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First published online June 12, 2003; 10.1104/pp.103.021766 Plant Physiology 132:1362-1369 (2003) © 2003 American Society of Plant Biologists Glycosylation Motifs That Direct Arabinogalactan Addition to Arabinogalactan-Proteins1Department of Chemistry and Biochemistry, Ohio University, Athens, Ohio 45701 (L.T., M.J.K.); and Biochemistry Department, Michigan State University, East Lansing, Michigan 48824 (J.F.L.)
Hydroxyproline (Hyp)-rich glycoproteins (HRGPs) participate in all aspects of plant growth and development. HRGPs are generally highly O-glycosylated through the Hyp residues, which means carbohydrates help define the interactive molecular surface and, hence, HRGP function. The Hyp contiguity hypothesis predicts that contiguous Hyp residues are sites of HRGP arabinosylation, whereas clustered noncontiguous Hyp residues are sites of galactosylation, giving rise to the arabinogalactan heteropolysaccharides that characterize the arabinogalactan-proteins. Early tests of the hypothesis using synthetic genes encoding only clustered noncontiguous Hyp in the sequence (serine [Ser]-Hyp-Ser-Hyp)n or contiguous Hyp in the series (Ser-Hyp-Hyp)n and (Ser-Hyp-Hyp-Hyp-Hyp)n confirmed that arabinogalactan polysaccharide was added only to noncontiguous Hyp, whereas arabinosylation occurred on contiguous Hyp. Here, we extended our tests of the codes that direct arabinogalactan polysaccharide addition to Hyp by building genes encoding the repetitive sequences (alanine [Ala]-proline [Pro]-Ala-Pro)n, (threonine [Thr]-Pro-Thr-Pro)n, and (valine [Val]-Pro-Val-Pro)n, and expressing them in tobacco (Nicotiana tabacum) Bright-Yellow 2 cells as fusion proteins with green fluorescent protein. All of the Pro residues in the (Ala-Pro-Ala-Pro)n fusion protein were hydroxylated and consistent with the hypothesis that every Hyp residue was glycosylated with arabinogalactan polysaccharide. In contrast, 20% to 30% of Pro residues remained non-hydroxylated in the (Thr-Pro-Thr-Pro)n, and (Val-Pro-Val-Pro)n fusion proteins. Furthermore, although 50% to 60% of the Hyp residues were glycosylated with arabinogalactan polysaccharide, some remained non-glycosylated or were arabinosylated. These results suggest that the amino acid side chains of flanking residues influence the extent of Pro hydroxylation and Hyp glycosylation and may explain why isolated noncontiguous Hyp in extensins do not acquire an arabinogalactan polysaccharide but are arabinosylated or remain non-glycosylated.
Glycoproteins play diverse roles in the eukaryotic extracellular matrices of both plants and animals. However, in the Hyp-rich glycoprotein (HRGP) superfamily, which is restricted to plants and green algae, Hyp-O-glycosylation forms unique glycomodules that contribute to molecular shape, size, rigidity, stability, solubility, and charge. Such glycomodules often create the bulk of the interactive molecular surface.
Peptide periodicity together with the extent and type of
O-glycosylation define three major HRGP families
(Kieliszewski and Lamport,
1994
Extensins exhibit more complex periodicity and glycosylation where
repetitive motifs typically of 5, 10, and 16 amino acid residues include
monogalactosylated Ser residues (Lamport
et al., 1973
Arabinogalactan-proteins (AGPs) are the least periodic and most highly
glycosylated HRGPs. However, AGPs do generally contain repetitive motifs like
Xaa-Hyp-Xaa-Hyp and Xaa-Hyp-Hyp (where Xaa is often Ser, Thr, or Ala;
Chen et al., 1994
RPRPs, extensions, and AGPs also contain members that are chimeras of an
HRGP fused to a non-HRGP. For example, extensin chimeras, such as potato
(Solanum tuberosum) lectin
(Kieliszewski et al., 1994
AGPs are of considerable current interest because of their apparent role in
virtually all aspects of plant development and their location as glycoproteins
tethered to the outer leaflet of the plasma membrane through
glycosylphosphatidyl inositol (GPI) anchors
(Youl et al., 1998
Earlier, we proposed a sequence-based glycosylation code based on Hyp
contiguity. This Hyp contiguity hypothesis postulates arabinosylation of
contiguous Hyp residues and galactosylation of noncontiguous Hyp residues
(Kieliszewski et al., 1992a
Gene Synthesis, Plasmid Construction, and Tobacco Cell Transformation
Head-to-tail polymerization of the oligonucleotide sets
(Fig. 1; Shpak et al., 1999
Fusion proteins AP-EGFP, TP-EGFP, and VP-EGFP eluted from the hydrophobic interaction chromatography column (HIC) in water (not shown). Further fractionation of the green fluorescent HIC fractions by reverse-phase chromatography on a Hamilton polymeric reverse-phase column (PRP-1, Hamilton Co., Reno, NV; Fig. 3) yielded fusion proteins suitable for sequence analyses. Partial protein sequences corroborated the DNA sequences and the amino acid compositions (Tables I and II). After PRP-1 fractionation, typical yields of the fusion glycoproteins from the most productive cell lines were: AP-EGFP, 30 mg L–1 medium; TP-EGFP, 10 mg L–1 medium; and VP-EGFP, 6 mg L–1 medium. Proteolysis of AP-EGFP, TP-EGFP, and VP-EGFP cleaved EGFP from the glycomodules, producing single symmetric peaks during gel filtration chromatography on Superose-12 (Amersham-Pharmacia, Piscataway, NJ; not shown).
Amino acid analyses of the isolated glycomodules indicated that 100% of the Pro residues were hydroxylated to form Hyp in AP, 80% in VP, and 70% in TP (Table II).
All the fusion glycoproteins contained Gal, Ara, Rha, and GlcUA (Table III). Judging by Hyp-glycoside profiles, 100% of the Hyp residues in AP-EGFP contained arabinogalactan polysaccharide adducts, compared with 50% to 60% in TP-EGFP and VP-EGFP, which also contained Hyp-arabinosides and non-glycosylated Hyp (Table IV). TP-EGFP was unique in that it contained an unknown Hyp derivative (Hyp-unknown, Table IV) that eluted later than non-glycosylated Hyp on the Chomobeads C cation exchange column, which meant it was more positively charged than non-glycosylated Hyp.
The amino acid composition of TP glycomodule after NaOH/sodium sulfite
treatment compared with the control (HF deglycosylated TP after the same
treatment) showed no loss of Thr or gain of the elimination/sulfite addition
product 2-amino-3-sulfonyl butyric acid
(Simpson et al., 1972
AP-EGFP, TP-EGFP, and VP-EGFP precipitated
(
Glycoproteins sculpt the surface of all eukaryotic cells. Intriguingly, plants have chosen HRGPs as their major surface glycoproteins; these glycoproteins have no animal homologs and utilize Hyp glycosylation to tailor the molecular properties of both membrane-bound AGPs and cross-linked extensins in the wall. With the Hyp contiguity hypothesis as a guide, further identification of the detailed rules that govern potential Hyp glycosylation, therefore, is essential for interpretation of genomic readouts and functional glycomics. This applies particularly to the plasma membrane proteome because the unusual characteristics of AGPs preclude their facile detection and identification by standard proteomics assays. Based on genomic readouts, the Hyp contiguity hypothesis predicts that arabinogalactan glycomodules are a pervasive feature of the plasma membrane proteome—of 210 GPI-anchored proteins identified in Arabidopsis, 40% contain sites putatively involved in arabinogalactan polysaccharide addition in protein families that include not only classical AGPs but also putative phytocyanins, COBRAs, glycerophosphodiesterases, fasciclins, aspartyl proteases, lipid transfer proteins, receptors, and other unidentified proteins (Borner et al., 2002  ).
Our data confirm that endogenous plant prolyl hydroxylase(s) recognize
common AGP motifs with flanking residues Ala, Val, or Thr when expressed as
repetitive VP, TP, or AP and show that the resulting Hyp residues serve as
arabinogalactan addition sites with the site occupancy ranging from
approximately 50% to 60% (VP and TP) to 100% (AP). Assuming that the
relatively low molecular size of VP did not significantly influence
hydroxylation or subsequent Hyp-glycosylation, this indicates that the
residues flanking Pro/Hyp influence both hydroxylation and the subsequent type
and amount of glycosylation, consistent with our earlier results showing that
noncontiguous Hyp residues can be arabinosylated or remain non-glycosylated
(Kieliszewski et al., 1995
What constitutes a noncontiguous Hyp cluster, and can lone occurrences of
Hyp be arabinogalactosylated? Although four to five intervening residues
permit arabinogalactan addition to noncontiguous Hyp in LeAGP-1
(Zhao et al., 2002
Thus, design of the repetitive TP and VP construct tested the possibility
that a flanking Thr or Val residue might suppress polysaccharide addition to
Hyp. Table I does show
incomplete hydroxylation of VP and TP motifs with subsequent polysaccharide
addition to only 45% to 60% of the Hyp residues
(Table III), the rest being
arabinosylated, remaining non-glycosylated, or as in the case of TP, with a
small amount of an unknown adduct (Table
IV). Known flanking residues that influence Pro hydroxylation
include Lys-Pro, Tyr-Pro, and Leu-Pro, which are never hydroxylated in
contrast to SP and AP motifs that are 100% hydroxylated and undergo 100%
polysaccharide addition. Significantly, we have never detected polysaccharide
addition to contiguous Hyp motifs (Shpak
et al., 2001
We can now deal with several issues that involve the identification of bona
fide AGPs. Extreme sequence variability and absence of an obvious single
signature motif (Schultz et al.,
2000
Creation of Plasmid pUC-SStom-SP-EGFP
We amplified the tomato (Lycopersicon esculentum) LeAGP-1 signal
sequence DNA (designated SStom here) as described earlier
(Li and Showalter, 1996
Construction of a given synthetic gene involved three sets of partially
overlapping, complementary oligonucleotide pairs
(Fig. 1) polymerized as
described earlier (Shpak et al.,
1999
pUC-SStom-AP-EGFP,
pUC-SStom-TP-EGFP, and
pUC-SStom-VP-EGFP were introduced into A.
tumefaciens strain LBA4404 by the freeze thaw method
(McCormick et al., 1986
We maintained transformed BY2 cells in liquid culture for 20 d at room temperature on an Innova gyrotary shaker (New Brunswick Scientific, Edison, NJ) rotating at 90 rpm. The culture medium was filtered from cells, concentrated via rotary evaporation at 28°C, and dialyzed against distilled water overnight. The dialyzed medium was further concentrated by rotary evaporation, and NaCl was added to a final concentration of 2 M. Insoluble material was pelleted by centrifugation (13,000g, 20 min, SS-34 rotor), then loaded on an HIC (Phenyl Sepharose 6 Fast Flow, 16 x 700 mm, Amersham-Pharmacia Biotech) equilibrated in 2 M NaCl. A decreasing step gradient was used to elute the fusion glycoproteins, starting with 2 M NaCl and followed by 1 M NaCl, then deionized distilled water. The flow rate was 1 mL min–1, and the fractions were monitored by eye for green fluorescence. We combined the fluorescent fractions and loaded them on a semipreparative polymeric C-18 reverse-phase column (10 µm, PRP-1, 7 x 305 mm, Hamilton) equilibrated with buffer A (0.1% [v/v] aqueous trifluoroacetic acid). Samples were eluted with a gradient of 100% (v/v) buffer A increasing to 70% (v/v) buffer B (80% [v/v] acetonitrile in 0.1% [v/v] aqueous trifluoroacetic acid) for 105 min. The flow rate was 1 mL min–1, and the eluate was monitored at 220 nm on a 1050 HPLC system (Hewlett-Packard, Novi, MI). The fusion glycoproteins eluted in 50% (v/v) buffer B and native AGPs in 30% (v/v) buffer B.
We dissolved 10 mg of AP-EGFP, TP-EGFP, and VP-EGFP in 0.5 mL of deionized, distilled water and heated the samples at 100°C for 5 min. After cooling, an equal volume of freshly made 4% (w/v) ammonium bicarbonate containing 2.5 mM CaCl2 and either pronase or trypsin (1:100 [w/w] final enzyme:substrate ratio). The mixture was incubated at room temperature for 24 h and then freeze dried, redissolved in 0.5 mL of Superose buffer (0.2 M aqueous sodium phosphate [pH 7] containing 0.05% [w/v] sodium azide), and further separated on a semipreparative Superose-12 gel filtration column equilibrated in Superose buffer (see below). The enzymes used were pronase with AP-EGFP and trypsin with TP-EGFP and VP-EGFP.
Dialyzed and concentrated tobacco cell medium or proteolyzed fusion glycoproteins were loaded on a semipreparative Superose-12 column (16 x 500 mm, Amersham-Pharmacia Biotech), equilibrated, and eluted with Superose buffer at a flow rate of 0.3 mL min–1 and monitored at 220 nm. Peaks absorbing at 220 nm were collected, dialyzed against deionized, distilled water, freeze dried, and then analyzed (see below) to determine which peaks corresponded to the TP, VP, and AP glycomodules.
Amino acid compositions were determined either at the Michigan State
University Macromolecular Facility (East Lansing) or in our laboratory on a
Beckman HPLC (Beckman Instruments, Fullerton, CA) using methods described
earlier (Bergman et al., 1986
Hyp-glycoside profiles were determined on 10 mg of base-hydrolyzed fusion
glycoproteins as described earlier
(Weathers et al., 1977
One hundred micrograms of each fusion glycoprotein was dissolved in 300
µL of distilled water. An equal volume of
(
TP-EGFP, VP-EGFP, or the AP, TP and VP glycomodules (3–5 mg) were
dissolved in 500 µL of anhydrous hydrogen fluoride containing 10% (v/v)
anhydrous methanol and stirred for 2 h at 0°C. The reaction was quenched
with 10 mL of cold, deionized, distilled water, followed by dialysis against
deionized distilled water and freeze drying
(Sanger and Lamport, 1983
The TP glycomodule (680 µg) was dissolved in 800 µL of 0.1
N HCl (pH 1) and heated for 1 h at 100°C to remove Ara
residues. The mixture was dialyzed against deionized distilled water and
freeze dried. The dearabinosylated glycomodule (0.30 mg) was heated at
50°C for 5 h in 500 µL of 0.2 N NaOH/1 M sodium
sulfite (Whistler and BeMiller,
1958
Endongenous AGPs from non-transformed BY2 cells were prepared as described
earlier (Shpak et al.,
1999
Upon request, all novel materials described in this publication will be made available in a timely manner for noncommercial research purposes, subject to the requisite permission from any third party owners of all or parts of the material. Obtaining any permissions will be the responsibility of the requestor.
We thank Mr. Mick Held for assistance with amino acid analyses, Dr. Elena Shpak for plasmid SStob-SP-EGFP, and Dr. Derek T.A. Lamport for comments on the manuscript. Received March 12, 2003; returned for revision April 2, 2003; accepted April 2, 2003.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.021766.
1 This work was supported by the National Science Foundation (grant no.
MCB–9874744), by Ohio University (Molecular and Cellular Biology Program
grants), and in part by the National Institutes of Health (grant no.
2–P41–RR05351–06 awarded to the Complex Carbohydrate
Research Center). * Corresponding author; e-mail kielisze{at}helios.phy.ohiou.edu; fax 740–597–1772.
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TOP
ABSTRACT
RESULTS
-Elimination of TP-EGFP
