Biosynthesis of Arabinogalactan-Protein in Lolium multiflorum (Ryegrass) Endosperm Cells

Abstract

The peptidyl prolyl hydroxylase responsible for the formation of hydroxyproline during arabinogalactan-protein biosynthesis in Lolium multiflorun (ryegrass) endosperm cells is a membrane-associated enzyme which will catalyze the hydroxylation of poly(L-proline) in the presence of oxygen, a-ketoglutarate, ferrous ion, and ascorbate. The Km for poly(L-proline) (8000 molecular weight) is 40 micromolar. The enzyme will also hydroxylate the protocolfagen analog (Pro-Pro-Gly)5.4H20.Fractionation of membranes from protoplast lysates on a discontinuous sucrose/sorbitol density gradient, followed by centrifugation on a linear sucrose gradient in the presence of Mg2e, leads to a clear separation of a number of membrane components. The membrane components have been tentatively identified using marker enzymes and assayed for peptidyl prolyl hydroxylase. It is concluded that the ryegrass prolyl hydroxylase is enriched in Golgi-derived membranes, but that significant amounts are also located in other subceliular fractions, including the rough endoplasmic reticulum.

Polyproline II Confirmation in the Protein Component of Arabinogalactan-Protein from Lolium multiflorum

“…Interpretation of the circular dichroism data for the ryegrass arabinogalactan-protein in terms of polyproline II conformation in the protein component of the macromolecule is consistent with the observation that the peptidyl prolyl hydroxylase involved in its biosynthesis is specific for synthetic peptides containing the same conformation (6).…”

Biosynthesis of arabinogalactan-protein in Lolium multiflorum (Italian ryegrass) endosperm cells. Subcellular distribution of galactosyltransferases

“…The identity of the ryegrass subcellular-membrane fractions was based on putative-markerenzyme distributions and on the density shifts caused by dissociation of ribosomes from RER to form SER at low Mg2 + concentrations (Lord et al, 1973;Quail, 1979). The results shown in Table 1 are in agreement with previous results (Cohen et al, 1983), although it was observed here that careful preparation and fractionation of the sucrose gradient led to a clear and reproducible resolution of two broad membrane bands in fraction L3. The lower-density component (fraction L3a) accounted for only a few per cent of total protein at a high Mg2 + concentration, but at a lower Mg2 + concentration it contained a much higher proportion of the protein (Table 1).…”

“…Ryegrass endosperm protoplasts, prepared by incubation of suspension-cultured cells with 1% Driselase (Kyowa Hakka Kogyo Co., Tokyo, Japan) (Keller & Stone, 1978) were coated with murine myeloma protein J539, lysed in 50mMpotassium phosphate buffer, pH 8.0 (containing 5mM-MgCl2), and the plasma-membrane fraction isolated by low-speed centrifugation of the lysate on a discontinuous sucrose/sorbitol gradient as described by Schibeci et al (1982). The fraction containing the bulk of the intracellular membranes and cytoplasmic components (fraction F1) was centrifuged at 1000OOg for 1 h, the pellet resuspended in 20mM-Mops/NaOH buffer, pH7.5 (containing 5mM-dithiothreitol) with or without 5mM MgCl2, and the suspension centrifuged at 180000g for 2h on linear 5-42% (w/w)-sucrose density gradients in a Beckman SW41 rotor (Cohen et al, 1983). Gradient fractions were collected by using a J-shaped Pasteur pipette and the membranes collected by centrifugation at 200000g for 1 h and finally resuspended in 20mM-Mops/NaOH buffer, pH7.5 (containing 5mMdithiothreitol).…”

“…A more detailed study of individual polysaccharide synthases responsible for arabinogalactanprotein synthesis was precluded by the presence of other membrane-bound enzymes competing for the UDP-galactose substrate. In the present study we have used the assay system of Mascara & Fincher (1982) in conjunction with recently developed membrane-fractionation procedures (Schibeci et al, 1982;Cohen et al, 1983;Henry et al, 1983). These procedures permitted the isolation of membrane fractions enriched in RER, SER, Golgi membranes and 'secretory vesicles', enabling us to purify partially enzymes capable of transferring galactose from UDP-galactose into 66% ethanol-insoluble polymers containing (1-.6)-,B-galactose residues, and to identify the subcellular location of the enzymes.…”

Characterization of a plant-produced recombinant human secretory IgA with broad neutralizing activity against HIV

“…Hydroxyproline conversion is typical of plant cell wall proteins, and indicates exposure of P2G12 SIgA to prolyl hydroxylase, an enzyme enriched in the Golgi apparatus and protein bodies. 53 Hydroxyproline conversion is often associated with the subsequent addition of galactose and arabinose sugar moieties, 54 although such plant-specific glycans were not identified here.…”