Glycoprotein methods protocols - biotechnology 048-9-143-155.pdf

Dimerization of Domains of Mucin 143

143

From: Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The Mucins

Edited by: A. Corfield © Humana Press Inc., Totowa, NJ

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Mucin Domains to Explore Disulfide-Dependent

Dimer Formation

Sherilyn L. Bell and Janet F. Forstner

1. Introduction

The viscoelastic properties needed for the protective functions of secretory mucins

are in part conditional on the capacity of mucin macromolecules to form linear poly￾mers stabilized by disulfide bonds. The individual mucin monomers have a distinctive

structure, consisting of a long central peptide region of tandem repeat sequences,

flanked by cysteine-rich regions at each end, which are presumed to mediate polymer￾ization. Secretory mucins contain approx 60–80% carbohydrate, with extensive O￾glycosylation in the central tandem repeat regions, and N-linked oligosaccharides in

the peripheral regions (1).

The ability of mucin peptides to form large polymers, combined with their exten￾sive posttranslational glycosylation and sulfation, results in complexes that reach

molecular masses in excess of 10,000 kDa (2). This leads to difficulties in resolving

mucins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

or agarose gel electrophoresis, because both unreduced and reduced mucin samples

are capable of only limited movement. A related difficulty inherent in analyzing

mucins lies in their strong negative charge owing to sialic acid and sulfate content (3).

Migration through polyacrylamide gels becomes more influenced by charge than by

mass. The result is that interpretations of the size of mucin from electrophoretic

mobility are not as straightforward as with other proteins.

Hypotheses concerning the regions of secretory mucins that could be involved in

the initial dimer formation have centered on the terminal cysteine-rich, poorly

glycosylated domains. Indirect evidence that these domains are involved has been

shown by treating mucins with proteolytic enzymes and reducing agents that act on

these regions, and noting a resultant decrease in the size of mucin and gel formation

(4–6). Intriguingly, more indirect evidence was found when database searches identi￾fied a functionally unrelated protein, von Willebrand factor (vWF) (which also forms

S-S–dependent polymers), to exhibit a mucinlike pattern of cysteine residue align-