Immunoprecipitation of recombinant LyzC with His-SUMO-Cab45 purified from HEK293 supernatant

[Abstract]

Recent studies demonstrate a novel sorting mechanism for a subset of secreted molecules. This process requires the activity of SPCA1 (secretory pathway Ca²⁺ ATPase 1) to pump calcium (Ca²⁺) ions in the lumen of the trans-Golgi Network (TGN). Local Ca²⁺ increase causes the Ca²⁺- binding protein 45 kD (Cab45) to oligomerize and bind secretory cargoes (clients). This process segregates cargo-client complexes from the bulk milieu of the TGN lumen and packs them into sphingomyelin-rich vesicles. We have developed and optimized a protocol for coimmunoprecipitation of His-SUMO-tagged Cab45 with its recombinant client lysozyme C (LyzC) to show Ca²⁺-dependent protein-protein interaction. We combined mild detergent concentrations with purified recombinant proteins obtained from cell supernatants to achieve conditions close to the physiological estate. This protocol can be applied to any immunoprecipitation experiments where protein-protein interactions are studied in a cation- dependent manner under mild conditions with high specificity.

Keywords: Cab45, IP, Calcium, protein purification, cargo sorting, TGN, oligomerization.
 

[Background]

Sorting and secretion of protein in Golgi apparatus is crucial to maintain cell homeostasis, morphogenesis, differentiation and integrity [Frantz et al., 2010]. After synthesis, glycosylation and folding in the endoplasmic reticulum (ER), soluble and transmembrane proteins become either ER residents or are targeted to the secretory pathway. They pass through the ER-Golgi- intermediate compartment (ERGIC), before entering the Golgi apparatus via COPII vesicles [Blobel, 1980]. Within the Golgi apparatus proteins undergo further modifications when they travel from the cis-Golgi to the trans-Golgi. Then, proteins enter the trans-Golgi-network (TGN), the main sorting hub of the Golgi apparatus, to be sorted and packed for vesicular transport to their final destinations [Glick and Nakano, 2009][De Matteis and Luini, 2008]. While the sorting of lysosomal hydrolases via Mannose-6-phosphate-receptors (M6PR) recognizing mannose-6- phosphate (M6P) modifications is well understood [Kornfeld and Mellman, 1989], the sorting of soluble secretory proteins remains poorly understood. A mechanism involving the Calcium- dependent protein Cab45 was proposed to sort a subset of soluble proteins (clients), such as LyzC [Crevenna et al., 2016]. Monomeric Cab45 in the TGN binds free Ca²⁺, which is pumped into the lumen by the TGN-resident secretory pathway Ca²⁺ATPase 1 (SPCA1) after its activation by cofilin and F-actin [von Blume et al., 2009; 2011][Kienzle et al., 2014]. This causes the oligomerization of Cab45, to result in a specific interaction with its client. The newly formed Cab45-client-complexes are retained in the TGN lumen by differential size. Recent findings show the activity of Golgi kinase Fam20C to be crucial in the specific break-up of these clusters by phosphorylation of Cab45, which are packed subsequently for exit from the TGN in sphingomyelin-rich vesicles [Deng et al., 2018][Hecht et al., 2020]. As the interaction between Cab45 and cargo is highly dependent on the presence of Ca²⁺, the oligomeric state and the cargo itself, established standard assays to study protein-protein interactions were adjusted to overcome the challenging conditions. Our protocol for immunoprecipitation of His-SUMO-Cab45 with recombinant LyzC is exemplary to show transient Ca²⁺-dependent protein-protein interactions. We combined mild detergent concentrations and recombinant proteins purified from cell culture supernatant to achieve the configuration of Cab45 to be close to physiological conditions. This protocol can be applied to any immunoprecipitation experiments where protein-protein- interactions are studied under mild conditions and high specificity.
 

Materials and Reagents

1. Amicon®Ultra 0.5mL, Ultracel® - 10K (Milipore, catalog: UFC501096)
2. Ni²⁺-agarose bead-slurry (Thermo Fisher Scientific, catalog: #25214), store at +4°C
3. 1.7mL tubes (Posi-Click, catalog: 1149K01)
4. NaCl (sigma, catalog: AB13198-01000),
5. Tris (sigma, catalog: AB14044)
6. Triton X-100 (sigma, catalog: AB02025-00500)
7. cOmplete Protease Inhibitor Tablets (Roche, catalog: 11836170001), store +4°C
8. Recombinant LyzC protein (Sigma-Aldrich, catalog: L1667), store at -20°C
9. CaCl₂ solution
10. 2x Laemmli sample buffer (Bio Rad, catalog: #161-0737)
11. Purified His-SUMO-Cab45 variants, store at -80°C
     

Equipment

1. Cooling centrifuge (ThermoScientific,Sorvall (™), Legend(™) Micro21R Centrifuge, model: 75002446)
2. Rotator (Tube Revolver, ThermoScientific, model: 8888-1001)
3. Pipettes and pipette tips (10, 200 and 1000uL).
    

Procedure

All steps should be performed on ice, 4°C.

1. Preparation
   1. Equilibration and buffer exchange of protein
   Equilibrate one Amicon®Ultra (0.5mL, Milipore, Ultracel® - 10K, catalog: UFC501096) with 300 µl Immunoprecipitation buffer with 100 µM CaCl₂, centrifuge at 15.000 rcf for 1 minute, 4°C, discard flow-through.
   For freshly purified protein, skip the following step and thaw protein samples on ice.
   (For frozen and stored protein samples) In order to remove glycerol from the proteins (if applied), perform buffer exchange with equilibrated Amicon®Ultra with 200 uL Immunoprecipitation buffer with 100 µM CaCl₂, wash by centrifugation at 15.000 rcf for 5 minutes, 4°C, discard flow- through. Repeat 2 times.
   Prepare recombinant LyzC protein by thawing on ice, take a sample as input 2.
    
2. Immunoprecipitation
   2. Set-up of Immunoprecipitation
   Measure the protein concentration (optionally use Nanodrop), calculate the amount of protein for each sample to achieve the concentration of protein about 150 µg/mL in the same end volume of 500 µL Immunoprecipitation buffer with 100 µM CaCl₂. This is the working sample. Take 50 µL of each as input 1 sample.
   3. Preparation of Agarose beads
   Using cutted pipette tip to add 25 µL Ni²⁺-agarose bead-slurry to 1.5mL Eppendorf tubes. Add 1 mL Immunoprecipitation buffer with 100 µM CaCl₂ to the beads, wash by centrifugation at 1.000 rcf for 2 min, RT and discard the supernatant. Repeat twice. Carefully remove the supernatant, avoid touching the beads. Place the tubes with beads on ice.
   4. Incubation of working samples with the prepared beads
   Add 300 uL of working sample (from step 3) to each pre-chilled tube with washed beads from step 3. Incubate the tubes containing the protein-bead mixture for 2 hours on a rotator at 4°C.
   5. Removal of unbound protein from the beads
   Remove the protein-bead mixture from the turning wheel and wash 3 times with 1 mL Immunoprecipitation buffer with 100 µM CaCl₂buffer, centrifuge for 3 min, 1.000 rcf, 4°C between each step with the removal of the supernatant. (Save a sample of the supernatant as unbound 1, or wash 1 respectively).
   6. Incubation with LyzC protein
   Incubate the washed protein-on-beads with LyzC protein in 300 uL of Immunoprecipitation buffer with 100 µM CaCl₂ for 2 hours at 4°C on a turning wheel.
   7. Reduction of non specifically bound LyzC protein
   After incubation, wash three times with 1 mL Immunoprecipitation buffer with 100 µM CaCl₂, with centrifugation 1000 rcf for 5 min, 4°C between each step. Discard the supernatant (Take a sample as unbound 2 or wash 2 respectively, if desired). Carefully remove the supernatant completely without touching the beads.
   8. Elution of proteins and possible protein complexes
   Elute the proteins from the beads, by adding 40 uL of 1x Laemmli buffer and incubation at 95°C for 10 minutes. Spin at 15000 rcf for 5 minutes to pellet the beads. Collect the supernatant containing the proteins and store it as an IP sample.
    
3. Analysis of protein interaction
   9. Protein interaction analysis with SDS-Gel-Electrophoresis
   Perform SDS-Gel Electrophoresis following standard protocol, stain with Coomassie solution until bands are visible.
    

Recipes

Immunoprecipitation buffer (50 mM Tris, 100 mM NaCl, and 0.1% TritonX-100):

To make 100mL of Immunoprecipitation buffer add 2 mL of 5M NaCl (AB13198-01000), 5 mL of 1M Tris pH 7.4 (AB14044) and 100uL of Triton X0-100(AB02025-00500). Keep at +4°C. Before using, dissolve 2 tablets of cOmplete Protease Inhibitor Tablets (Roche 11836170001).

Immunoprecipitation buffer with 100 µM CaCl₂ (50 mM Tris, 100 mM NaCl,and 0.1% TritonX-100). 
Add 5µL of 1M CaCl₂ solution to 50mL of Immunoprecipitation buffer. Keep at +4°C. Before using dissolve 1 tablets of cOmplete Protease Inhibitor Tablets (Roche 11836170001).
 

Acknowledgments

J. von Blume was funded by the Deutsche Forschungsgemeinschaft Perspective Program (Boehringer Ingelheim Foundation; project grant BL 1186/4-1) and the National Institutes of Health National Institute of General Medical Sciences (award number GM134083-01) and Yale DRC (P30 DKO45735)

Competing interests

The author declare no competing interests.

References

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Kienzle, C., Basnet, N., Crevenna, A.H., Beck, G., Habermann, B., Mizuno, N., and von Blume, J.. (2014). Cofilin recruits F- actin to SPCA1 and promotes Ca2+-mediated secretory cargo sorting. J Cell Biol. 206:635-654.

Deng, Y., Pakdel, M., Blank, B., Sundberg, E.L., Burd, C.G., von Blume, J. (2018). Activity of the SPCA1 Calcium Pump Couples Sphingomyelin Synthesis to Sorting of Secretory Proteins in the Trans-Golgi Network. Dev Cell. 47:464-478 e468.

Hecht, T.K., Blank, B., Steger, M., Lopez, V., Beck, G., Ramazanov, B., Mann, M., Tagliabracci, V., von Blume, J. (2020). Fam20C regulates protein secretion by Cab45 phosphorylation. J Cell Biol. 219.