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

• 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.

Ämnesord

MEDICIN OCH HÄLSOVETENSKAP  -- Medicinska och farmaceutiska grundvetenskaper (hsv//swe)

MEDICAL AND HEALTH SCIENCES  -- Basic Medicine (hsv//eng)

Nyckelord

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

CHO Cells

Publikations- och innehållstyp

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