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. 1996 Apr;8(4):673–685. doi: 10.1105/tpc.8.4.673

An antibody Fab selected from a recombinant phage display library detects deesterified pectic polysaccharide rhamnogalacturonan II in plant cells.

M N Williams 1, G Freshour 1, A G Darvill 1, P Albersheim 1, M G Hahn 1
PMCID: PMC161128  PMID: 8624441

Abstract

Rhamnogalacturonan II (RG-II) is a structurally complex, low molecular weight pectic polysaccharide that is released from primary cell walls of higher plants by treatment with endopolygalacturonase and is chromatographically purified after alkaline deesterification. A recombinant monovalent antibody fragment (Fab) that specifically recognizes RG-II has been obtained by selection from a phage display library of mouse immunoglobulin genes. By itself, RG-II is not immunogenic. Therefore, mice were immunized with a neoglycoprotein prepared by covalent attachment of RG-II to modified BSA. A cDNA library of the mouse IgG1/kappa antibody repertoire was constructed in the phage display vector pComb3. Selection of antigen-binding phage particles resulted in the isolation of an antibody Fab, CCRC-R1, that binds alkali-treated RG-II with high specificity. CCRC-R1 binds an epitope found primarily at sites proximal to the plasma membrane of suspension-cultured sycamore maple cells. In cells deesterified by alkali, CCRC-R1 labels the entire wall, suggesting that the RG-II epitope recognized by CCRC-R1 is masked by esterification in most of the wall and tha such RG-II esterification is absent near the plasma membrane.

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Selected References

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  1. Adderson E. E., Shackelford P. G., Quinn A., Wilson P. M., Cunningham M. W., Insel R. A., Carroll W. L. Restricted immunoglobulin VH usage and VDJ combinations in the human response to Haemophilus influenzae type b capsular polysaccharide. Nucleotide sequences of monospecific anti-Haemophilus antibodies and polyspecific antibodies cross-reacting with self antigens. J Clin Invest. 1993 Jun;91(6):2734–2743. doi: 10.1172/JCI116514. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Anand N. N., Dubuc G., Phipps J., MacKenzie C. R., Sadowska J., Young N. M., Bundle D. R., Narang S. A. Synthesis and expression in Escherichia coli of cistronic DNA encoding an antibody fragment specific for a Salmonella serotype B O-antigen. Gene. 1991 Apr;100:39–44. doi: 10.1016/0378-1119(91)90347-e. [DOI] [PubMed] [Google Scholar]
  3. Austin F. W., Corstvet R. E. Purification of a Pasteurella haemolytica serotype 1-specific polysaccharide epitope by use of monoclonal antibody immunoaffinity. Am J Vet Res. 1993 May;54(5):695–700. [PubMed] [Google Scholar]
  4. Barbas C. F., 3rd, Kang A. S., Lerner R. A., Benkovic S. J. Assembly of combinatorial antibody libraries on phage surfaces: the gene III site. Proc Natl Acad Sci U S A. 1991 Sep 15;88(18):7978–7982. doi: 10.1073/pnas.88.18.7978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Baschong W., Wrigley N. G. Small colloidal gold conjugated to Fab fragments or to immunoglobulin G as high-resolution labels for electron microscopy: a technical overview. J Electron Microsc Tech. 1990 Apr;14(4):313–323. doi: 10.1002/jemt.1060140405. [DOI] [PubMed] [Google Scholar]
  6. Freshour G., Clay R. P., Fuller M. S., Albersheim P., Darvill A. G., Hahn M. G. Developmental and Tissue-Specific Structural Alterations of the Cell-Wall Polysaccharides of Arabidopsis thaliana Roots. Plant Physiol. 1996 Apr;110(4):1413–1429. doi: 10.1104/pp.110.4.1413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Garrard L. J., Yang M., O'Connell M. P., Kelley R. F., Henner D. J. Fab assembly and enrichment in a monovalent phage display system. Biotechnology (N Y) 1991 Dec;9(12):1373–1377. doi: 10.1038/nbt1291-1373. [DOI] [PubMed] [Google Scholar]
  8. Hoogenboom H. R., Griffiths A. D., Johnson K. S., Chiswell D. J., Hudson P., Winter G. Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains. Nucleic Acids Res. 1991 Aug 11;19(15):4133–4137. doi: 10.1093/nar/19.15.4133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hughes J., McCully M. E. The use of an optical brightener in the study of plant structure. Stain Technol. 1975 Sep;50(5):319–329. doi: 10.3109/10520297509117082. [DOI] [PubMed] [Google Scholar]
  10. Huse W. D., Sastry L., Iverson S. A., Kang A. S., Alting-Mees M., Burton D. R., Benkovic S. J., Lerner R. A. Generation of a large combinatorial library of the immunoglobulin repertoire in phage lambda. Science. 1989 Dec 8;246(4935):1275–1281. doi: 10.1126/science.2531466. [DOI] [PubMed] [Google Scholar]
  11. Kabat E. A., Liao J., Osserman E. F., Gamian A., Michon F., Jennings H. J. The epitope associated with the binding of the capsular polysaccharide of the group B meningococcus and of Escherichia coli K1 to a human monoclonal macroglobulin, IgMNOV. J Exp Med. 1988 Aug 1;168(2):699–711. doi: 10.1084/jem.168.2.699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kang A. S., Barbas C. F., Janda K. D., Benkovic S. J., Lerner R. A. Linkage of recognition and replication functions by assembling combinatorial antibody Fab libraries along phage surfaces. Proc Natl Acad Sci U S A. 1991 May 15;88(10):4363–4366. doi: 10.1073/pnas.88.10.4363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kieliszewski M. J., Kamyab A., Leykam J. F., Lamport D. T. A Histidine-Rich Extensin from Zea mays Is an Arabinogalactan Protein. Plant Physiol. 1992 Jun;99(2):538–547. doi: 10.1104/pp.99.2.538. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kobayashi M., Matoh T., Azuma Ji. Two Chains of Rhamnogalacturonan II Are Cross-Linked by Borate-Diol Ester Bonds in Higher Plant Cell Walls. Plant Physiol. 1996 Mar;110(3):1017–1020. doi: 10.1104/pp.110.3.1017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Koch J. L., Nevins D. J. Tomato fruit cell wall : I. Use of purified tomato polygalacturonase and pectinmethylesterase to identify developmental changes in pectins. Plant Physiol. 1989 Nov;91(3):816–822. doi: 10.1104/pp.91.3.816. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  17. Mainhart C. R., Potter M., Feldmann R. J. A refined model for the variable domains (Fv) of the J539 beta(1,6)-D-galactan-binding immunoglobulin. Mol Immunol. 1984 Jun;21(6):469–478. doi: 10.1016/0161-5890(84)90062-2. [DOI] [PubMed] [Google Scholar]
  18. Marks J. D., Hoogenboom H. R., Bonnert T. P., McCafferty J., Griffiths A. D., Winter G. By-passing immunization. Human antibodies from V-gene libraries displayed on phage. J Mol Biol. 1991 Dec 5;222(3):581–597. doi: 10.1016/0022-2836(91)90498-u. [DOI] [PubMed] [Google Scholar]
  19. McNeil M., Darvill A. G., Fry S. C., Albersheim P. Structure and function of the primary cell walls of plants. Annu Rev Biochem. 1984;53:625–663. doi: 10.1146/annurev.bi.53.070184.003205. [DOI] [PubMed] [Google Scholar]
  20. Owen M., Gandecha A., Cockburn B., Whitelam G. Synthesis of a functional anti-phytochrome single-chain Fv protein in transgenic tobacco. Biotechnology (N Y) 1992 Jul;10(7):790–794. doi: 10.1038/nbt0792-790. [DOI] [PubMed] [Google Scholar]
  21. Pennell R. I., Janniche L., Kjellbom P., Scofield G. N., Peart J. M., Roberts K. Developmental Regulation of a Plasma Membrane Arabinogalactan Protein Epitope in Oilseed Rape Flowers. Plant Cell. 1991 Dec;3(12):1317–1326. doi: 10.1105/tpc.3.12.1317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Pope D. G. Relationships between Hydroxyproline-containing Proteins Secreted into the Cell Wall and Medium by Suspension-cultured Acer pseudoplatanus Cells. Plant Physiol. 1977 May;59(5):894–900. doi: 10.1104/pp.59.5.894. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Puhlmann J., Bucheli E., Swain M. J., Dunning N., Albersheim P., Darvill A. G., Hahn M. G. Generation of monoclonal antibodies against plant cell-wall polysaccharides. I. Characterization of a monoclonal antibody to a terminal alpha-(1-->2)-linked fucosyl-containing epitope. Plant Physiol. 1994 Feb;104(2):699–710. doi: 10.1104/pp.104.2.699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. REYNOLDS E. S. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol. 1963 Apr;17:208–212. doi: 10.1083/jcb.17.1.208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Reuhs B. L., Carlson R. W., Kim J. S. Rhizobium fredii and Rhizobium meliloti produce 3-deoxy-D-manno-2-octulosonic acid-containing polysaccharides that are structurally analogous to group II K antigens (capsular polysaccharides) found in Escherichia coli. J Bacteriol. 1993 Jun;175(11):3570–3580. doi: 10.1128/jb.175.11.3570-3580.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Rose D. R., Przybylska M., To R. J., Kayden C. S., Oomen R. P., Vorberg E., Young N. M., Bundle D. R. Crystal structure to 2.45 A resolution of a monoclonal Fab specific for the Brucella A cell wall polysaccharide antigen. Protein Sci. 1993 Jul;2(7):1106–1113. doi: 10.1002/pro.5560020705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Ruff-Jamison S., Campos-González R., Glenney J. R., Jr Heavy and light chain variable region sequences and antibody properties of anti-phosphotyrosine antibodies reveal both common and distinct features. J Biol Chem. 1991 Apr 5;266(10):6607–6613. [PubMed] [Google Scholar]
  28. Talbott L. D., Ray P. M. Molecular size and separability features of pea cell wall polysaccharides : implications for models of primary wall structure. Plant Physiol. 1992 Jan;98(1):357–368. doi: 10.1104/pp.98.1.357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Tavladoraki P., Benvenuto E., Trinca S., De Martinis D., Cattaneo A., Galeffi P. Transgenic plants expressing a functional single-chain Fv antibody are specifically protected from virus attack. Nature. 1993 Dec 2;366(6454):469–472. doi: 10.1038/366469a0. [DOI] [PubMed] [Google Scholar]
  30. Thomas J. R., McNeil M., Darvill A. G., Albersheim P. Structure of Plant Cell Walls : XIX. Isolation and Characterization of Wall Polysaccharides from Suspension-Cultured Douglas Fir Cells. Plant Physiol. 1987 Mar;83(3):659–671. doi: 10.1104/pp.83.3.659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Whitcombe A. J., O'Neill M. A., Steffan W., Albersheim P., Darvill A. G. Structural characterization of the pectic polysaccharide, rhamnogalacturonan-II. Carbohydr Res. 1995 Jul 10;271(1):15–29. doi: 10.1016/0008-6215(94)00002-w. [DOI] [PubMed] [Google Scholar]
  32. Williamson R. A., Burioni R., Sanna P. P., Partridge L. J., Barbas C. F., 3rd, Burton D. R. Human monoclonal antibodies against a plethora of viral pathogens from single combinatorial libraries. Proc Natl Acad Sci U S A. 1993 May 1;90(9):4141–4145. doi: 10.1073/pnas.90.9.4141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Ye Z. H., Varner J. E. Tissue-Specific Expression of Cell Wall Proteins in Developing Soybean Tissues. Plant Cell. 1991 Jan;3(1):23–37. doi: 10.1105/tpc.3.1.23. [DOI] [PMC free article] [PubMed] [Google Scholar]

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