Chemistry - A European Journal
10.1002/chem.201602197
COMMUNICATION
β-1,4-linked galactans, and has been generated by
immunization of rats with a β-1,4-Gal4-BSA neoglycoprotein.[24]
The data in Table 1 clearly shows that LM5 exclusively binds
the linear galactans 1-3. Branching is not tolerated and the fact
that a terminal arabinan (oligosaccharide 4) eliminates binding
shows that the LM5 epitope most probably involves the reducing
end galactose residue. LM6, another rat monoclonal raised by
immunization with a BSA neoglycoconjugate and popular for
immunofluorescent microscopy of plant tissue is selective for
arabinans and is included here as a control to rule out unspecific
binding during the array screening. [25] It has been demonstrated
that LM6 requires at least three consecutive arabinose residues
for binding and as expected, no binding to any of the galactans
1–9 can be detected with LM6, whereas binding to the linear
arabinans is clearly detected.
Keywords: Carbohydrate • Plant Cell Wall • Glycosylation •
Oligosaccharide • Rhamnogalacturonan
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Table 1. Microarray data for binding of LM5 antibody and CBM61 to the
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synthesised linear and branched galactans
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1-9 as compared to controls
Compound
LM5
LM6
CBM61
1
2
3
4
0
0
0
5
0
0
0
6
0
0
0
7
0
0
0
8
0
0
0
9
0
0
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Ara3 Ara5 Ara8
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0
0
86 80 100
0
0
0
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CBM61, a carbohydrate-binding module from a Thermotoga
maritima endo--(14)-galactanase, has previously been shown
to bind an unusual epitope related to a helical conformation of β-
(14)-D-galactan.[26] Our studies show that neither linear nor
branched oligogalactans are recognized by CBM61 when
conjugated and immobilized on a microarray surface. The lack of
binding is likely a result of the helical conformation being lost by
branching and/or surface presentation.
In conclusion, a convergent strategy for (16)-branched
-(14)-D-galactans was developed and a small library of
hepta- and octasaccharides has been prepared. The late-stage
introduction of branching significantly reduces the overall
number of steps and makes it possible to use different donors.
The substrates have been used for the preparation of oligo-
saccharide microarrays and in addition to the results presented
here, the oligosaccharides are expected to give new insight
regarding the structure and function of pectic polysaccharides in
the future.
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Acknowledgements
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We acknowledge financial support from the Danish Council for
Independent Research and the Danish Strategic Research
Council (GlycAct and SET4Future projects), from the Villum
Foundation (PLANET project) and the Novo Nordisk Foundation
(Biotechnology-based Synthesis and Production Research).
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T. Willats, A. B. Boraston, J. Biol. Chem. 2010, 285, 35999–36009.