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

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Abstract
Antibodies were raised against carrot (Daucus carota) cell wall .beta.-fructosidase that was either in a native configuration (this serum is called anti-.beta.F1) or chemically deglycosylated (anti-.beta.F2). The two antisera had completely different specificities when tested by immunoblotting. The anti-.beta.F1 serum reacted with .beta.-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 .beta.-fructosidase. The anti-.beta.F1 serum also reacted with the bean vacuolar protein, phytohemagglutinin, but not with deglycosylated phytohemagglutinin. The anti-.beta.F2 serum reacted with both normal and deglycosylated .beta.-fructosidase but not with other proteins. These results indicate that the .beta.F2 antibodies recognize the .beta.-fructosidase polypeptide, while the .beta.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)(GlcNAc)2Fuc, Man3(Xyl)(GlcNAc)2, and Man(Xyl) (GlcNAc)2 glycans, but not with Man3(GlcNAc)2. Treatment of phytohemagglutinin, a glycoprotein with a Man3(Xyl)(GlcNAc)2Fuc glycan, with Charonia lampas .beta.-xylosidase (after treatment with jack-bean .alpha.-mannosidase) greatly diminished the binding between the antibodies and phytohemagglutinin. We conclude, therefore, that the antibodies bind primarily to the xylose.beta., 1.fwdarw.2mannose structure commonly found in the complex glycans of plant glycoproteins.