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Title: The biochemical identification and quantitation of mucins in human mucous secretions.
Author: Khan, Nagma.
ISNI:       0000 0001 3598 3762
Awarding Body: University of Manchester : University of Manchester
Current Institution: University of Manchester
Date of Award: 1999
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Mucins fonn the polymer matrix of mucous gels and give them their unique lubricating and protective properties. Most mucous secretions contain a heterogeneous population of mucins. The identification and quantitation of mucin phenotypes obtained from mucous secretions is a problem due to the density of protein glycosylation in these molecules. With the detennination of large portions or in some cases the complete primary sequence of a number of mucins. we now have precise protein compositional data. In particular it is becoming clear from the sequences of the heavily glycosylated domains that they contain distinctive ratios of the amino acids Ser, Thr and Pro. There is also evidence to suggest that mucin molecules have a unique pattern of glycosylation. These criteria could be used as a means for biochemically identifying mucins purified from mucous gels. The aims of this study were three-fold. Firstly to provide an approach for assessing the heterogeneity of mucin mixtures. Secondly to provide criteria by which the different mucins may be identified. Thirdly to establish methods for measuring their mass contribution to the mixture. The benefits of attaching mucins to PVDF membrane supports in order to simplify chemical analyses are described. j3-elimination, aqueous hydrazinolysis and anhydrous hydrazinolysis methods for the removal of O-linked glycan chains and fluorophore labelling and PAGE techniques for obtaining a chemical signature of the glycan chains are discussed. A strategy is presented for isolating, purifying and analysing mucins. using the previously uncharacterised high molecular weight salivary mucin MG 1 as a model. Intact mucins were extracted in 4 M GuHCI and separated using gel filtration chromatography and isopycnic density gradient centrifugation. The mucins were reduced and thereafter fractionated using ion exchange chromatography. Mucins were analysed using agarose gel electrophoresis and Western blotting. A broad and heterogeneous distribution of salivary mucin subunits was detected by both separation methods. Subunit fractions were trypsin digested, generating large glycopeptides and small peptide fragments, which were separated by gel chromatography. Amino acid analysis of the large glycopeptides from subunit fractions across the Mono Q yielded distinctive and identical Ser:Thr:Pro:Ala ratios. A chemical "fingerprint" of the small tryptic fragments was obtained by a combination of reverse phase chromatography and MALDI-TOF MS analysis. The composition and peptide mapping data were almost identical to that obtained from the MUCSB mucin from respiratory tract mucus. These data indicated that the MG 1 mucin in saliva is composed almost entirely of the MUCSB mucin. It was concluded that the physical heterogeneity observed must have been a result of variable glycosylation. Application of this strategy was used to show that MUCSB is the major large secreted mucin in nonnal cervical mucous secretions and many pathological respiratory mucous secretions. It was concluded that the approaches outlined above may prove valuable in three respects:- the assessment of mucin heterogeneity. the identification of their underlying polypeptide and the characterisation of the glycan chains from mucins.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available