Characterization of the hydroxyproline-rich protein core of an arabinogalactan-protein secreted from suspension-cultured Lolium multiflorum (Italian ryegrass) endosperm cells

Abstract

An arabinogalactan-protein (AGP) purified from the filtrate of liquid-suspension-cultured Italian-ryegrass (Lolium multiflorum) endosperm cells by affinity chromatography on myeloma protein J539-Sepharose was deglycosylated with trifluoromethanesulphonic acid to remove polysaccharide chains that are covalently associated with hydroxyproline residues in the peptide component of the proteoglycan. The protein core, which accounts for less than 10% (w/w) of the intact proteoglycan, was purified by h.p.l.c. It has an apparent Mr of 35,000, but reacts very poorly with both Coomassie Brilliant Blue R and silver stains. Amino-acid-sequence analysis of the N-terminus of the h.p.l.c.-purified protein core and of tryptic peptides generated from the unpurified protein reveals a high content of hydroxyproline and alanine. These are sometimes arranged in short (Ala-Hyp) repeat sequences of up to six residues. Polyclonal antibodies raised against the protein core do not cross-react with native AGP, the synthetic peptide (Ala-Hyp)4, poly-L-hydroxyproline or poly-L-proline. The results suggest that the polysaccharide chains in the native AGP render the protein core of the proteoglycan inaccessible to the antibodies and that the immunodominant epitopes include domains of the protein other than those rich in Ala-Hyp repeating units.

A Novel Hydroxyproline-Deficient Arabinogalactan Protein Secreted by Suspension-Cultured Cells of Daucus carota (Purification and Partial Characterization)

“…There is also the variable appearance of a minor band (at approximately 60 kD) that we believe is either human cytokeratin or a partially deglycosylated core protein. These data support the previous study of a secreted "classical/Hyp-rich" AGP isolated from Lolium multiflorum culture media (Gleeson et al, 1989), whose molecular mass decreased from 200 to 35 kD, suggesting the possible presence of a single core protein.…”

“…The secreted proteoglycan AGPs typically contain 2 to 10% protein by weight, which have an amino acid composition that is characteristically rich in Ser, Hyp, and Ala. N-terminal amino acid sequences of purified plasma membrane and secreted AGPs from Lolium and Rosa species have demonstrated the presence of a highly conserved N-terminal AlaHyp repeat sequence (Gleeson et al, 1989;Komalavilas et al, 1991). The carbohydrate moiety of these molecules typically consists of a 1,3-linked B-galactan backbone with side branches of 1,6-linked B-galactosyl residues that carry terminal residues typically of a-arabinofuranosides and minor amounts of uronic acids, Glc, rhamnose, Xyl, and Fuc.…”

Domain conservation in several volvocalean cell wall proteins

“…There are also a number of XPro doublets found in the flower cell wall proteins of tobacco [59]. Since serine and alanine codons differ only at the first nucleotide position, the (SerPro)x repeats in Chlamydomonas might be evolutionary precursors of the (AlaHyp)x repeats that are found in the higher plant arabinogalactan proteins [13,14] and that have recently been found in a cell wall extensin from maize [24] and Douglas fir [ 11]. There is no potential isodityrosine motif in either WP6 or VSP-3; in fact, no tyrosines at all are found within the repetitive domains.…”

A Histidine-Rich Extensin from Zea mays Is an Arabinogalactan Protein

“…This HRGP is unusually rich in His, hence an HHRGP, and is an apparent extensin AGP "chimera," i.e. it shares sequence homology and glycosylation patterns with both extensins (17,18,30,31,34) and AGPs (7,9,24). However, we suggest that HHRGP is not a chimera oftwo different proteins per se but, instead, occupies an intermediate position between extremes of the extensin family whose members are a phylogenetic series ranging from highly repetitive basic minimally glycosylated RPRPs to the highly glycosylated acidic AGPs.…”