Two Different Families of Hydroxyproline-Rich Glycoproteins in Melon Callus: Biochemical and Immunochemical Studies

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Two different families of hydroxyproline-rich glycoproteins, HRGP1 and HRGP2, have been isolated from melon callus and separated by ion exchange chromatography on CM-sepharose. HRGP1 corresponds to an arabinogalactan protein. The sugar portion of HRGP1 accounts for 94% of the molecule and contains galactose (66%) and arabinose (34%); these residues are present as polysaccharide side chains attached to hydroxyproline. Hydroxyproline is the main amino acid residue (46%) of the protein moiety. The arabinogalactan protein nature of HRGP1 has been checked by its ability to positively react with the β-glucosyl Yariv antigen; the 3H-labeled deglycosylated HRGP1 also called HRP1 migrates upon electrophoresis as a single band of molecular weight 76,000. HRGP2 was fractionated by affinity chromatography on heparin-Ultrogel into three different glycoproteins, HRGP2a,2b and 2c. Two of these glycoproteins behave as polycations (HRGP2b and 2c) and are chemically distinct from HRGP2a. HRGP2b is the most abundant component and contains 41% protein and 50% sugar. Hydroxyproline, lysine, tyrosine, and arabinose are the most prominent residues of their respective moiety. The glycosylation pattern of hydroxyproline indicates that HRGP2b is related to and possibly a precursor of the wall HRGP; as in melon cell wall HRGP, Hyp-Ara3 predominates, and small amounts of a putative Hyp-Ara5 a hitherto unreported hyp-arabinoside, are recorded. The molecular weight of HRP2b, the protein portion of HRGP2b is 55,000 ± 5,000, as estimated after deglycosylation of the molecule with trifluoromethane sulfonic acid. Antibodies have been raised against HRGP2b and HRP2b. Immunodiffusion shows that each antigen (HRGP2b or HRP2b) reacts with its own IgG, and cross-reacts with the heterologous IgG, thereby indicating the presence of common (unglycosylated) and specific (glycosylated and deglycosylated) epitopes. The arabinogalactan protein HRGP1 is not recognized by either antibody and HRGP2b does not react with the Yariv antigen. Immunoprecipitation of 3H-labeled HRP1 and HRP2b in the presence of goat antirabbit IgG, followed by gel electrophoresis, allows to recover HRP2b only. Again, HRP2b is immunoprecipitated by the two antisera.

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