring add 5 mL of the milk solution for 1 hr.

12. Remove milk solution and add an additional 5 mL of milk with 1 μL of anti-AAT rabbit polyclonal antibody.

13. The next day wash the membrane 3 times with 5 mL of milk for 5 min before adding 5 mL of milk with 1 μL of fluorescently labeled anti-rabbit IgG secondary for 1 hr and incubate at RT.

14. Wash away the secondary 3 times with 5 mL of PBST and image membrane using an infrared imaging system, such as the Li-Cor Odyssey and quantifying the bands using software that quantifies the pixel intensity, such as ImageJ (see Note 20).

15. Monomeric, non-complexed, AAT is detected at ~50 kDa while $\text{AAT:HNE}$ is detected at ~80 kDa. The amount of AAT in complex with HNE is defined as the amount of active protein as this population is folded correctly and can interact to form the complex with HNE. The percentage of active AAT is calculated dividing the amount of $\text{HNE:AAT}$ by the total amount of protein in the HNE treated lanes (see Note 23) (Fig. 2)

3.5. Measuring effectiveness of proteostasis activators on AAT secretion in cells

1. Replace the media in the 3-cm plate for the C2C12 with low serum media (no more than 5% FBS) 2 hr after transfection (see 3.1.2) (see Note 11).

2. After replacing the media add the desired proteostasis regulators at the correct concentration (Table 2) and incubate with the drugs for 24 hr before downstream analysis of lysate and media fractions (see Note 24).

3. Collect media and clear it of cell debris by centrifuging it at 20,000xg for 5 min at RT.

4. Process the media as noted in 3.4 to assess the amount secreted as well as the activity of the protein from each treated plate.

5. Wash the cells bound to the plate once with 1 mL of cold PBS and remove.

6. Add 500 μL of cold lysis buffer completed with protease inhibitor and 20 mM NEM and scrape the cells off the plate. Pipette cells into a fresh 1.5 mL tube.

7. Shake the cells vigorously for 10 min prior to spinning the lysate for 10 min at 4 °C at 20,000xg.

8. Collect the supernatant and precipitate the protein using 10% (v/v) TCA, vortex, and spin at 20,000xg for 10 min at 4 °C.

9. Remove the supernatant and wash the pellet with 1 mL of ice-cold acetone. Remove the acetone by spinning samples at 20,000xg for 10 min at 4 °C and aspirating the supernatant.

10. Dry pellets at RT for at least an hr.

11. Add 30 μL of gel loading buffer containing 100 mM of DTT and heat at 95 °C for 10 min before shaking at RT for 10 min (see Note 22).

12. Resolve samples by running them on a 9% SDS-PAGE gel at a constant 20 mA/gel and max voltage until the dye front has run off the gel.

13. Set up a Western blot to transfer resolved protein to a PVDF membrane. After transferring add 5 mL of milk solution for 1 hr.

14. Remove blocking milk and add 5 mL of fresh milk. Blot the membrane with various ER markers to assess the effect of proteostasis activators in the tested cell line. Ensure the correct dilution factor is used for each protein. Common proteins include, but are not limited to: HDAC7, Sel1L and BiP. GAPDH should be used as a loading control for sample normalization.

15. Incubate overnight at 4 °C with gentle rocking.

16. The next day wash 3 times with milk solution and add 1 μL of correct secondary antibody dependent on the primary antibody used.

17. Incubate the membrane with the secondary antibody for 1 hr at RT.

18. Wash away the secondary antibody 3 times with 5 mL of PBST. Image the membrane using an infrared imaging system, such as the Li-Cor Odyssey and quantifying the bands using software that quantifies the pixel intensity, such as ImageJ (see Note 20).

3.6. Endoglycosidase assay to assess cellular localization

1. Prior to assessing the glycan composition to assess localization within cells (Fig. 3A) transfect cells (see 3.1.2) or transduce C2C12 myotubes (see 3.2) with AAT cDNA.

2. Collect the media from transfected or transduced cells and clear cellular debris by centrifuging for 5 min at 20,000xg at RT.

3. Aliquot 300 μL of cleared media into 3 tubes (1 untreated control, 1 for treatment with PNGaseF and 1 for treatment with EndoH)

4. Additionally, create another set of tubes for the purified AAT as a positive control by adding 1 μL (3.06 mg/mL) AAT of into each tube.

5. Add 10% (v/v) of denaturation buffer to all samples and treat tubes at 95 °C for 10 min.

6. After denaturation spin down condensate and prep the tubes as followed:

   1. PNGaseF (available from PNGaseF kit from New England Biolabs)

      1. 10% (v/v) Glycobuffer 2
      2. 10% (v/v) 10% NP40
      3. 1 μL of PNGaseF
   2. Untreated

      1. 10% (v/v) Glycobuffer 2
      2. 10% (v/v) 10% NP40
      3. 1 μL water
   3. EndoH (available from EndoH kit from New England Biolabs)

      1. 10% (v/v) Glycobuffer 3
      2. 1 μL EndoH

7. Incubate the tubes at 37 °C for 1 hr under gentle agitation (see Note 25).

8. After incubation, precipitate the protein in the sample using 10% (v/v) TCA and vortex.

9. Precipitate the samples using 10% (v/v) TCA, vortex, and spin at 20,000xg for 10 min at 4 °C.

10. Remove the supernatant and wash the pellets with 1 mL of ice-cold acetone. Remove the acetone by spinning samples at 20,000xg for 10 min at 4 °C and aspirating the supernatant

11. Dry pellets at RT for at least 1 hr.

12. Add 30 μL of gel loading buffer containing 100 mM of DTT and heat at 95 °C for 10 min before shaking at RT for 10 min (see Note 22).

13. Resolve samples by running them on a 9% SDS-PAGE gel at a constant 20 mA/gel and max voltage until the dye front has run off the gel.

14. Set up a Western blot to transfer resolved protein to a PVDF membrane. After transferring block with 5 mL of milk solution for 1 hr.

15. Remove blocking solution and add an additional 5 mL of milk with 1 μL of anti-AAT rabbit polyclonal antibody overnight.

16. The next day wash 3 times with 5 mL of milk for 5 min before adding 5 mL of milk and 1 μL of fluorescently labeled αrabbit IgG secondary.

17. Incubate for 1 hr at RT.

18. Wash away the secondary antibody 3 times with 5 mL of PBST. Image the membrane using the Li-Cor system and quantify the bands using software that quantifies the pixel intensity, such as ImageJ (see Note 20).

19. PNGaseF removes most glycans from proteins whereas EndoH will only remove those that are added within the endoplasmic reticulum and are high mannose in composition. Insensitivity to EndoH results from modification of the glycan as the protein is trafficked through the Golgi (Fig. 3B).

Figure 3. Trafficking and analysis of proteins traversing the secretory pathway.

A) Secreted proteins are first synthesized and translocated into the endoplasmic reticulum (ER) (1) where the addition of high mannose glycans and protein maturation occurs (blue protein). Once properly folded the protein is trafficked through the ER-Golgi intermediate complex (ERGIC) (2) before entering the cis-Golgi. As the protein traverses the Golgi the glycans are modified by various transferases and hydrolases to form complex and hybrid glycans (green protein). The protein migrates further through the Golgi to the trans-Golgi (4) before being secreted into the extracellular space (5). B) Endoglycosidase activity determines localization of protein within the secretory pathway. EndoH removes high mannose glycans, indicative of ER localization (top left) but is unable to remove complex or hybrid forms (bottom left). PNGaseF can remove most glycans regardless of composition (right). EndoH sensitivity results from proteins residing within the ER, whereas insensitivity results from glycan modification made in the Golgi. Figure created using BioRender.

4. Notes

1. There can be variations in cell growth depending on lot and manufacture of FBS. If the recommended FBS is not available cells may be adapted to grow in different kinds of FBS. Adaptation can be accomplished by slowly introducing the new FBS into the culture media (10% at a time). At each increase allow cells to recover their doubling time before increasing the levels of the new FBS. Not all cells, however, can be adapted and this must be determined experimentally.

2. Other transfection reagents can be used but the efficiency of transgene incorporation may be reduced. If alternative reagents are used the optimal amounts to use must be determined experimentally to achieve the highest levels of protein expression with minimal cell death.

3. Ensure PVDF membrane is activated with 100% methanol prior to use for Western blot.

4. The O-ring on a cryovial can shrink at prolonged storage temperatures, resulting in liquid nitrogen leaking into the vial. Ensure no liquid nitrogen is in the vial prior to warming otherwise tube might explode.

5. Antibiotics, such as PenStrep, can be added at a 1:100 dilution if contamination is a concern. Cell growth and doubling time may slow as a result and could affect downstream experiments.

6. This time is approximate and should be adequate to obtain a 90% confluent plate. Cells should be inspected every day to ensure proper growth and that they are free from contamination.

7. More or fewer cells can be plated depending on experimental design and timelines. Plates will become confluent faster if more cells are used and will need to be split sooner.

8. Use of cold Opti-MEM will drastically reduce transfection efficiency and amount of recombinant protein produced by cells.

9. Combined DNA:transfection reagent tube should appear cloudy after 20 min of incubation and indicates the complex has formed correctly.

10. If antibiotics are being used, they must be removed prior to adding the transfection mixture to the cell. Transfecting in the presence of antibiotics will result in substantial cell death.

11. Opti-MEM contains 5% FBS and is better suited for downstream protein precipitation compared to culture media with 10% FBS. A high percentage of serum will affect Western blot resolution. Cells remain viable up to 48-72 hr using Opti-MEM.

12. If cells do not look elongated, as in Fig. 1, another 3-day incubation with DMEM + 2% HS can be done. Remove the media from the cells and replace with fresh differentiation media.

13. The presence of FBS in the media can reduce the transduction efficiency, resulting in poor protein expression. Allow cells to incubate with rAAV for at least 2 hr prior to reintroducing FBS or Opti-MEM.

14. MOI must be determined experimentally based on the cell type being transduced, the plasmid construction, and protein being exogenously introduced. It is suggested to try a few MOIs prior to running the experiment to determine optimum number of viral particles to add per cell.

15. Pulse time can be modified depending on desired outcome. Longer pulse time will result in more protein being labeled. Shorter pulse periods will reduce the population labeled with [³⁵S].

16. Chase times can be altered, and more time points can be added for finer resolution of AAT secretion.

17. Recipe for Protein-A master mix can be scaled according to sample. Always make enough for 1 extra sample as 100% recovery of the mix is not possible.

18. If using a different tag for protein immunoprecipitation the amount of antibody to use must be determined experimentally.

19. Length of incubation of the phosphor screen can be extended if higher signal from immunoprecipitated protein is desired. The signal from any background proteins will also be amplified.

20. Any software that quantifies band pixel intensity can be used. ImageJ is free and is provided by the National Institutes of Health (NIH).

21. As an example of this calculation, if the quantified amounts of protein in the media, lysate and insoluble fraction were 7,000, 2,000, and 1,000, respectively the total amount of protein would be quantified as 10,000. To calculate the percent secreted divide the media fraction (7,000) by the total protein (10,000) $\frac{7,000}{10,000}$ or 0.7 or 70% when multiplied by 100.

    $$\frac{Amountinmedia}{Totalprotein(media+lysate+insolublefraction)}\ast 100=PercentSecreted$$

22. If the gel loading buffer turns yellow after adding to the pellet add 1 μL of 0.5 M sodium hydroxide in water. The yellow color indicates a low pH due to residual TCA and the bromophenol blue in the loading buffer. Correctly adjusting the pH is necessary for running the SDS-PAGE gel.

23. As an example of this calculation, if in the HNE treated samples the $\text{HNE:AAT}$ band was quantified as 1,000 and the AAT alone was quantified as 9,000 the total AAT secreted would be 10,000 and the percent of active protein would be $\frac{1,000}{10,000}$ or 0.1 or 10% when multiplied by 100, as this is the percentage of AAT able to trap and bind HNE.

    $$\frac{AmountofHNE:AAT}{Totalprotein(HNE:AAT+AAT)}\ast 100=PercentActive$$

24. These concentrations are dependent on the type and number of cells. If varying from the above protocol the optimal drug concentration and incubation time must be determined experimentally.

25. If using endoglycosidases that are different from the ones mentioned in 2.7, a longer incubation time may be required to fully deglycosylate the protein.