NoteSynthetic methyl hexagalacturonate hapten inhibitors of anti-homogalacturonan monoclonal antibodies LM7, JIM5 and JIM7
A range of synthetic methyl hexagalacturonates were used as potential hapten inhibitors in competitive-inhibition enzyme-linked immunosorbent assays (ELISAs) with anti-homogalacturonan monoclonal antibodies LM7, JIM5 and JIM7. The selective inhibition of these antibodies by different haptens provides insight into the structures of the partially methyl-esterified pectin epitopes of these widely used monoclonal antibodies.
Introduction
Homogalacturonan (HG) is an abundant polymer of the matrix of primary plant cell walls and is the major component of commercial pectic polymers.1., 2. Current evidence indicates that HG is usually synthesized in a largely methyl-esterified form in the Golgi apparatus and that it can be de-esterified in the cell wall by the action of a class of enzymes known as pectin methylesterases (PMEs).1., 2. PMEs form a large multigene family in the model plant species Arabidopsis.3 The reason for existence of large numbers of PMEs in plants is far from clear but presumably reflects functional requirements for HG with varying degrees and patterns of methyl-esterification. Determining the functional significance of HG methyl-esterification in cell walls is important for understanding plant biology and also for understanding the functionality of commercial pectin preparations.
In recent years, a number of anti-HG monoclonal antibodies have been generated and used in studies of HG functions.1., 4. Antibodies to completely de-esterified epitopes of HG, such as 2F45 and PAM16 have been relatively easy to characterize due to the availability of defined fully unesterified oligogalacturonides. However, the current understanding of the epitope structures recognized by the widely used JIM5, JIM7 and LM7 are less clear.7., 8., 9. Studies using model pectins with varying degrees and patterns of methyl-esterification indicated that the HG epitopes bound by JIM5, JIM7 and LM7 are all partially methyl-esterified.8., 9. However, partially methyl-esterified hapten inhibitors that allow a more complete characterization of the epitopes recognized by these antibodies have not previously been available.
We now report the use of a panel of synthetic methyl hexagalacturonates with varying patterns of methyl-esterification that can act as inhibitors of binding of LM7, JIM5 and JIM7. These compounds provide direct insight into the structure of methyl-esterified HG epitopes for the first time.
Section snippets
Results and discussion
The five methyl hexagalacturonates with varying occurrences of methyl-ester groups (compounds 1–5) used in this study are shown in Fig. 1. Their structures were verified by MS10 and their synthesis is published elsewhere.11
The effectiveness of the methyl hexagalacturonates in inhibiting the binding of the antibodies was determined using competitive-inhibition ELISAs in which an antigen containing all epitopes (the model pectin F318) was used to coat microtitre plates and was therefore the
Experimental
Competitive-inhibition ELISAs were performed as described elsewhere.9
Acknowledgements
Financial support for MHC from the Danish Research Agency Center Contract Program is gratefully acknowledged.
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