Glycoprotein Methods and Protocols - P6

Separation and Identification of Mucin 77

7

Separation and Identification

of Mucins and Their Glycoforms

David J. Thornton, Nagma Khan, and John K. Sheehan

1. Introduction

This chapter describes a strategy for the separation and identification of the mucins

present in mucous secretions or from cell culture, focusing primarily on those mucins

involved in gel formation. At present, the mucins MUC2, MUC5AC, MUC5B, and

MUC6 are known to be gel-forming molecules (1–4). These mucins share common

features in that they are oligomeric in nature and consist of a variable number of mono￾mers (subunits) linked in an end-to-end fashion via the agency of disulfide bonds. In

addition, their polypeptides comprise regions of dense glycosylation interspersed with

“naked” cysteine-rich domains (4–7).

Histological and biochemical investigations suggested that mucous-producing tis￾sues and their secretions contained a complex mixture of mucin-type glycoproteins.

However, until recently and with the advent of the new mucin-specific probes arising

from cDNA cloning studies, this theory was not definitively proven. In situ hybridiza￾tion and Northern blotting studies have shown that more than one gel-forming MUC

gene product can be expressed in a single mucus-producing epithelia, i.e., MUC5AC

and MUC5B in the respiratory tract and MUC5AC and MUC6 in the stomach (4,8).

Subsequent biochemical studies on human airway mucus have shown that these two

mucin genes are not only expressed but that their glycosylated products are present in

respiratory tract secretions (2,3). A further more recent insight into the complex nature

of the mucin component of mucous has been the demonstration that a mucin gene

product from a single epithelium can have a different oligosaccharide decoration and

thus exist in what are termed glycoforms. For example, the MUC5B mucin in the res￾piratory tract can exist in two distinctly charged states (3). Thus, these studies demon￾strate the need to have techniques available to dissect these complex mixtures to

ascertain mucin type, amount, and glycoform. Such investigations may lead to the

identification of novel members of this growing family of molecules.

Owing to the extreme size and polydispersity (Mr = 5–50 × 106) of the gel-forming

mucins in particular, there are few separation techniques available to use with these

77

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

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