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Unfolding dynamics of the mucin SEA domain probed by force spectroscopy suggest that it acts as a cell-protective device.

Journal article
Authors Thaher Pelaseyed
Michael Zäch
Asa C Petersson
Frida Svensson
Denny G A Johansson
Gunnar C. Hansson
Published in The FEBS journal
Volume 280
Issue 6
Pages 1491-501
ISSN 1742-4658
Publication year 2013
Published at Institute of Biomedicine, Department of Medical Biochemistry and Cell Biology
Pages 1491-501
Language en
Links dx.doi.org/10.1111/febs.12144
Keywords Animals, Biomechanics, CHO Cells, Cell Membrane, chemistry, Cricetinae, Enzyme-Linked Immunosorbent Assay, Microscopy, Atomic Force, methods, Models, Molecular, Mucin-1, chemistry, genetics, Mutagenesis, Site-Directed, Protein Conformation, Protein Stability, Protein Structure, Tertiary, Protein Unfolding, Proteolysis, Recombinant Proteins, chemistry, genetics, Stress, Mechanical, Temperature, Transfection
Subject categories Basic Medicine

Abstract

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.

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