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Cellular and Structural Biology

Characterization of a Xylose-Specific Antiserum That Reacts with the Complex Asparagine-Linked Glycans of Extracellular and Vacuolar Glycoproteins ¹

Department of Biology C-016, University of California, San Diego, La Jolla, California 92093-0116

Antibodies were raised against carrot (Daucus carota) cell wall -fructosidase that was either in a native configuration (this serum is called anti-F₁) or chemically deglycosylated (anti-F₂). The two antisera had completely different specificities when tested by immunoblotting. The anti-F₁ serum reacted with -fructosidase and many other carrot cell wall proteins as well as with many proteins in extracts of bean (Phaseolus vulgaris) cotyledons and tobacco (Nicotiana tabacum) seeds. It did not react with chemically deglycosylated -fructosidase. The anti-F₁ serum also reacted with the bean vacuolar protein, phytohemagglutinin, but not with deglycosylated phytohemagglutinin. The anti-F₂ serum reacted with both normal and deglycosylated -fructosidase but not with other proteins. These results indicate that the F₂ antibodies recognize the -fructosidase polypeptide, while the F₁ antibodies recognize glycan sidechains common to many glycoproteins. We used immunoadsorption on glycoprotein-Sepharose columns and hapten inhibition of immunoblot reactions to characterize the nature of the antigenic site. Antibody binding activity was found to be associated with Man₃(Xyl)(GIcNAc)₂Fuc, Man₃(Xyl)(GIcNAc)₂, and Man(Xyl) (GIcNAc)₂ glycans, but not with Man₃(GIcNAc)₂. Treatment of phytohemagglutinin, a glycoprotein with a Man₃(Xyl)(GIcNAc)₂Fuc glycan, with Charonia lampas -xylosidase (after treatment with jack-bean -mannosidase) greatly diminished the binding between the antibodies and phytohemagglutinin. We conclude, therefore, that the antibodies bind primarily to the xylose, 1 2mannose structure commonly found in the complex glycans of plant glycoproteins.

³ On leave from Laboratoire de Physiologie des Organes Végétaux, C.N.R.S., 4 ter Route des Gardes, 92190 Meudon, France.

⁴ On leave from the Department of Biology, San Diego State University, San Diego, CA.

⁵ Present address: Friedrich Miescher-institute, P. O. Box 2543, CH-4002 Basel, Switzerland.

¹ Supported in part by a grant to M. L. from the Ministère de la Recherche et de la Technologie (France) and by grants to M. J. C. from the National Science Foundation (Metabolic Biology), the U.S. Department of Energy and the Universitywide Energy Research Group.

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