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

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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

The FEBS journal

1742-4658 (eISSN)

Vol. 280 6 1491-501

Subject Categories

Basic Medicine

DOI

10.1111/febs.12144

PubMed

23331320

More information

Created

10/6/2017