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

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.

Oncogenic KRAS, Mucin 4, and Activin A‐Mediated Fibroblast Activation Cooperate for PanIN Initiation

“…[ 10 , 12 ] Although the precise mechanism remains to be explored, the mutations in Muc 4 may trigger alternative splicing of Muc 4, resulting in an increase in Muc4 /X and Muc4 /Y for PanIN initiation. Thus, targeting MUC4/X and MUC4/Y using specific recombinant β subunit of human MUC4 (rMUC4β) [ 41 ] would be a worthwhile endeavor for treating PDAC.…”

Genetic variants of MUC4 are associated with prevalence and mortality of colorectal cancer and exhibit synergistic effects with LDL-C levels

“…For example, MUC1 and MUC2, which are members of the mucin gene family, have been shown to exacerbate carcinogenesis 7,21 . MUC4 is also a member of the mucin gene family, and it has been suggested to be related to carcinogenesis in multiple types of epithelial tissue 22 . In addition, MUC4 has been indicated to be associated with several carcinogenesis-related signaling pathways including the epidermal growth factor receptor (EGFR) and Wnt/β-catenin pathways 23 .…”

Silver–Gold Alloy Nanoparticles (AgAu NPs): Photochemical Synthesis of Novel Biocompatible, Bimetallic Alloy Nanoparticles and Study of Their In Vitro Peroxidase Nanozyme Activity

“…Breast cancer cells (MCF7) (ICLC number: HTL95021) were maintained in 75 cm 2 flasks (Sarstedt, Hildesheim, Germany) in high-glucose Dulbecco's Modified Eagle Medium (DMEM), supplemented with 10% (v/v) fetal bovine serum, 1% (v/v) 10,000 U/mL Penicillin and 10,000 U/mL Streptomycin, and 2 mM L-glutamine +1% non-essential amino acids [44]. For all the experiments, the cells were maintained under standard cell culture conditions (5% CO 2 , 95% humidity, and 37 • C temperature) and harvested every 3 days.…”