DETECTION OF MUCIN POLYPEPTIDES IS THE KEY TO THE CHARACTERIZATION AND QUANTIFICATION OF MUCINS

Mucins are very large, very heavily O-glycosylated proteins, and as such are difficult to study by conventional biochemical techniques. Their size is usually far above 1,000,000 Da, they often show profound heterogeneity in both size, due to their O-glycosylation, and in their negative charge due to variable degrees of sialylation and sulfation. The combination of size, heavy O-glycosylation and negative charge makes them relatively easy to detect, and in practice they can only be mistaken for proteoglycans (from which they can be easily distinguished by the very different carbohydrate composition). However, due to their similar biochemical characteristics, different mucins are very difficult to distinguish from one another. This becomes more and more important since at least 9 human mucin genes have been identifled, most of which are expressed in the gastrointestinal (GI) tract.

Carbohydrate structures are usually not specific for any mucin, and are not suited to distinguish mucins.
Measuring mRNA levels of specific mucins might be a good method to quantify mucin gene expression, but in our view this method has two major drawbacks :
1. Since the efficiency of translation is not known, clear-cut relations between mRNA levels and actual mucin synthesis are not unequivocal;
2. Identifying mRNA expression does not help us in distinguishing the various mucin molecules in a preparation.

Although we use mRNA detection as a means to independently verify our results on mucin biosynthesis, these data have only circumstantial value. Thus, the polypeptide chain holds the key to identification and quantification of mucins, and antibodies against the polypeptides.

There is one crucial feature of mucins enabling us to develop these methods : all mucins contain regions, usually at the N- and C-terminus of the polypeptide, that are un- or low-glycosylated. Thus, antibodies raised against isolated mucin preparations, recognizing these polypeptide regions, are able to identify any form of a given mucin, independent of its state of glycosylation. It is important to realize that inhibition of protease activity is crucial both in preparing these antigenic mucins as well as in analysis of mucins and their precursors using these raised antisera.

Methods for analyzing mucins are based on :

1. the availability of antibodies against the mucin polypeptide to immunochemically recognize specific mucins;
2. metabolic labeling experiments, in which the mucin polypeptide is labeled with radioactive amino acids;
3. analysis of mucins on low percentage SDS-PAGE;
4. the ability to perform these studies on gastrointestinal biopsies of only a few mm3 .

In our terminology, the mucin precursor is the first identifiable form of a mucin, that is the primary translation product present in the rough endoplasmic reticulum (RER) of mucin-producing cells. Since these precursors contain only some N-glycans, but are not yet O-glycosylated, they behave like "normal" proteins. This has two important implications :

1. Each mucin gene is responsible for the production of one mucin precursor that can be distinguished in mixtures from other mucin precursors by its unique molecular mass on SDS-PAGE;
2. Since the precursors of each mucin can be identified separately, the polypeptide synthesis can be quantified by assessing the incorporation of radioactive amino acids in metabolic labeling experiments with gastrointestinal biopsies.

Usually the mucin precursor oligomerizes in the RER and is transported to the Golgi complex where it is heavily O-glycosylated and sulfated to yield the final biosynthetic form : the mature mucin. Most of the mucin is stored in the cells and a part is constitutively secreted. The stored mucin is secreted in a regulated fashion in response to secretagogues to add extra protection for epithelia in situations of stress. Using antibodies against the un- or low-glycosylated regions of the polypeptide, the mature mucin can be equally well recognized (as well as quantified) as the mucin precursor. Thus, the total amounts of intra- and extracellular mucin can be measured using Western blot experiments. Moreover, the dynamics of biosynthetic conversion of the precursor to the mature mucin and the dynamics of storage and secretion of the mature mucin can be assessed by metabolic labeling experiments with biopsies.

In summary, by employing this methods :

1. To identify the mucins expressed in a each region of the Gl tract, and
2. To quantify the biosynthesis, storage and secretion of each of these mucins.

MUCINS IN PATHOLOGY : CHICKEN OR EGG ?

Aberrant mucin synthesis or structure has been reported over the past decades in relation to various pathological conditions, like cystic fibrosis, ulcerative colitis, Crohn's disease and cancer. One always wonders if the changes in mucin are the cause or the result of the disease. In cystic fibrosis, for instance, increased mucin production and alterations in mucin structure seem totally secondary to a deficient chloride secretion. But even these secondary effects on mucin production may be very relevant in pathogenesis, since patients often die of infections due to this altered mucin production.

In our work we are interested primarily in the role of mucin production in ulcerative colitis (UC), a chronically recurring inflammation of the colon mucosa. Many studies indicated a lower mucin synthesis in UC compared to controls, which could lead to a higher susceptibility of the colonic mucosa to inflammation. Virtually all studies on mucin production in pathological (or experimental) conditions had one major drawback: by using techniques like total mucin isolation or histochemistry the effects could not be assigned to specific mucin gene products. The latter seems essential, knowing that there are at least nine mucin genes expressed in the various human epithelia. Employing the techniques as described above, we are able to identify and quantify specific mucin gene products, and to detect changes in mucin production in UC, or any other situation.

We isolated mucins from normal and active UC colonic tissue, which appeared biochemically very similar. When antisera were raised, they showed extensive cross-reactivity towards both normal and UC colonic mucin. Moreover, both antisera recognized only one mucin precursor and the corresponding mature mucin which, in both normal and UC colonic biopsies, appeared to be identical to MUC2. Analysis of mRNA levels confirmed the predominance of MUC2 in both healthy and UC tissue. Knowing that MUC2 was very abundant (although it is not the only mucin in the colon - BJW Van Klinken, HA Buller, J Dekker, AWC Einerhand, submitted), we quantified the MUC2 mRNA levels, MUC2 precursor synthesis and the amounts of MUC2 present in colonic biopsies of both normal and active UC patients. It appeared that MUC2 mRNA levels were unaltered in active UC, while both the MUC2 precursor synthesis and the overall levels of MUC2 were significantly decreased in active UC (KMAJ Tytgat, JWG Van der Wal, AWC Einerhand, HA Buller, J Dekker, submitted). Involuntarily, these data demonstrate an important advantage of our method: if only MUC2 mRNA had been quantified, this difference between health and disease would have passed unnoticed.
These potentially important observations could only be made after evaluation of MUC2 synthesis in UC at the protein level.

Now that we have shown that MUC2 synthesis is reduced in active UC, we await the task of establishing whether this causes the recurrence of the active inflammation (primary effect) or whether it is caused by the disease (secondary effect). We will approach this ever-recurring question in the study of pathogenesis of complex diseases by investigating the effects of MUC2 depletion in the susceptibility of animals to experimentally induced colitis, using the same techniques as in the human biopsy study.

Application of antibodies in the identification and quantification of gastrointestinal mucins

C. The antibodies are used to identify the mucins produced in a specified region of the Gl tract. All analyses are based on the separation of mucin-containing preparations on very low percentage SDS-PAGE (4% running gel, or 3%-10% gradient running gel). Mature mucins are identified in homogenates of biopsies from this specified region by Western blotting.
Mucin precursors are identified by immunoprecipitation from homogenates of biopsies, that were metabolically labeled for a short period of time ("pulse-labeling", 0-45 min) with radioactive amino acids to label the mucin polypeptide. Since the mucin precursors are N-glycosylated, the precursor status of these molecules can be checked by the presence of "high mannose" N-glycans. The precursor-product relationship between the putative precursor and the mature mucin is established in metabolic pulse-chase experiments, which demonstrate the conversion over time ("chase-incubation", taking about 4-6 h) of the precursor into the mature mucin, and the eventual secretion of the mature mucin. Identification of the mucins expressed in a particular part of the Gl tract enables the quantification of these mucins. The total amount of mucin in biopsy tissue and mature mucin secreted into the milieu can be quantified using Western blot-type of assays. The amount of precursor synthesis can be measured by the amount of radioactive amino acids incorporated into the polypeptide within a specified time period. The dynamics of formation and secretion of the mature mucin are determined in metabolic pulse-chase analysis.
To identify the roles of mucins in pathogenesis, we are now able to measure the effects on mucin synthesis and secretion in biopsies obtained from pathologic tissues or from tissues under experimentally modified conditions. The antisera allow us to detect changes in mucin gene expression under these conditions (which mucins), and to quantify these mucins (how much of each mucin).

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