Purification and Partial Characterization of a Hydroxyproline-Rich Glycoprotein in a Graminaceous Monocot, Zea mays

Abstract

Graminaceous monocots generally contain low levels of hydroxyproline-rich Glycoproteins (HRGPs). As HRGPs are often at the cell surface, we used the intact cell elution technique (100 millimolar AlCl(3)) to isolate soluble surface proteins from Zea mays cell suspension cultures. Further fractionation of the trichloroacetic acid-soluble eluate on the cation exchangers phospho-cellulose and BioRex-70 gave several retarded, hence presumably basic fractions, which also contained hydroxyproline (Hyp). One of these fractions yielded a pure HRGP after a final purification step involving Superose-6 gel filtration. As this HRGP was unusually rich in threonine, (25 mole%) we designated it as a threonine-hydroxyproline-rich glycoprotein (THRGP); it contained about 27% carbohydrate occurring exclusively as arabinosylated Hyp, predominantly as the monosaccharide (15%), and trisaccharide (25%) with 48% Hyp nonglycosylated-a characteristically graminaceous monocot profile. Amino acid analysis confirmed the basic character, and gave a low alanine content. Reaction with Yariv artificial antigen was negative. These characteristics show that the THRGP is not an arabinogalactan protein. On the other hand, antibodies raised against tomato extensin P1 cross-reacted significantly with the THRGP; this cross-reactivity and the above analytical data provide the best evidence to date for the presence of extensin in a graminaceous monocot.