Two different families of hydroxyproline-rich glycoproteins in melon callus: biochemical and immunochemical studies

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, HRGP_2a,2b and _2c. Two of these glycoproteins behave as polycations (HRGP_2b and _2c) and are chemically distinct from HRGP_2a. HRGP_2b 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 HRGP_2b 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 HRP_2b, the protein portion of HRGP_2b is 55,000 ± 5,000, as estimated after deglycosylation of the molecule with trifluoromethane sulfonic acid. Antibodies have been raised against HRGP_2b and HRP_2b. Immunodiffusion shows that each antigen (HRGP_2b or HRP_2b) 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 HRGP_2b does not react with the Yariv antigen. Immunoprecipitation of ³H-labeled HRP₁ and HRP_2b in the presence of goat antirabbit IgG, followed by gel electrophoresis, allows to recover HRP_2b only. Again, HRP_2b is immunoprecipitated by the two antisera.