Contiguous Hydroxyproline Residues Direct Hydroxyproline Arabinosylation in Nicotiana tabacum*

Elena ShpakDagger , Elisar BarbarDagger , Joseph F. Leykam§, and Marcia J. KieliszewskiDagger

From the Dagger  Department of Chemistry and Biochemistry, Ohio University, Athens, Ohio 45701 and the § Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824

Received for publication, December 15, 2000, and in revised form, January 11, 2001



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Hydroxyproline (Hyp) O-glycosylation characterizes the hydroxyproline-rich glycoprotein (HRGP) superfamily of the plant extracellular matrix. Hyp glycosylation occurs in two modes: Arabinosylation adds short oligoarabinosides (Hyp-arabinosides) while galactosylation leads to the addition of larger arabinogalactan polysaccharides (Hyp-polysaccharides). We hypothesize that sequence-dependent glycosylation of small peptide motifs results in glycomodules. These small functional units in combination with other repetitive peptide modules define the properties of HRGPs. The Hyp contiguity hypothesis predicts arabinosylation of contiguous Hyp residues and galactosylation of clustered noncontiguous Hyp residues. To determine the minimum level of Hyp contiguity that directs arabinosylation, we designed a series of synthetic genes encoding repetitive (Ser-Pro2)n, (Ser-Pro3)n, and (Ser-Pro4)n. A signal sequence targeted these endogenous substrates to the endoplasmic reticulum/Golgi for post-translational proline hydroxylation and glycosylation in transformed Nicotiana tabacum cells. The fusion glycoproteins also contained green fluorescence protein, facilitating their detection and isolation. The (Ser-Pro2)n and (Ser-Hyp4)n fusion glycoproteins yielded Hyp-arabinosides but no Hyp-polysaccharide. The motif (Ser-Pro3)n was incompletely hydroxylated, yielding mixed contiguous/noncontiguous Hyp and a corresponding mixture of Hyp-arabinosides and Hyp-polysaccharides. These results plus circular dichroic spectra of the glycosylated and deglycosylated (Ser-Pro2)n, (Ser-Pro3)n, and (Ser-Pro4)n modules corroborate the Hyp contiguity hypothesis and indicate that Hyp O-glycosylation is indeed sequence-driven.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Hydroxyproline-rich glycoproteins (HRGPs)1 participate in the plant extracellular matrix as networks, exudates, and glycocalyx, comprising a superfamily that includes extensins (1), proline-rich proteins (PRPs) (2) and arabinogalactan-proteins (AGPs) (3). The three major families are distinguished by characteristic repetitive structural motifs: Ser-Hyp4 in extensins, Pro-Hyp-Val-Tyr-Lys repeats and variants in PRPs, and Xaa-Hyp-Xaa-Hyp repeats plus the presence of arabinogalactan polysaccharide in AGPs.

The major post-translational modifications of HRGPs, proline hydroxylation and subsequent O-Hyp glycosylation, determine the properties of HRGPs to a greater or lesser extent. Carbohydrate accounts for as much as 95% of the hyperglycosylated AGPs and about 60% of extensins, thus forming the interactive molecular surface. In the lightly glycosylated PRPs, however, sugar may contribute as little as 1% of the mass.

O-Hyp glycosylation occurs in two distinct modes, Hyp arabinosylation (4) and Hyp galactosylation (5), respectively. Hyp arabinosylation of virtually all HRGPs results in short (usually 1-4 residues), neutral, linear homooligosaccharides of L-arabinofuranose (Hyp-arabinosides). Hyp galactosylation, which is restricted to the AGPs, results in addition of much larger arabinogalactan heteropolysaccharides (Hyp-polysaccharides) (5). These consist of a beta -1right-arrow3-linked galactan backbone (6) with 1right-arrow6-linked side chains (7) containing galactose, arabinose, and often rhamnose and glucuronic acid.

Each HRGP possesses its own unique Hyp-glycoside profile based on the number of Hyp residues glycosylated and the type of carbohydrate side chains. It is therefore of interest to know what directs the glycosylation. Do the Hyp glycosyltransferases recognize precise sequences or a general conformation?

Previously identified peptide sequences and corresponding Hyp-glycoside profiles of selected HRGPs indicated that arabinosylation is correlated with Hyp contiguity. This led to the Hyp contiguity hypothesis (1) that predicts arabinosylation of contiguous Hyp residues (8) and, as its corollary, the galactosylation of clustered noncontiguous Hyp residues, hence the view of an HRGP as an assemblage of glycomodules. These are small, repetitive functional units putatively involved in molecular recognition and wall self-assembly, ultimately contributing to higher level functions like cell wall porosity, tensile strength, and cell extension.

Examples of contiguous Hyp arabinosylation range from dipeptidyl Hyp in the Pro-Hyp-Hyp-Val-Tyr-Lys repetitive glycomodule of a PRP (8, 9) to the tetraHyp blocks in the Ser-Hyp4 glycomodule of extensins (10, 11).

Until recently, evidence for polysaccharide addition to clustered noncontiguous Hyp, typified by the Xaa-Hyp-Xaa-Hyp motif of AGPs, was only correlative. Therefore, as a direct test we constructed two synthetic genes encoding simple glycomodule repeats for stable expression in transgenic cultures of tobacco cells (12). The first result confirmed the predicted O-Hyp galactosylation in the repetitive sequence (Ser-Hyp)32; there was exclusive addition of arabinogalactan polysaccharide to all of the Hyp residues yielding a hyperglycosylated neo-AGP that coprecipitated with the AGP-specific Yariv reagent, thus supporting the glycomodule status of repetitive Ser-Hyp. The second synthetic gene showed that the introduction of contiguous Hyp between the glycomodules of clustered noncontiguous Hyp also introduced arabinosides (12).

To further test the predictive value of the Hyp contiguity hypothesis and the likelihood that other small, conserved repeats direct Hyp glycosylation, such as the commonly occurring Xaa-Hyp-Hyp motif of many AGPs, we designed another set of synthetic genes encoding the putative glycomodules. Here we describe the construction and expression of synthetic genes encoding the repetitive series: Ser-Pro-Pro (SP2), Ser-Pro-Pro-Pro (SP3), and Ser-Pro-Pro-Pro-Pro (SP4), assuming that, targeted for secretion, they would be post-translationally hydroxylated in tobacco cells. Arabinosylation of about half of the Hyp residues in the dipeptidyl Hyp blocks and almost 100% of the Hyp of the tetraHyp blocks confirmed the predictive value of this simple contiguity code. However, the repetitive SP3 motif gave an expression product, nominally Ser-Hyp-Hyp-Hyp, but with incompletely hydroxylated Pro residues, which resulted in a mixture of contiguous and noncontiguous Hyp residues. Consistent with the Hyp contiguity hypothesis, the corresponding Hyp-glycoside profile contained both Hyp-arabinosides and Hyp-polysaccharides. Furthermore, circular dichroic spectra of the glycosylated and deglycosylated modules suggested that Hyp arabinosides facilitate the polyproline II conformation of HRGPs, whereas Hyp polysaccharides favor a less ordered conformation.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Synthetic Gene and Plasmid Construction-- Construction of a given synthetic gene involved three sets of partially overlapping, complementary oligonucleotide pairs (Fig. 1) polymerized as described earlier (12, 13). The entire signal sequence-synthetic gene-enhanced green fluorescence protein (EGFP) constructs were then subcloned into the plant vector pBI121 (CLONTECH), as BamHI-SstI fragments in place of the glucuronidase reporter gene. All constructs were under control of the 35 S cauliflower mosaic virus promoter. The oligonucleotides were synthesized by Life Technologies (Grand Island, NY) and by Integrated DNA Technologies (Coralville, IA). DNA sequencing was performed at the Ohio Agricultural Research and Development Center, The Ohio State University Wooster campus.



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Fig. 1.   Oligonucleotide sets used to construct the synthetic genes encoding (A) SP2-EGFP and (B) SP3-EGFP, and SP4-EGFP. Internal repeat oligonucleotide sets were polymerized head-to-tail in the presence of the 5'-end linker sets. After ligation, the 3'-end linker sets were added. The genes were first subcloned into pUC18 as BamHI-SacI fragments (the restriction sites are boldfaced and italicized) and subsequently subcloned as XbaI-NcoI fragments into a pUC18-derived plasmid between a tobacco extensin signal sequence and the enhanced green fluorescence protein gene (EGFP, CLONTECH) as described earlier (12).

Agrobacterium and Tobacco Cell Transformation and Selection of Cell Lines-- The pBI121-based plasmids containing the synthetic gene constructs were delivered into Agrobacterium tumefaciens strain LBA4404 by the freeze-thaw method (14), then suspension-cultured tobacco cells (Nicotiana tabacum, BY2) were transformed with the Agrobacterium as described earlier (15). Transformed cells were grown on solid and liquid Schenk-Hildebrandt medium and selected for kanamycin resistance (12). EGFP fluorescence was visualized using a Molecular Dynamics Sarastro 2000 confocal laser-scanning fluorescence microscope equipped with an fluorescein isothiocyanate filter set comprising a 488-nm laser wavelength filter, a 510-nm primary beam splitter, and a 510-nm barrier filter.

Isolation of the Fusion Glycoproteins-- Culture medium from transformed cells was harvested 16-21 days after subculture, concentrated by rotary evaporation, then dialyzed against water and concentrated again by rotary evaporation. Sodium chloride was added to a 2 M final concentration and 40-50 ml of the medium was injected onto a hydrophobic-interaction chromatography column (Phenyl-Sepharose 6 Fast Flow, 16 × 700 mm, Amersham Pharmacia Biotech) equilibrated in 2 M sodium chloride. We used a decreasing sodium chloride step gradient (2, 1, and 0 M, 150 ml of each) to elute the column at a flow rate of 1.3 ml/min. Collected fractions (4 ml) were monitored for fluorescence by a Hewlett-Packard 1100 Series flow-through fluorometer (488-nm excitation; 520-nm emission) or by a hand-held UV lamp (365 nm). The fluorescent fractions were pooled, concentrated by freeze-drying, redissolved in 1-2 ml of water, and injected onto a Hamilton semipreparative polymeric reverse phase column (10-µm PRP-1, 7 × 305 mm) equilibrated with start buffer (0.1% aqueous trifluoroacetic acid). Proteins were gradient-eluted in 0.1% trifluoroacetic acid/80% (v/v) aqueous acetonitrile (0-70%/120 min) at a flow rate of 0.75 ml/min. The fusion glycoprotein (Ser-Hyp)32-EGFP and endogenous tobacco AGPs were isolated as described earlier (12). Protein sequence analysis was performed at the Michigan State University Macromolecular Facility on a 477-A Applied Biosystems Inc. gas phase sequencer.

Coprecipitation with beta -Glucosyl Yariv Reagent-- We assayed the ability of the fusion glycoproteins, as well as earlier reported (Ser-Hyp)32-EGFP and tobacco AGPs (12), to coprecipitate with the beta -glucosyl Yariv reagent (16). Absorbency was read at 420 nm.

Carbohydrate Analyses-- Hyp-glycoside profiles were determined on 2-4 mg of isolated fusion glycoprotein as described earlier by Lamport and Miller (17) and Shpak et al. (12). We monitored the automated postcolumn hydroxyproline assay at 560 nm. Neutral sugars were analyzed as alditol acetates (18) by gas chromatography using a 6-foot × 2-mm polyethylene glycol succinate 224 column programmed from 130 to 180° at 4 °C/min for neutral sugars. Data capture was achieved by Hewlett-Packard Chem station software. One hundred micrograms of glycoprotein was used for each analysis. We assayed the uronic acid content of 70 µg of each fusion glycoprotein via the specific colorimetric assay based on reaction with m-hydroxydiphenyl (19). Galacturonic acid was the standard.

Pronase Digestion of the Fusion Glycoproteins-- Each fusion glycoprotein (10-20 mg, 10 mg/ml aqueous) was heat-denatured in boiling water for 2 min, cooled, and then incubated in an equal volume of freshly prepared 2% (w/v) ammonium bicarbonate containing 2.5 mM calcium chloride and Pronase (substrate:enzyme ratio 100:1, w/w). The digestion proceeded at room temperature overnight, then the peptides were freeze-dried, dissolved in 0.5 ml of Superose buffer, and separated via semipreparative Superose-12 gel filtration (see below).

Superose-12 Gel Filtration Chromatography of Tobacco Cell Medium and Proteolyzed Fusion Glycoproteins-- We injected 0.5 ml of tobacco medium from transformed cells (three lines of each construct) onto a Superose-12 analytical gel filtration column (10 × 300 mm) eluted at a flow rate of 0.2 ml/min and monitored via flow-through detection by a Hewlett-Packard 1100 series fluorometer (excitation, 488 nm; emission, 520 nm). The column was calibrated with molecular weight standards (bovine serum albumin, insulin, catalase, and sodium azide) and absorbency was read at 220 nm. Pronased fusion glycoproteins were injected onto a semipreparative Superose-12 column (16 × 500 mm) and eluted at a flow rate of 1 ml/min. For both the analytical and semipreparative Superose-12 columns, the elution buffer was 0.2 M sodium phosphate (pH 7.0) containing 0.01% sodium azide.

Anhydrous Hydrogen Fluoride (HF) Deglycosylation-- We deglycosylated 4-5 mg of each fusion glycoprotein for 1 h in anhydrous HF as described earlier (20). The proteins were dialyzed against deionized, distilled water for 2 days at 4 °C and then freeze-dried. The glycomodules were isolated from Pronase digestions of the fusion glycoproteins as described above, then HF-deglycosylated 1 h at 4 °C. The HF was blown off under nitrogen gas, and the deglycosylated modules (designated dSP2, dSP3, dSP4) were then rerun on the reverse-phase column as described above.

Circular Dichroism (CD)-- We recorded CD spectra of poly-L-hydroxyproline (5-20 kDa, Sigma Chemical Co.) and the isolated glycoprotein modules before and after deglycosylation on a Jasco-715 spectropolarimeter (Jasco Inc., Easton, MD). Spectra were averaged over two scans with a bandwidth of 1 nm, and step resolution was 0.1 nm. All spectra are reported in terms of mean residue ellipticity with the 180- to 250-nm region using a 1-mm pathlength. The modules were dissolved in water at the following concentrations: poly-L-hydroxyproline, 18.4 µM; SP, 9.2 µM; SP2, 18.4 µM; SP3, 19.6 µM; and SP4, 30.4 µM. Spectra were obtained from 18.4 µM of each deglycosylated module.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Synthetic Gene and Plasmid Construction-- We built three plasmids, each encoding a tobacco signal sequence, a synthetic gene, and EGFP placed in one transcriptional frame. The synthetic genes encoded an SP2-EGFP fusion protein containing 24 Ser-Pro2 repeats, an SP3-EGFP glycoprotein containing 15 Ser-Pro3 repeats, and an SP4-EGFP glycoprotein containing 18 Ser-Pro4 repeats. For brevity, from here forward the fusion glycoproteins encoded by the synthetic genes will be referred to as SP2-EGFP, SP3-EGFP, and SP4-EGFP, although most Pro residues were hydroxylated in the glycoproteins. The earlier reported fusion glycoprotein (Ser-Hyp)32-EGFP (12) will be referred to as SP-EGFP.

Tobacco Cell Transformation-- Agrobacterium-mediated transformation of tobacco cell cultures gave stably transformed lines, judging by the fact that the cells continue to produce the gene products more than 1 year after transformation. We isolated three lines each of SP2-EGFP, SP3-EGFP, and SP4-EGFP by observing the characteristic fluorescence of EGFP in both the growth medium and the cytoplasm of the transformed cells (not shown), which was similar to that published earlier (12). The three lines of each construction were assayed for the fusion glycoproteins by Superose-12 gel permeation chromatography. All lines produced fluorescent products that coeluted with the products visualized in Fig. 2. Two lines of each construction were selected for further characterization of the fusion glycoproteins by Hyp-glycoside profiles. One line of each construction was characterized by protein sequence analysis, amino acid composition, neutral sugar analyses, and circular dichroism. Yields of purified glycoproteins from the most productive cell lines were: SP2-EGFP: 10 mg/liter; SP3-EGFP: 36 mg/liter; SP4-EGFP: 23 mg/liter.



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Fig. 2.   Superose-12 gel permeation chromatography with fluorescence detection of concentrated culture medium containing the fusion glycoproteins. Gel permeation chromatography on analytical Superose-12 corroborated the predicted glycosylation of the fusion proteins in the growth medium (A-D) as they eluted much earlier than predicted for the nonglycosylated products, which would have eluted near the EGFP standards (E and F) (i.e. EGFP is 27 kDa and the synthetic genes sans carbohydrate were estimated to be 34-37 kDa). Depending on the transgene expressed, the glycoproteins produced by separate culture lines yielded products that co-chromatographed. Controls included 10 µg of EGFP standard from CLONTECH (F) and EGFP targeted to the tobacco extracellular space by the tobacco extensin signal sequence (E).

Coprecipitation with Yariv Reagent-- The Yariv reagent did not precipitate SP2-EGFP or SP4-EGFP, but it did precipitate SP3-EGFP, although to a much lesser extent than endogenous tobacco AGPs or SP-EGFP (Table I).


                              
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Table I
Coprecipitation of the Ser-Pron-EGFP fusion glycoprotein series with Yariv reagent
The glycoproteins were precipitated with Yariv reagent, then the precipitate was dissolved in base and the absorbance was measured.

Carbohydrate Analyses-- Hyp-glycoside profiles of SP2-EGFP, SP3-EGFP, and SP4-EGFP showed that, although all the glycoproteins contained Hyp-arabinosides, SP3-EGFP glycoprotein also contained Hyp-arabinogalactan polysaccharides (Table II). Arabinose was the only saccharide component of SP2-EGFP and the major saccharide component of SP4-EGFP, which contained a small amount of galactose as well (Table III). In contrast, the SP3-EGFP contained mainly galactose and arabinose, with lesser amounts of rhamnose, glucose, and uronic acid (Table III). Saccharide accounted for 17% of SP2-EGFP, 40% of SP3-EGFP, and 41% of SP4-EGFP on a dry weight basis.


                              
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Table II
Hyp-glycoside profiles of recombinant proteins SP2-EGFP, SP3-EGFP, and SP4-EGFP
The fusion glycoproteins isolated from each of two lines (A and B) were characterized.


                              
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Table III
Glycosyl composition of fusion glycoproteins SP-EGFP, SP2-EGFP, SP3-EGFP, and SP4-EGFP

Anhydrous HF Deglycosylation-- Weight loss after HF deglycosylation agreed with the sugar analyses. Thus, from 3.9 mg of SP2-EGFP we recovered 3.3 mg of deglycosylated SP2-EGFP (15% weight loss); from 4.8 mg of SP3-EGFP we recovered 2.6 mg of deglycosylated SP3-EGFP (46% weight loss); and 3.9 mg of SP4-EGFP yielded 2.2 mg of deglycosylated SP4-EGFP (44% weight loss).

Amino Acid Analysis and Sequence Analysis-- Amino acid analyses (Table IV) showed that almost all the proline of the SP2 and SP4 glycomodules had been hydroxylated to form Hyp. However, 27% of the proline residues (20 mol % of the amino acids) in the SP3 glycomodule remained nonhydroxylated. Edman degradation (Fig. 3) confirmed the identity of the gene products.


                              
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Table IV
Amino acid compositions of isolated glycomodules SP2, SP3, and SP4



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Fig. 3.   N-terminal partial amino acid sequences of SP2-EGFP, SP3-EGFP, and SP4-EGFP. A, SP2-EGFP contained 47 Hyp residues and 3 Pro residues, one of which occurred at the N terminus judging by this sequence as well as the gene sequences (not shown) and the amino acid composition of the module (Table IV). B, Edman degradation of SP3-EGFP indicated incomplete hydroxylation at each Pro residue except at the extreme N terminus. Thus the SP3-EGFP was a population of molecules containing a mixture of contiguous and noncontiguous Hyp residues. X denotes a blank cycle that yielded no signal during Edman degradation. The SP3 glycomodule contained about 28 Hyp residues and 22 Pro residues overall. C, Edman degradation of SP4-EGFP indicated the N terminus was completely hydroxylated and the amino acid composition of the isolated SP4 module indicated it contained 74 Hyp residues and 2 Pro residues.

Calculated Molecular Masses of the Fusion Glycoproteins SP2-EGFP, SP3-EGFP, and SP4-EGFP-- Both SDS-polyacrylamide gel electrophoresis and gel permeation chromatography tend to overestimate HRGP molecular mass due to the biased amino acid compositions, extended structures, and extensive glycosylation (21). Therefore, we calculated the molecular masses of our fusion glycoproteins based on the gene sequences of the constructions (not shown) and their Hyp-glycoside profiles (Table II), amino acid compositions (Table IV), neutral sugar analyses (Table III), and protein sequences (Fig. 3). We calculated the following masses for the glycosylated and deglycosylated proteins: glycosylated SP2-EGFP, 43.9 kDa; dSP2-EGFP, 34.8 kDa; glycosylated SP3-EGFP, 63-77 kDa; dSP3-EGFP, 33.9 kDa; glycosylated SP4-EGFP, 66.4 kDa; and dSP4-EGFP, 37.3 kDa.

Circular Dichroism of the Glycosylated and Deglycosylated Modules-- CD spectra of the deglycosylated modules (Fig. 4A) show that both negative (~206 nm) and positive (~226 nm) ellipticities increased in the order dSP (light blue) < dSP2 (yellow) < dSP3 (lavender) < dSP4 (dark green). The glycosylated SP2 and SP3 modules (orange and dark blue, respectively) had virtually identical spectra (Fig. 4B). Glycosylated SP had a "random coil" conformation (Fig. 4, B and C, red), which adopted more structure after deglycosylation (Fig. 4C, light blue). The negative ellipticities of SP2 and SP4 increased markedly and shifted to a lower wavelength (~201 nm) when arabinosylated (Fig. 4, D and F, orange and light green, respectively), whereas SP3 showed virtually no change in conformation after deglycosylation (Fig. 4E, dark blue). Samples containing Hyp-polysaccharide substituents had a second minimum at 180 nm (Fig. 4, B, C, and E, red and dark blue).



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Fig. 4.   CD spectra of isolated SP, SP2, SP3, and SP4 modules before and after deglycosylation. The glycomodules were isolated from Pronase digestions of the intact fusion glycoproteins. The black curve in each panel with a minimum at 206 nm and a maximum at 225 nm corresponds to the standard, polyhydroxyproline in the polyproline II conformation, a left-handed helix with 3 residues/turn, and a pitch of 0.94 nm (50). A, the amount of polyproline II conformation in the deglycosylated modules increased with increased Hyp contiguity. The colored lines correspond to the deglycosylated modules as follows: dSP, light blue; dSP2, yellow; dSP3, lavender; and dSP4, dark green. B, glycosylated SP, red; SP2, orange; SP3, dark blue; and SP4, light green. C, a comparison of SP (red) to dSP (light blue); D, a comparison of SP2 (orange) to dSP2 (yellow); E, a comparison of SP3 (dark blue) to dSP3 (lavender); and F, a comparison of SP4 (light green) to dSP4 (dark green). The spectra shown in B, D, and F indicate that arabinosylation of SP2 and SP4 deepened the minimum and shifted it from 206 to 201 nm, suggesting that the arabinosides interact with the polypeptide backbone, altering the polyproline II conformation. Surprisingly, deglycosylated SP (C) has more "structure" than glycosylated SP. Thus, arabinogalacatan polysaccharide addition to each Hyp in the SP module favors a "random coil conformation" rather than a polyproline II conformation enhanced by Hyp arabinosylation. The addition of both arabinosides and arabinogalactan polysaccharides to SP3 (E) had little effect on the conformation of SP3, presumably due to the opposing conformational effects of the two different saccharide substituents.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

HRGP glycosylation involves proline-rich sequences targeted to the endoplasmic reticulum/Golgi for initial cotranslational hydroxylation of the proline residues followed by O-Hyp glycosylation. Generally, the multiplicity and similar properties of HRGPs make them difficult to purify. However, addition of the hydrophobic fluorescent EGFP reporter protein facilitated the chromatographic separation of HRGP-EGFP fusion proteins from the other endogenous, hydrophilic HRGPs. Thus, the design of new HRGPs composed of single glycomodule repeats and expressed as HRGP-EGFP fusion proteins provided bulk quantities of the glycomodules for structural analysis. This approach has allowed us to elucidate the major determinants of Hyp glycosylation and currently is enabling us to determine the detailed structures of the glycosyl substituents. Moreover, as endogenous substrates, these new HRGPs containing only simple repeats can define the enzymic specificity of prolyl hydroxylase(s) and glycosyltransferases under in vivo conditions and prior to isolation of the enzymes.

Because proline/hydroxyproline-rich polypeptides often adopt characteristic conformations (Fig. 4) one must ask whether it is primarily peptide conformation or peptide sequence that directs hydroxylation and glycosylation.

Prolyl hydroxylase does not hydroxylate all HRGP proline residues, the most notable example being the repetitive "insertion sequence" Val-Lys-Pro-Tyr-His-Pro of tomato P1 extensin. Indeed, in all known HRGP sequences, Lys-Pro always occurs nonhydroxylated, whereas His-Pro is hydroxylated in some HRGPs (22) but not in others (23), and Pro-Val is invariably hydroxylated (1). The Pro residues in the SP2 and SP4 modules (Fig. 3 and Table IV) show almost complete hydroxylation of Pro residues. However, incomplete and inconsistent hydroxylation of SP3 proline residues indicates that the prolyl hydroxylase of Nicotiana has a low affinity for a tripeptidyl proline substrate. Not surprisingly, such sequences are rare in Nicotiana (24), although common in other species; for example, repetitive Ser-Hyp3 occurs in the gum arabic glycoprotein of Acacia senegal (25) and in maize extensins (26). Likewise, Ser-Pro3 motifs that may be completely hydroxylated occur frequently in potato cDNAs (27) and in an Arabidopsis extensin gene (28).

The above observations are consistent with sequence-specific hydroxylation rather than the earlier view of plant prolyl hydroxylase as specific for the polyproline II conformation (29). This included protocollagen (30), which has a polyproline II conformation, although collagen expressed in transgenic tobacco was not hydroxylated (31). Plant prolyl hydroxylase is therefore similar to that of animals in being sequence-specific, although it hydroxylates distinctly different sequences. It is also possible that plants, like some animals, have multiple prolyl hydroxylases (1) possessing a catalytic domain separate from a sequence-specific peptide substrate binding domain (32).

Other factors may also influence proline hydroxylation of a given substrate. These may include differences in the enzyme specificity of different plant species or the targeting of potential substrates to specific endoplasmic reticulum subdomains, as reported for rice storage proteins (33). This may explain why the repetitive Pro-Pro-Pro-Val-His-Leu motif of zein, the maize endosperm seed storage protein that shares sequence identity with the PRP HRGPs, is not hydroxylated (34).

Although the data show that Hyp is the major glycosylation site of HRGPs, it does not rule out the glycosylation of other hydroxyamino acids. Certainly single residues of galactose occur as O-galactosylserine in the Ser-Hyp4 glycomodules of extensin HRGPs (10, 35) and may account for the small amount of galactose in the SP4 glycoprotein (Table III), which contains Hyp-arabinosides but no Hyp-polysaccharide. Speculatively, one can suggest that, in concert with the Hyp-arabinosides, galactosylserine stabilizes the Ser-Hyp4 glycomodule.

Serine is often considered as a polysaccharide attachment site in the AGPs (36). Although some of the evidence is strongly suggestive (22, 37), it is not definitive (38) and is sometimes contradictory (38-41). On the other hand, addition of arabinogalactan polysaccharide to the clustered noncontiguous Hyp residues of (Ser-Hyp)n, as previously demonstrated (12), is a natural corollary of the Hyp contiguity hypothesis. Other clusters can also be defined by the design of synthetic gene constructs that test the efficacy of AGP motifs, such as (Ala-Pro)n, (Thr-Pro)n, and (Ala-Thr-Pro)n, to direct polysaccharide addition. Identification of a sequence motif that directs polysaccharide addition to serine or threonine residues might also be a possible outcome of this work in progress.

Because hydroxyproline residues can exist in any one of three states, nonglycosylated, arabinosylated, and galactosylated, a sequence code seems more likely than a purely conformational control of glycosylation. The Hyp contiguity hypothesis predicts the arabinosylation of contiguous Hyp residues, where contiguity begins with dipeptidyl Hyp (8), confirmed here by the arabinosylation mainly of a single Hyp residue of each SP2 module (Table II). Interestingly, not only the number of arabinosylated Hyp residues increase with the size of the contiguous Hyp block (about 50% in Ser-Hyp2 and nearly 100% in Ser-Hyp4), but also the size of the attached arabinooligosaccharide. Thus, the small amounts of penta-arabinoside occasionally reported (42) might be attributable to the uncommon Ser-Hyp5-6 motif.

Although both the SP3 and SP4 modules were arabinosylated, there was anomalous addition of polysaccharide to SP3, apparently due to incomplete hydroxylation of the proline residues resulting in some clustered noncontiguous Hyp. However, the polyproline II content of deglycosylated SP3 is higher than that of the deglycosylated SP2 and much higher than that of the deglycosylated SP (Fig. 4A). But SP3 contains both Hyp-Ara and Hyp-Gal, whereas SP2 contains Hyp-Ara exclusively, and SP (with the lowest polyproline II content) contains Hyp-Gal exclusively. Thus the CD data support sequence-directed Hyp-glycosylation rather than a simple conformational control based on polyproline II content.

The putative Hyp glycosyltransferases clearly distinguish between arabinosylation of contiguous Hyp and galactosylation of clustered noncontiguous Hyp residues. Thus it is now possible to predict the approximate glycosylation profile of an HRGP based on its primary structure, a crucial step toward predicting three-dimensional native structure. However, two exceptions suggest that certain flanking sequences may modify the simple code by suppressing the glycosylation of typical AGP motifs that occur in extensins. For example, the THRGP extensin isolated from maize contains no detectable Hyp arabinogalactan polysaccharide (43) but does have a prominent AGP motif flanked by residues that are either bulky, charged, or aromatic: Thr/Tyr-Thr-Hyp-Ser-Hyp-Lys/Pro (44). Similarly, an extensin isolated from the gymnosperm Douglas fir contained two AGP motifs, both flanked by bulky, charged, or aromatic residues: Hyp/Hyp-Ala-Hyp-Thr-Hyp-Val/Val and Lys/Pro-Ala-Hyp-Ala-Hyp-Tyr/Tyr (11); however, the glycoprotein contained no arabinogalactan polysaccharide.

The (Ser-Pro1-4)n series results in the addition of two very different types of substituent. Hydroxyproline arabinosides have a relatively simple linear structure (45) in contrast to the hydroxyproline arabinogalactan polysaccharide (often described as "highly branched") that consist of a beta -1right-arrow3-linked galactose backbone with short side chains, typically a pentasaccharide (7) attached by 1right-arrow6-linkages to the main chain (6). The role of these substituents is also quite different.

A monotonic increase in Hyp/Pro contiguity, represented by the SP, SP2, SP3, and SP4 repetitive modules gave a simple monotonic increase in polyproline II secondary structure, judging from the CD spectra of the deglycosylated modules (Fig. 4A). However, CD spectra of the intact glycomodules were more complicated. Addition of arabinosides to SP2 and SP4 (Fig. 4, D and F) increased their polyproline II helix content and included a shift of the minimum to a lower wavelength (Fig. 4, B, D, and F). This is particularly evident in SP4 and indicates that a direct interaction between arabinooligosaccharides and the polypeptide backbone facilitates the polyproline II conformation as a slightly modified version.

In contrast, Hyp-polysaccharide addition led to a more random coil structure for the SP glycomodule, whereas the addition of both arabinoside and polysaccharide to the SP3 glycomodule did not affect the polyproline II content, presumably because Hyp-arabinosylation favors polyproline II formation whereas the Hyp-polysaccharide opposes it. Polysaccharide addition also resulted in spectra having a new minimum at 180 nm (Fig. 4, B, C, and E) contributed by the polysaccharide substituent itself (Fig. 4).

Overall, these CD data confirm that Hyp-arabinosides enhance the polyproline II helix in the Ser-Hyp4 glycomodules of extensin HRGPs (5, 46, 47) as they do in the Ser-Hyp2 and Ser-Hyp4 modules here. Presumably, this underlies the contribution of extensin to the tensional integrity of the cell wall itself. On the other hand, judging from the CD spectra, Hyp-polysaccharide does not contribute appreciably to the polypeptide conformation, although the 1right-arrow6-linked side chains should greatly enhance the water-holding capacity of AGPs (48). Hyp-polysaccharide is also a prominent constituent of glypiated AGPs anchored to the plasma membrane where they make a major contribution (49) as a periplasmic hydrophilic buffer between plasma membrane and cell wall.


    ACKNOWLEDGEMENTS

We thank Li Tan for help with protein purifications and Dr. Derek Lamport for comments on the manuscript.


    FOOTNOTES

* This work was supported by Grant MCB-9874744 from the National Science Foundation and by grants from The Ohio Plant Biotechnology Consortium and The Ohio University Early Stage Development Fund.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Chemistry and Biochemistry, Ohio University, Clippinger Laboratories, Athens, OH 45701. Tel.: 740-593-9466; Fax: 740-593-0148 (ext. 4795); E-mail: kielisze@helios.phy.ohiou.edu.

Published, JBC Papers in Press, January 11, 2001, DOI 10.1074/jbc.M011323200


    ABBREVIATIONS

The abbreviations used are: HRGPs, hydroxyproline-rich glycoproteins; Hyp, hydroxyproline; PRPs, proline-rich proteins; AGPs, arabinogalactan proteins; CD, circular dichroism; EGFP, enhanced green fluorescence protein; BY2, Bright Yellow 2; SPn, Ser-Pron.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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