Unfolding dynamics of the mucin SEA domain probed by force spectroscopy suggest that it acts as a cell-protective device.
Journal article, 2013
MUC1 and other membrane-associated mucins harbor long, up to 1 μm, extended highly glycosylated mucin domains and sea urchin sperm protein, enterokinase and agrin (SEA) domains situated on their extracellular parts. These mucins line luminal tracts and organs, and are anchored to the apical cell membrane by a transmembrane domain. The SEA domain is highly conserved and undergoes a molecular strain-dependent autocatalytic cleavage during folding in the endoplasmic reticulum, a process required for apical plasma membrane expression. To date, no specific function has been designated for the SEA domain. Here, we constructed a recombinant protein consisting of three SEA domains in tandem and used force spectroscopy to assess the dissociation force required to unfold individual, folded SEA domains. Force-distance curves revealed three peaks, each representing unfolding of a single SEA domain. Fitting the observed unfolding events to a worm-like chain model yielded an average contour length of 32 nm per SEA domain. Analysis of forces applied on the recombinant protein revealed an average unfolding force of 168 pN for each SEA domain at a loading rate of 25 nN·s(-1). Thus, the SEA domain may act as a breaking point that can dissociate before the plasma membrane is breached when mechanical forces are applied to cell surfaces.
CHO Cells
Models
Protein Unfolding
Enzyme-Linked Immunosorbent Assay
Mucin-1
Cricetinae
Animals
Mutagenesis
chemistry
Cell Membrane
Stress
Protein Stability
chemistry
Molecular
Protein Structure
genetics
Microscopy
methods
Transfection
Tertiary
Protein Conformation
Proteolysis
Atomic Force
Mechanical
genetics
Temperature
Biomechanics
chemistry
Site-Directed
Recombinant Proteins
Author
Thaher Pelaseyed
University of Gothenburg
Michael Zäch
Chalmers, Applied Physics, Chemical Physics
Asa C Petersson
University of Gothenburg
Frida Svensson
University of Gothenburg
Denny G A Johansson
University of Gothenburg
Gunnar C. Hansson
University of Gothenburg
FEBS Journal
1742-464X (ISSN) 17424658 (eISSN)
Vol. 280 6 1491-501Subject Categories
Basic Medicine
DOI
10.1111/febs.12144
PubMed
23331320