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. 2021 Dec 9;11(1):23730.
doi: 10.1038/s41598-021-02860-5.

Characterization of recombinant β subunit of human MUC4 mucin (rMUC4β)

Prakash G Kshirsagar  1 , Mansi Gulati #  1 , Wade M Junker #  1   2 , Abhijit Aithal #  1 , Gaelle Spagnol  1 , Srustidhar Das  1 , Kavita Mallya  1 , Shailendra K Gautam  1 , Sushil Kumar  1 , Paul Sorgen  1 , Krishan K Pandey  3 , Surinder K Batra  4   5   6   7 , Maneesh Jain  8   9
Affiliations

Characterization of recombinant β subunit of human MUC4 mucin (rMUC4β)

Prakash G Kshirsagar et al. Sci Rep. .

Abstract

MUC4 is a transmembrane mucin expressed on various epithelial surfaces, including respiratory and gastrointestinal tracts, and helps in their lubrication and protection. MUC4 is also aberrantly overexpressed in various epithelial malignancies and functionally contributes to cancer development and progression. MUC4 is putatively cleaved at the GDPH site into a mucin-like α-subunit and a membrane-tethered growth factor-like β-subunit. Due to the presence of several functional domains, the characterization of MUC4β is critical for understanding MUC4 biology. We developed a method to produce and purify multi-milligram amounts of recombinant MUC4β (rMUC4β). Purified rMUC4β was characterized by Far-UV CD and I-TASSER-based protein structure prediction analyses, and its ability to interact with cellular proteins was determined by the affinity pull-down assay. Two of the three EGF-like domains exhibited typical β-fold, while the third EGF-like domain and vWD domain were predominantly random coils. We observed that rMUC4β physically interacts with Ezrin and EGFR family members. Overall, this study describes an efficient and simple strategy for the purification of biologically-active rMUC4β that can serve as a valuable reagent for a variety of biochemical and functional studies to elucidate MUC4 function and generating domain-specific antibodies and vaccines for cancer immunotherapy.

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Conflict of interest statement

SKB is one of the co-founders of Sanguine Diagnostics and Therapeutics, Inc. The other authors disclose no potential conflicts of interest.

Figures

Figure 1
Figure 1
Plasmid cloning strategy, expression, and purification of the human rMUC4β. (A) Schematic representation of the complete structure of MUC4 protein and construction of pET-28a-MUC4β plasmid: MUC4β sequence (from GDPH to C-terminal) was amplified with primers incorporated with ‘NdeI’ and XhoI’ restriction sites and cloned in-frame with the His6x tag and thrombin site (TS) encoded by pET-28a (+) vector. The figure was drawn by using Microsoft PowerPoint. (B) Expression profile of total cellular proteins from E. coli C41(DE3) strain: The pET-28a (empty vector) and pET-28a-MUC4β transformed C41(DE3) cells were induced with an indicated amount of IPTG. SDS-PAGE and immunoblotting analyses were performed on the uninduced (U) and IPTG-induced (I) culture supernatant. (C) Comparative assessment of rMUC4β expression profile in C41(DE3) and Rosetta 2(DE3) competent cells at different IPTG concentrations using SDS-PAGE and immunoblotting with anti-His tag antibody. (D) Effect of different post-induction incubation temperatures on rMUC4β expression in Rosetta 2(DE3): Total protein expression from each temperature condition was assessed by SDS-PAGE and immunoblotting. (E) Isolation and ÄKTA-FPLC affinity purification of rMUC4β. The quality of pre-and-post-ÄKTA fractions was assessed by Coomassie staining and immunoblot analyses.
Figure 2
Figure 2
Biophysical characterization and secondary structure analysis of rMUC4β. (A) Far-UV CD spectra of rMUC4β were plotted using GraphPad Prism (https://www.graphpad.com/ version 8). (B) Comparison of the percentage of secondary structure content estimated or predicted from CD spectrum, amino acid sequence, and I-TASSER analyses. The 2D multicolor pie chart is drawn using Origin 2019 software (https://www.originlab.com/ version 9.6), showing the structure compositions estimated using (C) CDSSTR and (D) Contin-LL (Provencher and Glockner) methods. (E) Predicted structure of rMUC4β generated with the I-TASSER tool (https://zhanggroup.org/I-TASSER/) and PyMOL Molecular Graphics System (https://pymol.org/2/ version 2.3.4), Schrödinger, LLC. The cartoon structure of MUC4β indicating N-terminus (blue) and C-terminus (red). Right panel: i-TASSER predicted structure of rMUC4β rotated 180° to visualize the three EGF-like domains. EGF- like domain I (Cyan) and EGF- like domain II (Magenta) appear to be typical β-strand structures, while EGF-like domain III (Red) is a loop.
Figure 3
Figure 3
Affinity pull-down and tandem mass spectrometry to determine the interacting partners of MUC4. (A) Schematic outline of affinity pull-down assays (I) Pre-clearing of total lysate [input] of MUC4 expressing (CD18/HPAF) and non-expressing (PANC-1) pancreatic cancer cell lines. (II) Affinity pull-down reactions were performed in the presence [rMUC4β (+)] or absence [rMUC4β (−), i.e., RIPA buffer] of rMUC4β (bait) added with washed Ni–NTA beads. Respective supernatants [i.e., Flow-through (FT) and last wash (W5)] were collected before the final elution. (III) An aliquot of rMUC4β-bound Ni-resin was eluted separately to serve as a positive control. All saved fractions (as shown in pink color, italic font) from CD18/HPAF (B) and PANC1 (C) were resolved on SDS-PAGE gel and stained with Coomassie Blue. The ~ 80 kDa band (arrowed) was identified, excised, and assessed by the tandem MS/MS and proteomic analyses.
Figure 4
Figure 4
Detection and confirmation of the new and known interacting partners of MUC4 via immunoblotting and IP analyses. (A) Expression profile of MUC4 in CD18/HPAF and PANC-1 cells determined by immunoblotting following 2% agarose gel electrophoresis and 10% SDS-PAGE. (B) Immunoblot analyses of Ni-NTA pull-down fractions from CD18/HPAF or PANC-1 cell lysates in the presence (+) or absence (−) of rMUC4β. The blots were probed with the indicated antibodies. Input, flow-through (FT), wash, and eluate are the same as described in Fig. 3A. (C) Immunoprecipitation using an anti-His tag Ab (clone 27E8) conjugated to magnetic beads following incubation of CD18/HPAF cell lysate in the presence and absence of rMUC4β. (D) Interaction of endogenous MUC4 with Ezrin. Immunoprecipitation was performed with protein A/G agarose beads on CD18/HPAF lysates following incubation with anti-MUC4β Ab.
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