Tandem Mass Spectrometry and Structural Elucidation of Glycopeptides from a Hydroxyproline-rich Plant Cell Wall Glycoprotein Indicate That Contiguous Hydroxyproline Residues Are the Major Sites of Hydroxyproline O-Arabinosylation (*)

Hydroxyproline-rich glycoproteins (HRGPs) occur in the extracellular matrix of land plants and green algae. HRGPs contain from 2 to 95% of their dry weight as carbohydrate, predominantly as oligoarabinosides and/or as heteropolysaccharides which are O-linked to the hydroxyproline residues. A glycosylation code that determines the presence or absence and extent of arabinosylation at each hydroxyproline residue is likely, as each HRGP has a unique arabinosylation profile. Previously we noted a positive correlation between the contiguity of hydroxyproline residues and the extent of HRGP O-arabinosylation (Kieliszewski, M., deZacks, R., Leykam, J. F., and Lamport, D. T. A.(1992) Plant Physiol. 98, 919-926); most arabinosylated hydroxyproline residues and the longer arabinofuranoside chains occur in HRGPs where Hyp residues occur as blocks of tetrahydroxyproline, while those with little or no contiguous Hyp exhibit very little Hyp arabinosylation. In order to test this Hyp contiguity hypothesis, we have for the first time determined the arabinosylation site specifics of an HRGP, namely the proline and hydroxyproline-rich glycoprotein (PHRGP) isolated from Douglas fir (Pseudotsuga menziesii). Pronase digests of PHRGP yielded a major peptide and three glycopeptides whose structures were determined directly from the unfractionated, underivatized Pronase digest by tandem mass spectrometry using collisionally induced dissociation. We corroborated the peptide and glycopeptide structures by Edman degradation, neutral sugar analyses, hydroxyproline arabinoside profiles, and further mass spectrometric analyses after purification of the major peptide and glycopeptides by a combination of hydrophilic interaction and reverse phase column chromatography. Consistent with the Hyp contiguity hypothesis, the structural analyses indicate that while the sequence Ile-Pro-Pro-Hyp is never arabinosylated and Lys-Pro-Hyp-Val-Hyp is only occasionally monoarabinosylated at Hyp-5, the peptide containing contiguous Hyp, Lys-Pro-Hyp-Hyp-Val, is always arabinosylated at Hyp-3, mainly by a triarabinoside. We also obtained precise molecular masses for both intact and anhydrous hydrogen fluoride-deglycosylated PHRGPs (73.113 and 53.834 kDa) via matrix-assisted laser desorption/ionization time of flight mass spectrometry, representing the first HRGP to be analyzed by this method.

[1]  J. Leykam,et al.  Potato lectin: a modular protein sharing sequence similarities with the extensin family, the hevein lectin family, and snake venom disintegrins (platelet aggregation inhibitors). , 1994, The Plant journal : for cell and molecular biology.

[2]  K L Williams,et al.  Glycosylation sites identified by solid-phase Edman degradation: O-linked glycosylation motifs on human glycophorin A. , 1993, Glycobiology.

[3]  C. G. Edmonds,et al.  Tandem mass spectrometry of very large molecules. 2. Dissociation of multiply charged proline-containing proteins from electrospray ionization. , 1993, Analytical chemistry.

[4]  J. J. Conboy,et al.  The determination of glycopeptides by liquid chromatography/mass spectrometry with collision-induced dissociation , 1992, Journal of the American Society for Mass Spectrometry.

[5]  B. Chait,et al.  Weighing naked proteins: practical, high-accuracy mass measurement of peptides and proteins. , 1992, Science.

[6]  A. Hallmann,et al.  A novel extensin that may organize extracellular matrix biogenesis in Volvox carteri. , 1992, The EMBO journal.

[7]  J. Leykam,et al.  A Histidine-Rich Extensin from Zea mays Is an Arabinogalactan Protein. , 1992, Plant physiology.

[8]  J. Leykam,et al.  A repetitive proline-rich protein from the gymnosperm douglas fir is a hydroxyproline-rich glycoprotein. , 1992, Plant physiology.

[9]  K. Biemann,et al.  A variable dispersion array detector for a tandem mass spectrometer , 1991 .

[10]  Derekt . A. Lamport,et al.  Gum arabic glycoprotein is a twisted hairy rope : a new model based on o-galactosylhydroxyproline as the polysaccharide attachment site. , 1991, Plant physiology.

[11]  A. Burlingame,et al.  Structure determination of O-linked glycopeptides by tandem mass spectrometry. , 1990, Biomedical & environmental mass spectrometry.

[12]  A. Burlingame,et al.  Characterization of O-glycosylation sites in recombinant B-chain of platelet-derived growth factor expressed in yeast using liquid secondary ion mass spectrometry, tandem mass spectrometry and Edman sequence analysis. , 1990, Biomedical & environmental mass spectrometry.

[13]  J. Klinman,et al.  A new redox cofactor in eukaryotic enzymes: 6-hydroxydopa at the active site of bovine serum amine oxidase. , 1990, Science.

[14]  J. Leykam,et al.  Structure of the Threonine-Rich Extensin from Zea mays. , 1990, Plant physiology.

[15]  B. Chait,et al.  Cinnamic acid derivatives as matrices for ultraviolet laser desorption mass spectrometry of proteins. , 1989, Rapid communications in mass spectrometry : RCM.

[16]  C. Lamb,et al.  Specific expression of a novel cell wall hydroxyproline-rich glycoprotein gene in lateral root initiation. , 1989, Genes & development.

[17]  A. Marcus,et al.  Characterization of two soybean repetitive proline-rich proteins and a cognate cDNA from germinated axes. , 1989, The Plant cell.

[18]  J. Schwartz,et al.  Unusual composition of peptidoglycan in Bordetella pertussis. , 1989, The Journal of biological chemistry.

[19]  M. Karas,et al.  Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. , 1988, Analytical chemistry.

[20]  B. T. Terhune,et al.  Characterization of native and modified extensin monomers and oligomers by electron microscopy and gel filtration. , 1988, Plant physiology.

[21]  Derekt . A. Lamport,et al.  Purification and Partial Characterization of a Hydroxyproline-Rich Glycoprotein in a Graminaceous Monocot, Zea mays. , 1987, Plant physiology.

[22]  Stephen Naylor,et al.  An approach towards the complete FAB analysis of enzymic digests of peptides and proteins , 1986 .

[23]  M. Esquerré-Tugayé,et al.  Hydroxyproline-rich glycoprotein accumulation in the cell walls of plants infected by various pathogens , 1986 .

[24]  L. Staehelin,et al.  Cross-linking patterns in salt-extractable extensin from carrot cell walls. , 1986, Plant physiology.

[25]  S. Martin,et al.  Protein conformation of potato (Solanum tuberosum) lectin determined by circular dichroism. , 1986, The Biochemical journal.

[26]  C. Lamb,et al.  Accumulation of hydroxyproline-rich glycoprotein mRNAs in response to fungal elicitor and infection. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[27]  Jychian Chen,et al.  An extracellular matrix protein in plants: characterization of a genomic clone for carrot extensin , 1985, The EMBO journal.

[28]  P. Roepstorff,et al.  Proposal for a common nomenclature for sequence ions in mass spectra of peptides. , 1984, Biomedical mass spectrometry.

[29]  D. Lamport,et al.  Isolation of extensin precursors by direct elution of intact tomato cell suspension cultures , 1984 .

[30]  L. Epstein,et al.  An intramolecular linkage involving isodityrosine in extensin , 1984 .

[31]  J. Varner,et al.  Reinforced Polyproline II Conformation in a Hydroxyproline-Rich Cell Wall Glycoprotein from Carrot Root. , 1984, Plant physiology.

[32]  G. Fincher,et al.  Arabinogalactan-Proteins: Structure, Biosynthesis, and Function , 1983 .

[33]  Y. Akiyama,et al.  13C-NMR Analysis of Hydroxy-proline Arabinosides from Nicotiana tabacum , 1980 .

[34]  Derekt . A. Lamport,et al.  Cell Surfaces in Plant-Microorganism Interactions: I. A Structural Investigation of Cell Wall Hydroxyproline-rich Glycoproteins Which Accumulate in Fungus-infected Plants. , 1979, Plant physiology.

[35]  A. Mort,et al.  Anhydrous hydrogen fluoride deglycosylates glycoproteins. , 1977, Analytical biochemistry.

[36]  D. G. Pope,et al.  Relationships between Hydroxyproline-containing Proteins Secreted into the Cell Wall and Medium by Suspension-cultured Acer pseudoplatanus Cells. , 1977, Plant physiology.

[37]  V. Marchesi,et al.  Amino-acid sequence and oligosaccharide attachment sites of human erythrocyte glycophorin. , 1975, Proceedings of the National Academy of Sciences of the United States of America.

[38]  D. T. Lamport,et al.  Galactosylserine in extensin. , 1973, The Biochemical journal.

[39]  D. Lamport,et al.  Hydroxyproline Heterooligosaccharides in Chlamydomonas , 1972, Science.

[40]  D. Lamport,et al.  Hydroxyproline arabinosides in the plant kingdom. , 1971, Plant physiology.

[41]  D. Lamport.  Cell Wall Metabolism , 1970 .

[42]  D. Lamport,et al.  Hydroxyproline-O-glycosidic Linkage of the Plant Cell Wall Glycoprotein Extensin , 1967, Nature.

[43]  Donald J. Nevins,et al.  A method for the analysis of sugars in plant cell-wall polysaccharides by gas-liquid chromatography , 1967 .

[44]  P. Meikle,et al.  A (1→3,1→4)‐β‐glucan‐specific monoclonal antibody and its use in the quantitation and immunocytochemical location of (1→3,1→4)‐β‐glucans , 1994 .

[45]  K. Biemann.  Mass spectrometry of peptides and proteins. , 1992, Annual review of biochemistry.

[46]  C. Costello,et al.  Tandem mass spectrometry of glycolipids. , 1990, Methods in enzymology.

[47]  J. C. Fenyo,et al.  Macromolecular distribution of Acacia senegal gum (gum arabic) by size-exclusion chromatography , 1985 .

[48]  P. Albersheim,et al.  Structure and function of the primary cell walls of plants. , 1984, Annual review of biochemistry.

[49]  D. Lamport,et al.  A microapparatus for liquid hydrogen fluoride solvolysis: sugar and amino sugar composition of Erysiphe graminis and Triticum aestivum cell walls. , 1983, Analytical biochemistry.

[50]  A. Neuberger,et al.  Structural studies of the carbohydrate moieties of lectins from potato (Solanum tuberosum) tubers and thorn-apple (Datura stramonium) seeds. , 1982, The Biochemical journal.