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Bioscience Methods
BM 2011, Vol.2, No.4, 21-30
http://bm.sophiapublisher.com
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mucins as a barrier against bacterial attachment to
epithelial cells (Alemka et al., 2010; Bergstrom et al.,
2010) and the mechanisms by which bacteria can
utilise these mucin glycoproteins to facilitate adhesion
and colonisation (Vieira et al., 2010; Linden et al.,
2009). Our results show that GalNAc and Gal inhibit
adhesion of Paer6294-GFP to cultured corneal
epithelial cells and suggest that the interaction
between bell mucin and
P. aeruginosa
is glycan-
mediated. This observation is consistent with previous
studies reporting that the sugars GalNAc and Gal,
typically associated with mucins, bind to
P. aeruginosa
pilus adhesions (Sheth et al., 1994; Ramphal and
Arora 2001). However, different
P. aeruginosa
strains
are known to exhibit different binding specificities to
mucins (Aristoteli and Willcox 2001) and some sugar
structures on mucins may be more favourable for
bacterial adhesion (Abbeele et al., 2009; Laparra and
Sanz 2009). Our results support this notion. Bovine
MUC1 (21% GalNAc, 35% sialic acid) (Sando et al.,
2009) showed very strong binding activity to enteric
bacteria such as
Escherichia coli
and
Samonella
typhimurium
(Parker et al., 2010) but bound less
effectively to
P. aeruginosa
than
C. mosaicus
bell
mucin (54% GalNAc, 5% sialic acid) (Table 2).
Understanding the interaction between specific
bacteria and mucins is required in order to optimise
their protective binding properties.
Jellyfish are an untapped resource of easily harvested
mucins and other bioactives such as collagen,
phospholipids and sphingophosphonolipids. Mucins
are found in abundance and in almost every organ of
jellyfish (Masuda et al., 2007) and we have
demonstrated here that a considerable amount of
mucin could be harvested from jellyfish (0.01% wet
weight for bell mucin). Interestingly, the crude exu-
date which requires very simple and low cost extra-
ction can yield a very high amount (0.1% wet weight)
of material (presumably complex oligosaccharides)
that have high bacterial adhesion inhibitory activity.
Our understanding of the biological functions of
jellyfish mucin is rudimentary. Jellyfish are known to
secret mucus to help clean their surface and to
discourage attacks by predators (Hanaoka et al., 2001).
In addition and like their mammalian counterparts,
mucins secreted by jellyfish together with their
interacting proteins may function to mediate various
cellular activities to enhance physical protection,
enhance tissue integrity and enhance non-immune host
defence (Senapati et al., 2010). These activities allow
the jellyfish to survive in their aqueous habitat.
In conclusion, a new and simple protocol was devised
to isolate mucins from jellyfish. Both the highly
purified material from bell and the crude exudate
mucins showed anti-bacterial adherence effects when
tested using an ocular isolate of
P. aeruginosa
. The
anti-
Pseudomonas
adhesion property of
C. mosaicus
bell mucin probably occurs via interactions involving
bacterial proteins and the carbohydrate moieties of the
mucin. Jellyfish may be rich sources of novel mucins
exhibiting many different biological and mechano-
physical activities.
3 Materials and Methods
3.1 Jellyfish
Mature healthy
Catostylus mosaicus
specimens were
collected from the clear waters of Moreton Bay,
Queensland during the height of their spawning cycle
(October/November). The jellyfish were drained of
excess sea water and transported to the laboratory
where the tentacles and the bell were separated prior
to freezing at
-
20 . External mucus
℃
-like fluid
(exudate) released from the jellyfish during transport
to the laboratory, was also collected and frozen at
-
20 .
℃
3.2 Mucin extraction from jellyfish bell
Two frozen bells (total weight 1 900 g) were thawed,
homogenised for 2 min using an Ultra-Turrax homo-
geniser (Janke and Kunkel laboratories, Germany)
and sonicated (Misonix Incorporated NY, USA) in
4×450 mL aliquots using a 7.0 mm probe for 30 s at
maximum power. To prevent proteolysis and microbial
growth, benzamidine hydrochloride (Sigma-Aldrich,
Australia), EDTA and sodium azide were added to the
homogenate to a final concentration of 1 mmol/L,
1 mmol/L and 0.04%, respectively and the sample
centrifuged at 70 000×g for 40 min at 4 . The super
℃
-
natant was concentrated by ultra filtration YM 30
(Millipore Corp, MA, USA) to 100 mL prior to exten-
sive dialysis against 20 mmol/L TrisHCl (pH 8.0). The
sample was again centrifuged as above to remove the