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Bioscience Methods
BM 2011, Vol.2, No.4, 21-30
http://bm.sophiapublisher.com
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may be impaired in various disorders affecting the
ocular surface including dry eye syndrome, Sjogren’s
syndrome as well as other types of ocular surface
trauma. For example, patients with Sjogren’s
syndrome showed a significant decrease in mucin
MUC5AC mRNA in a conjunctival epithelial sample
and associated reduction in MUC5AC protein in tear
samples (Argüeso et al., 2002).
Mucins have recently been extracted from jellyfish.
Masuda et al (Masuda et al., 2007) reported isolation
and characterization of a novel jellyfish mucin named
qniumucin or Q-mucin, with structural similarity to
the human MUC5AC mucin. Q-mucin contains
unique tandem repeat regions composed of 8 amino
acids with a consensus sequence of Val-Val-Glu-Thr-
Thr-Ala-Ala-Pro and is heavily glycosylated through
N-acetylgalactosamine (GalNAc)
O
-linkage to the two
Thr residues in the tandem repeat. Mucins in general
may have potential for wider commercial applications
due to their moisture retention, high viscosity and
lubrication abilities. For example, they are considered
to be potent protective coating substances for use in
the field of biomaterials (Chen et al., 2004; Sandberg
et al., 2009) due to their resistance to proteolytic
degradation and ability to aggregate and form
lubricating gels. They are also generally bio-
compatible and non-toxic. However, the extensive
post-translational modifications of mucins make their
synthesis through recombinant protein expression
systems very difficult. Moreover, current utilisation of
mucins is limited because of the difficulty in
extracting and isolating sufficient quantities from most
animals. These extractions also typically employ toxic
substances such as guanidium chloride (Berry et al.,
2003), acetone (Adikwu 2005) or a large amount of
ethanol (Masuda et al., 2007). Suitable extraction
procedures for marine mucins such as those from
jellyfish, molluscs and squids, which do not involve
the use of toxic substances and can be scaled up to
factory production level may pave the way for
utilising these mucins in the pharmaceutical, food and
cosmetics industries. Here we report the extraction,
composition analyses and microbial anti-adhesion
activity of
C. mosaicus
mucins.
1 Results
1.1 Isolation and purification of
C. mosaicus
mucins
Mucins were extracted from
C. mosaicus
bell tissue
(i.e. the umbrella-shaped body) of healthy jellyfish
(Figure 1A). Figure 1B summarizes the steps used in
the purification of bell mucin. Following tissue
homogenization and sonication, proteins in the supernatant
were concentrated by ultra-filtration followed by dialysis
against 20 mmol/L Tris-HCl (pH 8.0). During this
process, approximately 60% of the non-mucin
proteins and a blue pigment precipitated and were
subsequently removed by centrifugation (Figure 2,
lane B). Extensive glycosylation and the lack of
lysine and arginine (specificity determinants for
trypsin cleavage) in the repeat domain of previously
characterized Q-mucin (Masuda et al., 2007) suggested
that
C. mosaicus
mucin could be trypsin-resistant
whereas most other non-mucin-like proteins would
be digested by trypsin. Therefore, the supernatant
(Figure 1B, supernatant 2) was subjected to tryptic
digestion followed by hydrophobic interaction
chromatography (HIC). When the break-through
fractions from the HIC column were analysed by
sodium dodecyl sulfate-polyacrylamide gel electro-
phoresis (SDS-PAGE), a large trypsin- resistant
(120~300 kD) diffuse band was observed which was
strongly stained by Alcian blue (Figure 2, lane C) and
poorly with silver or Coomassie blue (data not shown).
These staining properties are typical of mucins (Sando
et al., 2009). The diffuse nature of the SDS-PAGE
band may be due to anomalous electrophoretic
behavior caused by substantial glycosylation, which is
also characteristic of mucins.
We also attempted to purify mucin proteins from the
mucus-like exudate released from the jellyfish when
suspended in air (Figure 1C). This exudate was
characterized by high viscosity and high lubrication
abilities (data not shown). Initial dialysis of the
concentrated exudate against Tris-HCl (pH 8.0),
150 mmol/L sodium chloride produced a pale light
blue precipitate constituting of approximately 30%~
40% of the total protein in the sample, which was
removed by centrifugation. The supernatant was
subjected to tryptic digestion followed by
ultrafiltration (Figure 1C). The presence of protein