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Monoclonal antibodies to rhamnogalacturonan I backbone

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Abstract

Monoclonal antibodies were raised against rhamnogalacturonan I backbone, a pectin domain, using Arabidopsis thaliana seed mucilage-derived rhamnogalacturonan I oligosaccharides—BSA conjugates. Two monoclonal antibodies, designated INRA-RU1 and INRA-RU2, selected for further characterization, were specific for the backbone of rhamnogalacturonan I, displaying no binding activity against the other pectin domains i.e. homogalacturonans, galactans or arabinans. A range of oligosaccharides was prepared by enzymatic digestion of rhamnogalacturonan I isolated from Arabidopsis thaliana seed mucilage and from sugar beet pectin, purified by low-pressure chromatography and characterized by high-performance anion-exchange chromatography and mass spectrometry. These rhamnogalacturonan I oligomers were used to characterize the binding site of the two monoclonal antibodies by competitive inhibition. Both INRA-RU1 and INRA-RU2 showed maximal binding to the [→2)-α-l-rhamnosep-(1→4)-α-d-galacturonic acid p-(1→]7 structural motif but differed in their minimum binding requirement. INRA-RU2 required at least two disaccharide (rhamnose–galacturonic acid) repeats for the antibody to bind, while INRA-RU1 required a minimum of six disaccharide repeats. Furthermore, the binding capacity of INRA-RU1 decreased steeply as the number of disaccharide repeats go beyond seven. Each of these antibodies reacted with hairy regions isolated from sugar beet pectin. Immunofluorescence microscopy indicated that both antibodies can be readily used to detect rhamnogalacturonan I epitopes in various cell wall samples.

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Abbreviations

BSA:

Bovine serum albumin

Dp:

Degree of polymerization

ESI-IT-MS:

Electrospray ion trap mass spectrometry

G:

galactose

HG:

Homogalacturonan

HG0:

Non-methylated HG

HG-93:

HG with a degree of methylesterification of 93

HG-B69:

Base-deesterified HG with a degree of methylesterification of 69

HG-B20:

Base-deesterified HG with a degree of methylesterification of 20

HG-P36:

Plant PME-deesterified HG with a degree of methylesterification of 36

HG-TBA:

Homogalacturonan in tetrabutyl ammonium hydroxide form

HPAEC:

High-performance anion exchange chromatography

HR:

Hairy regions

HR-H:

Acid-treated hairy regions

HRP:

Horseradish peroxidase

Ig:

Immunoglobulin

LRW:

London Resin White

mAb:

Monoclonal antibody

MS:

Mass spectrometry

PBS:

Phosphate buffer saline

R:

Rhamnose

RG-I:

Rhamnogalacturonan I

RG-II:

Rhamnogalacturonan II

RU-pool-BSA:

Selected rhamnogalacturonan oligosaccharides pool conjugated to bovine serum albumin

RU:

[→2)-α-l-Rhap-(1→4)-α-d-GalpA-(1→]

RU-pool-OVA:

Selected rhamnogalacturonan oligosaccharides pool conjugated to ovalbumin

TBA:

Tetrabutyl ammonium hydroxide

U:

Galacturonic acid

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Acknowledgments

We thank Marie-Jeanne Crépeau for assistance in the isolation and characterization of oligosaccharides, hairy regions and homogalacturonans; Valerie Echasserieau for performing the cell cultures and the isolation of antibodies; Lauren Helary for carrying out the immunochemical characterization, the preparation of plant material, the characterization of antibody binding to sections and the acquisition of micrographs.

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Authors and Affiliations

  1. INRA, UR1268 Biopolymères Interactions Assemblages, 44300, Nantes, France

    M.-C. Ralet, O. Tranquet, D. Poulain, A. Moïse & F. Guillon

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  1. M.-C. Ralet
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  2. O. Tranquet
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  3. D. Poulain
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  4. A. Moïse
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  5. F. Guillon
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Correspondence to F. Guillon.

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Ralet, MC., Tranquet, O., Poulain, D. et al. Monoclonal antibodies to rhamnogalacturonan I backbone. Planta 231, 1373–1383 (2010). https://doi.org/10.1007/s00425-010-1116-y

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