Presence of a Glycosylphosphatidylinositol Lipid Anchor on Rose Arabinogalactan Proteins

Abstract

Arabinogalactan proteins constitute a class of plant cell surface proteoglycans with widespread occurrence and suggested functions in various aspects of plant growth and development, including cell proliferation, expansion, marking, and death. Previous investigations of subcellular fractions from suspension-cultured cells of “Paul's Scarlet” rose (Rosa sp.) have revealed extensive structural similarity between some soluble arabinogalactan proteins from the cell wall space and some plasma membrane-associated arabinogalactan proteins, thus inspiring the present investigation of the mechanism through which these inherently water-soluble molecules are held on the plasma membrane. Several lines of evidence gained through a combination of methods including reversed-phase chromatography, treatment with phosphatidylinositol-specific phospholipase C, and chemical structural analysis now show that some rose arabinogalactan proteins carry a ceramide class glycosylphosphatidylinositol lipid anchor. The predominant form of the ceramide is composed of tetracosanoic acid and 4-hydroxysphinganine. Plasma membrane vesicles readily shed arabinogalactan proteins by an inherent mechanism that appears to involve a phospholipase. This finding has significance toward understanding the biosynthesis, localization, and function of arabinogalactan proteins and toward stimulating other studies that may expand the currently very short list of higher plant proteins found to carry such membrane lipid anchors. Arabinogalactan proteins constitute a class of plant cell surface proteoglycans with widespread occurrence and suggested functions in various aspects of plant growth and development, including cell proliferation, expansion, marking, and death. Previous investigations of subcellular fractions from suspension-cultured cells of “Paul's Scarlet” rose (Rosa sp.) have revealed extensive structural similarity between some soluble arabinogalactan proteins from the cell wall space and some plasma membrane-associated arabinogalactan proteins, thus inspiring the present investigation of the mechanism through which these inherently water-soluble molecules are held on the plasma membrane. Several lines of evidence gained through a combination of methods including reversed-phase chromatography, treatment with phosphatidylinositol-specific phospholipase C, and chemical structural analysis now show that some rose arabinogalactan proteins carry a ceramide class glycosylphosphatidylinositol lipid anchor. The predominant form of the ceramide is composed of tetracosanoic acid and 4-hydroxysphinganine. Plasma membrane vesicles readily shed arabinogalactan proteins by an inherent mechanism that appears to involve a phospholipase. This finding has significance toward understanding the biosynthesis, localization, and function of arabinogalactan proteins and toward stimulating other studies that may expand the currently very short list of higher plant proteins found to carry such membrane lipid anchors. Arabinogalactan proteins (AGPs) 1The abbreviations used are: AGP, arabinogalactan protein; (β-d-Glc)3 Yariv phenylglycoside, 1,3,5-tri-(p-β-glucosyloxyphenylazo)-2,4,6-trihydroxybenzene; CM, culture medium; CW, cell wall; GC, gas chromatography; GPI, glycosylphosphatidylinositol; MSD, mass-selective detector; phytosphingosine, 4-hydroxysphinganine; PI, phosphatidylinositol; PI-PLC, phosphatidylinositol-specific phospholipase C; PM, plasma membrane; RPC, reversed-phase chromatography; TMS, trimethylsilyl. 1The abbreviations used are: AGP, arabinogalactan protein; (β-d-Glc)3 Yariv phenylglycoside, 1,3,5-tri-(p-β-glucosyloxyphenylazo)-2,4,6-trihydroxybenzene; CM, culture medium; CW, cell wall; GC, gas chromatography; GPI, glycosylphosphatidylinositol; MSD, mass-selective detector; phytosphingosine, 4-hydroxysphinganine; PI, phosphatidylinositol; PI-PLC, phosphatidylinositol-specific phospholipase C; PM, plasma membrane; RPC, reversed-phase chromatography; TMS, trimethylsilyl. form a class of structurally complex proteoglycans that are present at cell surfaces throughout the plant kingdom from bryophytes to angiosperms (1Nothnagel E.A. Int. Rev. Cytol. 1997; 174: 195-291Crossref PubMed Google Scholar). Although stylar secretions and gum exudates are particularly rich sources of AGPs (2Clarke A.E. Anderson R.L. Stone B.A. Phytochemistry. 1979; 18: 521-540Crossref Scopus (387) Google Scholar), it is likely that every plant cell produces AGPs. Despite this widespread occurrence, AGP functions, particularly at the molecular level, remain to be established. Various forms of evidence, however, have pointed to roles of AGPs in several aspects of growth and development (1Nothnagel E.A. Int. Rev. Cytol. 1997; 174: 195-291Crossref PubMed Google Scholar, 3Du H. Clarke A.E. Bacic A. Trends Cell Biol. 1996; 6: 411-414Abstract Full Text PDF PubMed Scopus (77) Google Scholar, 4Knox J.P. Smallwood M. Knox J.P. Bowles D.J. Membranes: Specialized Functions in Plants. Bios Scientific, Oxford, United Kingdom1996: 93-102Google Scholar). Developmentally regulated expression of AGPs has been demonstrated by biochemical or immunological means in floral development (5Sommer-Knudsen J. Clarke A.E. Bacic A. Sex. Plant Reprod. 1997; 10: 253-260Crossref Scopus (34) Google Scholar), pollen growth (6Cheung A.Y. Zhan X.-Y. Wang H. Wu H.-M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3853-3858Crossref PubMed Scopus (28) Google Scholar), somatic embryogenesis (7McCabe P.F. Valentine T.A. Forsberg L.S. Pennell R.I. Plant Cell. 1997; 9: 2225-2241Crossref PubMed Google Scholar), vascular differentiation (8Casero P.J. Casimiro I. Knox J.P. Planta. 1998; 204: 252-259Crossref Scopus (62) Google Scholar), and various other aspects of reproductive and vegetative development. Evidence of a more direct nature regarding AGP action has been obtained through perturbation experiments. Application of exogenous AGPs altered the course of somatic embryogenesis, thus suggesting an active role of AGPs in this process (9Kreuger M. van Holst G.-J. Planta. 1995; 197: 135-141Crossref Scopus (103) Google Scholar, 10Toonen M.A.J. Schmidt E.D.L. van Kammen A. de Vries S.C. Planta. 1997; 203: 188-195Crossref Scopus (75) Google Scholar). Another perturbation approach of growing popularity involves use of (β-d-Glc)3 Yariv phenylglycoside, a synthetic molecule that binds and precipitates AGPs. Experiments in which (β-d-Glc)3 has been applied to living plants or cells have yielded evidence of AGP action in cell proliferation (11Serpe M.D. Nothnagel E.A. Planta. 1994; 193: 542-550Crossref Scopus (162) Google Scholar), cell expansion (12Willats W.G.T. Knox J.P. Plant J. 1996; 9: 919-925Crossref PubMed Scopus (193) Google Scholar, 13Ding L. Zhu J.-K. Planta. 1997; 203: 289-294Crossref PubMed Scopus (112) Google Scholar), somatic embryogenesis (14Thompson H.J.M. Knox J.P. Planta. 1998; 205: 32-38Crossref Scopus (68) Google Scholar), pollen tube growth (15Roy S. Jauh G.Y. Hepler P.K. Lord E.M. Planta. 1998; 204: 450-458Crossref PubMed Scopus (124) Google Scholar), and cell death (16Langan K.J. Nothnagel E.A. Protoplasma. 1997; 196: 87-96Crossref Scopus (52) Google Scholar). One line of research in this laboratory has focused on the localization and structure of AGPs at defined sites at the plant cell surface. The aims have been to determine whether different AGPs are located at different sites (thereby implying different functions), to determine how AGPs are held at these sites, and to elucidate possible precursor-product relationships between the various AGPs. The model system for this work has been suspension-cultured cells of “Paul's Scarlet” rose (Rosa sp.). Through biochemical purification and chemical structural analysis, PM-AGPs bound to the plasma membrane, CW-AGPs bound to the cell wall, and soluble CM-AGPs of the cell wall space/culture medium have been characterized in this model system (17Komalavilas P. Zhu J.-K. Nothnagel E.A. J. Biol. Chem. 1991; 266: 15956-15965Abstract Full Text PDF PubMed Google Scholar, 18Serpe M.D. Nothnagel E.A. Plant Physiol. 1995; 109: 1007-1016Crossref PubMed Scopus (51) Google Scholar, 19Serpe M.D. Nothnagel E.A. Plant Physiol. 1996; 112: 1261-1271Crossref PubMed Scopus (28) Google Scholar). Two major AGPs, plus other minor forms, were found at each of these three This and of the major PM-AGPs from at the other sites, and major from or the other of the major PM-AGPs structural with of the major CM-AGPs (1Nothnagel E.A. Int. Rev. Cytol. 1997; 174: 195-291Crossref PubMed Google Scholar). The present investigation by this of between a and a the to determine how the held on the membrane. of the PM-AGPs that were more of CM-AGPs (1Nothnagel E.A. Int. Rev. Cytol. 1997; 174: 195-291Crossref PubMed Google Scholar), a membrane of a the that such a has been by several of AGPs H. Clarke A.E. Bacic A. Trends Cell Biol. 1996; 6: 411-414Abstract Full Text PDF PubMed Scopus (77) Google Scholar, Plant Biol. 1996; PubMed Scopus Google Scholar). the of this investigation on the of that the of a a of and that it have from AGP such in a or other lipid M.D. Nothnagel E.A. Plant Physiol. 1996; 112: 1261-1271Crossref PubMed Scopus (28) Google Scholar). now several lines of evidence that a lipid on some AGPs. of these have been in J. Nothnagel E.A. Biol. Cell. 1996; Scholar). PM-AGPs were from suspension-cultured cells of rose (Rosa sp.) M.D. Nothnagel E.A. Plant Physiol. 1996; 112: 1261-1271Crossref PubMed Scopus (28) Google Scholar). plasma membrane vesicles were from the by for rose (17Komalavilas P. Zhu J.-K. Nothnagel E.A. J. Biol. Chem. 1991; 266: 15956-15965Abstract Full Text PDF PubMed Google Scholar). The PM-AGPs were from the plasma membrane vesicles with M.D. Nothnagel E.A. Plant Physiol. 1996; 112: 1261-1271Crossref PubMed Scopus (28) Google Scholar), and (β-d-Glc)3 Yariv to the to AGPs. (β-d-Glc)3 binds AGPs. The complex is soluble in precipitates in of or higher (1Nothnagel E.A. Int. Rev. Cytol. 1997; 174: 195-291Crossref PubMed Google Scholar). The AGPs thus from the were through three of in and in The complex in and to the AGPs the (β-d-Glc)3 in the the in and to the by with PM-AGPs thus obtained were of proteins of 19Serpe M.D. Nothnagel E.A. Plant Physiol. 1996; 112: 1261-1271Crossref PubMed Scopus (28) Google Scholar). CM-AGPs were from the medium of the cell (17Komalavilas P. Zhu J.-K. Nothnagel E.A. J. Biol. Chem. 1991; 266: 15956-15965Abstract Full Text PDF PubMed Google with (16Langan K.J. Nothnagel E.A. Protoplasma. 1997; 196: 87-96Crossref Scopus (52) Google Scholar). Plasma membrane vesicles by were in at from the vesicles of were in with and by at were and with of in in of the for at in the of a of including and the the membrane vesicles were by at from the and were in of of from the and from were to (17Komalavilas P. Zhu J.-K. Nothnagel E.A. J. Biol. Chem. 1991; 266: 15956-15965Abstract Full Text PDF PubMed Google in Yariv phenylglycoside, which precipitates and AGPs through the used a of or PM-AGPs to of in to were on a and with of and acid M.D. Nothnagel E.A. Plant Physiol. 1996; 112: 1261-1271Crossref PubMed Scopus (28) Google Scholar). were and for by the M.D. Nothnagel E.A. Plant Physiol. 1996; 112: 1261-1271Crossref PubMed Scopus (28) Google Scholar). treatment of AGPs to or PM-AGPs of were with of in of for at the of the the the of AGPs to an of the of 1994; PubMed Scopus Google Scholar), of AGPs were in of and in an of were at for at the the of the which to of of with and to by obtained from and applied to AGPs to in of from J. Biol. Chem. Full Text PDF PubMed Google Scholar). of of AGPs and of were in of and for at the in a for molecular and for in a that the of in the at the of the in which the of AGPs were with of were in that molecular of AGP were and at for with in the of in with (17Komalavilas P. Zhu J.-K. Nothnagel E.A. J. Biol. Chem. 1991; 266: 15956-15965Abstract Full Text PDF PubMed Google Scholar). analysis of acid to to a and the three each with of The three were in and in the were and The were in for the the analysis of the analysis of with of of a of were in with the of in an such the in The were a of in a of and and and were and were in a and tetracosanoic acid of to for and yielded regarding other from from from from from from from for and yielded regarding other from in a Two were in with the of in an such the in The were and in of AGPs, a used for to The AGPs were in of and of were of were at and for an at The at and the by and of three of to the to The nature of the of to AGPs by AGP in of and at for The to in and three with applied to other molecules lipid this in of and in the 1994; PubMed Scopus Google Scholar). The by and with The and the and were and the The and and were (17Komalavilas P. Zhu J.-K. Nothnagel E.A. J. Biol. Chem. 1991; 266: 15956-15965Abstract Full Text PDF PubMed Google Scholar, 18Serpe M.D. Nothnagel E.A. Plant Physiol. 1995; 109: 1007-1016Crossref PubMed Scopus (51) Google Scholar). of analysis by were and by with M.D. Nothnagel E.A. Plant Physiol. 1996; 112: 1261-1271Crossref PubMed Scopus (28) Google Scholar). of and in AGPs use of and were on to and of the were by to in the of and by with the model for tetracosanoic phytosphingosine, were obtained from acid used an of for for which these in to the each for were obtained from of were at the at the The that PM-AGPs a lipid by the of AGPs from plasma membrane vesicles treatment with or plasma membrane vesicles were and the were for AGP by that AGP from the of and from the of and of were in each this demonstrated an of AGPs to other proteins in the treatment of plasma membrane vesicles with Although the from the and from the treatment of AGPs, from with various of revealed a between the of and the by which the AGP of the treatment the AGP of the The AGP of the treatment to of AGPs of at The of AGPs in the of the treatment and in the of the suggested that an inherent mechanism to AGPs from the plasma membrane. Although a of used in these the of these the inherent or the of AGPs from plasma membrane vesicles for the action of an PI-PLC, inherent of AGPs plasma membrane vesicles were in or Although has on the from H. Scopus Google Scholar), is of a phospholipase from P. U. J. Biol. Chem. Full Text PDF PubMed Google Scholar). that a of AGP the of rose plasma membrane H. Scopus Google Scholar, A. 197: Scopus (62) Google and P. U. J. Biol. Chem. Full Text PDF PubMed Google AGP in the of rose plasma membrane vesicles in the of This to of PI-PLC, however, of vesicles and proteins, in a that to Chem. Scopus Google and P. U. J. Biol. Chem. Full Text PDF PubMed Google inherent of AGPs from rose plasma and the inherent of AGPs from plasma membrane higher to of the inherent of AGPs and a were used of plasma membrane vesicles with the exogenous The that PM-AGPs a lipid by RPC, the of be to the of AGPs from the that PM-AGPs a complex at plus at and The major with work that that rose cells major of which from the at M.D. Nothnagel E.A. Plant Physiol. 1996; 112: 1261-1271Crossref PubMed Scopus (28) Google Scholar). that it that the major CM-AGPs of rose from the at that the major PM-AGPs were more the major that the three from the with (β-d-Glc)3 Yariv that treatment with exogenous a in the of PM-AGPs on The treatment with in extensive of and a of The of the in with exogenous PI-PLC, and on the of the at a to the of in that treatment with the of with the that this AGP a lipid anchor. structural of to were to that these AGPs and to evidence regarding the of a lipid anchor. The of to are in and the of and are in the and of were with this a of the major rose PM-AGPs M.D. Nothnagel E.A. Plant Physiol. 1996; 112: 1261-1271Crossref PubMed Scopus (28) Google Scholar). The and that were very to that of with an that in and higher in and these that have a more the of from and that this in on the of the to the that a lipid the structural to be were the lipid and the between the lipid and the the to the of some plasma membrane proteins of and the has the structure lipid 1994; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). some the of this has been found to carry or more of or and analysis, three of the fractions in were found to and the of the tetracosanoic in three of the in the to including and were in some the of these were that of tetracosanoic acid The of in the molecules in the the of the major PM-AGPs of rose M.D. Nothnagel E.A. Plant Physiol. 1996; 112: 1261-1271Crossref PubMed Scopus (28) Google Scholar). from this the of were to AGP this of of of and of tetracosanoic acid of to of of of and of tetracosanoic acid of were more or in the of to be with the that and the and some of were with the lipid This regarding the of lipid with of these fractions and treatment and for these regarding the of on PM-AGPs however, of this revealed in the of tetracosanoic acid and other in the three Experiments to elucidate the of these revealed that of complex with PM-AGPs through (β-d-Glc)3 Yariv of AGPs from of plasma membrane vesicles and through the of (β-d-Glc)3 and AGPs in M.D. Nothnagel E.A. 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PubMed Scopus Google J. 10: Full Text PDF PubMed Google Scholar). of the in several were to or that the of the of these however, in the the of the of a lipid and in a with the it that of in be evidence of a lipid on these AGPs. PM-AGPs be obtained in for of for of the from the AGPs. to this suggested by the on the the plasma membrane of which a with lipid and a structurally L. P. M.A.J. J. 1995; PubMed Scopus Google found that of from the plasma membrane a in which the of the the lipid of the membrane the of from the membrane however, to the lipid of the membrane. that some AGPs with lipid be found in the culture medium of rose the in this and analysis of CM-AGPs in of of tetracosanoic and The major of rose cells has and (1Nothnagel E.A. Int. Rev. Cytol. 1997; 174: 195-291Crossref PubMed Google Scholar). these a for these to of acid of and acid were in the the of in this however, these were found to be laboratory use of the and with acid or other The of the lipid were that by of for such regarding the or of be a of however, on acid were to be of of and of acid of at AGP, tetracosanoic acid the acid in of rose CM-AGPs by is in The at of the of at higher from the three were and found to with (β-d-Glc)3 Yariv treatment with exogenous PI-PLC, the of CM-AGPs from the by of and of this and were to and the of were for the and in may have in these were and to and that and with PI-PLC, on to that of a to the treatment with of PI-PLC, at the of and at the of were with the that a lipid of in treatment with an to that of from with and to the in the at the in for the of in the with applied the of This to that which in the of for the the of however, or were in the This the of the from the thus a chemical analysis of the structural of the were to that these AGPs and to evidence regarding the of a lipid anchor. and thus of the to in some experiments. This to at higher the from that for the in The major present in were with this a of the major CM-AGPs for rose cells (16Langan K.J. Nothnagel E.A. Protoplasma. 1997; 196: 87-96Crossref Scopus (52) Google Scholar). to the at higher higher and the in in which the by a higher and to by and the higher for for and The of these AGPs were in some more and a higher and tetracosanoic acid of to for and yielded regarding other for and yielded regarding other in a The minor were with to the of a lipid anchor. from the analysis of and that of and an AGP of and (1Nothnagel E.A. Int. Rev. Cytol. 1997; 174: 195-291Crossref PubMed Google Scholar), the in were to of of and of of of of and of of the of the CM-AGPs or more of of AGP of the of in the of 1994; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar), to the that AGPs in in Although thus to AGP sites (1Nothnagel E.A. Int. Rev. 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J. 1991; PubMed Scopus Google Scholar). evidence of of and to other CM-AGPs were with of the and were with the CM-AGPs to The were for lipid by and the of tetracosanoic acid the major acid in acid at in and acid in were in or a and a the of were to of tetracosanoic acid of and of tetracosanoic acid of This of tetracosanoic acid in a purification from which of tetracosanoic acid of This of purification by with the that and for and of CM-AGPs of the tetracosanoic acid in CM-AGPs in and to of of AGP, the of that the of and were more and the of were to be with the that through the by and of however, and some of were with lipid to the This regarding the of lipid with the of these fractions and treatment and were to the nature of the lipid of the on rose AGPs. be in the and of of the and tetracosanoic acid and acid were This to to that the were to AGPs through an evidence of obtained with of the or from of and were more to in the of the of in AGPs. of and acid obtained through of an The were however, to be including of the more were the and of the were to of the that the were The used with and J. M. J. Biol. Chem. Full Text PDF PubMed Google with of the to to of this in of phytosphingosine, in the of of This the of acid a likely to of the lines of evidence in this the that rose AGPs a some of these with a lipid treatment with exogenous the of AGPs from plasma membrane of PM-AGPs by revealed the of AGP with exogenous PI-PLC, to a more on and were obtained with CM-AGPs and and of from other were in and fractions at with in a chemical of PM-AGPs were however, by the of the of lipid anchors. particularly tetracosanoic were at the in AGPs that were to to forms by exogenous The of these to AGPs an a thus the lipid a The predominant of the ceramide Although membrane proteins of and have been demonstrated to lipid PubMed Scopus Google Scholar, M.A.J. of Oxford, United Scholar, I. Int. J. Cell Biol. 1998; PubMed Scopus Google Scholar), the of such proteins in higher a that the of this Bacic A. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google demonstrated that and cell AGPs with that have been at the in the for a and with and were minor of the AGPs, lipid Several other plant proteins have been to lipid by to by of such plant proteins an in the plant H. H. 1996; PubMed Scopus Google Scholar), in the plasma of and M. J. U. Plant Physiol. 1997; Scholar), and several plasma membrane proteins in 1997; PubMed Scopus Google Scholar). finding of the present work is the inherent of AGPs from plasma membrane vesicles in This a of soluble AGPs in the treatment of plasma membrane vesicles with exogenous and in the that a of PM-AGPs a in of were in AGP from rose plasma membrane vesicles This that an be with the rose plasma membrane and of the lipid anchor. has been from P. U. J. Biol. Chem. Full Text PDF PubMed Google Scholar). regarding AGP from the plasma membrane is that some PM-AGPs are lipid and are bound to the plasma membrane through a an membrane of for of AGPs from plasma membrane vesicles in of the such and in the present to the that some AGPs are a anchor. the evidence, however, it is likely that rose AGPs are with lipid and the of these AGPs from is through action of a or The of lipid on a of however, that AGPs with lipid are to from the plasma membrane by for the of L. major L. P. M.A.J. J. 1995; PubMed Scopus Google Scholar). lipid found in various the of lipid structure and have been found M.A.J. of Oxford, United Scholar). The ceramide class lipid of rose AGPs, found in this is to the predominant class of lipid lipid in this are of acid and phytosphingosine, at has been found with a lipid acid in a or a structure A. M.A.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The of complex in the The of these with the PM-AGPs to be through of plasma membrane three of with (β-d-Glc)3 Yariv phenylglycoside, and of in and with M.D. Nothnagel E.A. Plant Physiol. 1996; 112: 1261-1271Crossref PubMed Scopus (28) Google Scholar). the of the of from the by to of the applied to the the are to an of molecule R.L. PubMed Scopus Google Scholar, A. J. 10: Full Text PDF PubMed Google and PM-AGPs are to an of M.D. Nothnagel E.A. Plant Physiol. 1996; 112: 1261-1271Crossref PubMed Scopus (28) Google Scholar), the of to be of of This the that the been that were from AGPs to the which to and in a on evidence, however, this The chemical of the particularly the of to the ceramide found by PubMed Scopus Google or the ceramide found by R.L. PubMed Scopus Google it to the lipid 1994; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). the of and in AGP fractions and the action of a or which these with the AGP of with the lipid the of and were of and however, it possible that some from AGPs were present in the structural studies are to elucidate the between the complex and the lipid anchors. The of lipid on AGPs has toward understanding the biosynthesis, the subcellular localization, and the functions of these complex in each of these may from this structural of AGPs.