©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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 (*)

(Received for publication, September 8, 1994; and in revised form, November 22, 1994)

Marcia J. Kieliszewski (1) (2)(§) Malcolm O'Neill (1) Joseph Leykam (3) Ron Orlando (1) (2)

From the  (1)Complex Carbohydrate Research Center and (2)Biochemistry Department, University of Georgia, Athens, Georgia 30602 and the (3)Biochemistry Department, Michigan State University, East Lansing, Michigan 48824

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
CONCLUSIONS
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

Hydroxyproline O-glycosylation is a posttranslational modification unique to plants and Chlorophycean algae(1, 2) . The hydroxyproline-rich glycoproteins (HRGPs) (^1)so modified occur predominantly at the cell surface, where they are implicated in diverse roles ranging from network formation (3, 4) and disease resistance (5, 6) to cell differentiation and morphogenesis(7, 8) . HRGPs are characteristically extended rodlike molecules(9, 10, 11, 12, 13) with highly repetitive peptide motifs extensively posttranslationally modified by hydroxylation of proline(3, 14) , O-glycosylation of serine and hydroxyproline residues(15, 16) , and both intra- and intermolecular cross-linking(4, 17) . Thus, questions about HRGP function must deal not merely with the primary amino acid sequence as deduced from clones, but also with the mature glycoprotein whose three-dimensional structure, conformation, and arrangement in the cell wall all depend on extensive posttranslational modifications. Of these, hydroxyproline O-glycosylation figures prominently, accounting for up to 95% of an HRGP's dry weight (13, 14, 18, 19, 20, 21) , and ranges from the addition of a single arabinose or galactose residue up to a 75-residue arabinogalactan(2, 13, 18, 19) .

Most commonly, hydroxyproline substituents are short arabinofuranoside chains (degree of polymerization: 1-5 residues), usually isolated as hydroxyproline arabinosides (Hyp-arabinosides); they occur in every type of HRGP examined thus far, including the Ser-Hyp(4)-containing extensins(1, 2, 10, 20) , arabinogalactan-proteins (AGPs)(2, 18, 22) , gum arabic glycoprotein (13) , repetitive proline-rich proteins (RPRPs)(12, 14, 21) , and the solanaceous lectins(23, 24) . Each HRGP possesses its own unique Hyp-arabinoside profile based on the number of substituted Hyp residues and the relative proportion of each oligoarabinoside chainlength. Hence, four questions arise about the site specificity of Hyp-arabinosylation in any given HRGP: what determines (a) the total number of Hyp residues substituted, (b) the size of each oligoarabinoside substituent, (c) the precise arrangement of the different oligoarabinosides, and (d) the positions of the Hyp-arabinosides relative to the larger arabinogalactan polysaccharide substituents of the AGPs and gums? Elucidation of the rules for HRGP O-glycosylation will help enable us to predict mature glycoprotein structure from genomic/cDNA sequences and help us understand how glycosylation contributes to HRGP molecular topography and, hence, to the possible roles of HRGPs in molecular recognition and wall self-assembly.

Recently, we discovered that Hyp-arabinosylation is not random, but correlated with the contiguity of the Hyp residues(21) . Thus, the tetrahydroxyproline blocks of the Ser-Hyp(4)-containing extensins represent highly contiguous Hyp that is also highly arabinosylated mostly with the larger (3-4 residues) rather than the smaller (1-2 residues) oligoarabinosides. RPRPs represent the other extreme, where Hyp residues are largely interspersed with other residues, i.e. there is little contiguous Hyp (25) and correspondingly little arabinosylated Hyp(14, 21) .

Thus the Hyp contiguity hypothesis approaches the problem of Hyp arabinosylation coding by predicting that Hyp arabinosylation increases with Hyp contiguity rather than with Hyp mole percent, and that noncontiguous Hyp residues are rarely, if ever, arabinosylated. In order to test and further refine this hypothesis, we set out to determine the arabinosylation site specifics for one of the simpler cases of HRGP glycosylation, notably the proline-rich HRGP (PHRGP) from Douglas fir (Pseudotsuga menziesii). Its Hyp residues are only lightly arabinosylated, arabinogalactan polysaccharide is absent, and the polypeptide backbone is largely made up of the simple tandem repeat: Lys-Pro-Hyp-Val-Hyp-Val-Ile-Pro-Pro-Hyp-Val-Val-Lys-Pro-Hyp-Hyp-Val-Tyr (21) , with low Hyp contiguity.

Unique problems arise, however, in attempts to identify the precise sites of Hyp-arabinosylation as these involve a base-stable linkage to a -carbon(1, 2, 15) , which is unlike glycosylation of seryl or threonyl residues, whose base-labile beta-linkage permits beta-elimination of the carbohydrate with the concomitant conversion of Ser or Thr to another amino acid(26) . Nor can direct Edman degradation determine the site specifics of arabinosylation, for unlike other glycosylated amino acid residues, which can often be inferred from blank cycles(27) , the trifluoroacetic acid-phenylthiohydantoin cleavage step hydrolyzes the labile arabinofuranosides removing the distinction between arabinosylated and nonarabinosylated Hyp. These complications led us to consider using tandem mass spectrometry (MS/MS) to elucidate the arabinosylation site specifics of the PHRGP.

MS/MS using collisionally induced dissociation (CID) has become increasingly popular for characterizing the posttranslational modifications of proteins and peptides(28, 29, 30, 31, 32, 33) ; however, it also has drawbacks, as the more hydrophilic components of a sample, such as glycopeptides, frequently are not detected due to a low ion yield (32, 33, 34) . Furthermore, glycopeptides preferentially cleave at O-glycosidic bonds, while the peptide backbone remains intact(29, 33) , which precludes the precise identification of glycosylation sites when more than one potential glycosylation site is present. Thus, the sequencing of glycopeptides by MS/MS typically requires chemical degradation or modification of the glycopeptide before analysis. Recently, however, Medzihradszky et al.(29) reported that MS/MS identified both the peptide sequence and carbohydrate attachment site of a purified, underivatized glycopeptide containing a single hexose residue. This demonstrated the potential of MS/MS for the structural characterization of intact, underivatized glycopeptides, which we have extended to the unfractionated Pronase digests of the gymnosperm glycoprotein, PHRGP.


MATERIALS AND METHODS

Preparation of PHRGP from Douglas Fir Suspension Cultures

We prepared PHRGP from suspension-cultured cells of Douglas fir as described previously(21) .

Deglycosylation of PHRGP with Anhydrous Hydrogen Fluoride (HF)

We deglycosylated 500 µg of PHRGP (henceforth dPHRGP) with HF using methods described previously(24, 35) .

Digestion of Glycosylated PHRGP with Pronase

PHRGP (4-55 mg quantities) was digested overnight with Pronase (2% sodium bicarbonate, pH 8, 10 mM CaCl(2); substrate:enzyme ratio, 100:1). Aliquots of the digest were freeze-dried and then directly analyzed by electrospray ionization and fast atom bombardment mass spectrometry.

Hydrophilic Interaction Liquid Chromatography (HILIC)

We fractionated PHRGP Pronase digests on a polyhydroxyethyl A column (200, inner diameter, times 9.4 mm; 30 nm pore, PolyLC) at 0.75 ml/min, using gradient elution with the mobile phase solvents A (30 mM triethylamine/phosphoric acid buffer (TEAP), pH 3.4, 40% (v/v) aqueous acetonitrile) and B (30 mM TEAP, pH 3.4). The gradient began at 100% A and increased to 50% B in 40 min. The arabinosides were unstable during concentration in the TEAP buffer; therefore, after elution of each glycopeptide from the column, we stabilized the arabinosides by precipitating the phosphoric acid with saturated Ba(OH)(2) (cf.Table 1, columns 1-3).



Peptide and Glycopeptide Purification by Reverse Phase Liquid Chromatography (RPLC)

The HILIC-fractionated peptides and glycopeptides were purified on a Hamilton polymeric reversed-phase column (PRP-1; 4.1, inner diameter, times 150 mm) using conditions described previously(21) . Hyp-arabinoside profiles indicated the arabinosides were stable in the 0.1% trifluoroacetic acid buffers used for these experiments (data not shown).

Amino Acid Composition and Sequence Analysis of the PHRGP Peptides and Glycopeptides

We determined amino acid compositions of the peptides and glycopeptides using methods described previously (21) . Peptides were sequenced at the Michigan State University Biochemistry Department Macromolecular Facility on an Applied Biosystems, Inc. 477A gas phase sequencer.

Hydroxyproline Arabinoside and Neutral Sugar Analyses of the PHRGP and of the PHRGP Peptides and Glycopeptides

Hyp-arabinoside profiles and sugar compositions of the PHRGP glycoprotein and glycopeptides were determined by methods described previously (21, 36) .

Electrospray Ionization Mass Spectrometry (ESIMS) of the PHRGP Pronase Digest and of the Purified Glycopeptides

ESIMS spectra were acquired on an API-III Biomolecular analyzer (Perkin-Elmer Sciex Instruments) operated in the positive ion mode with an ion spray voltage of 5000 V and orifice potential of 35 V. Solutions of peptides or glycopeptides (1 µg/µl) in 50% aqueous acetonitrile containing 0.1% formic acid and 2 mM ammonium formate were introduced into the ES source at 2 µl/min using a Harvard 22 syringe infusion pump. The mass range was scanned from 100 to 1500 atomic mass units. Ten scans were collected and averaged. For ESIMS/MS the appropriate protonated molecular ion ([M + H]) was selected in the first quadrupole, then MS/MS spectra were obtained using CID with argon as the collision gas (collision gas thickness 300 times 10/cm^2) in the opened-structured quadrupole collision cell. The fragment ions generated by CID were separated in the third quadrupole. A total of 50-100 scans (100-1000 atomic mass units) were collected and averaged.

Continuous Flow-Fast Atom Bombardment Mass Spectrometry (CF-FABMS) of the PHRGP Pronase Digest

We acquired CF-FABMS and CF-FABMS/MS spectra with a JEOL HX/HX110A tandem four-sector mass spectrometer operated at 10 kV accelerating potential in combination with a JEOL complement data system. Ions were produced by bombardment with xenon using a JEOL FRIT-FAB ion source and a JEOL FAB gun operated at 6 kV. MS1 was calibrated with CsI. MS1 spectra were acquired from 300 to 1500 atomic mass units (scan rate: m/z 1-6000/min). The filtering rate was 300 Hz, and the resolution was 1000 m/Deltam. Samples were dissolved in a 1:1 mixture of water-methanol (1 mg/ml) containing 5% trifluoroacetic acid and 4% thioglycerol as the CF-FAB matrix. Samples were introduced into the FRIT-FAB source using a fused silica capillary tube (60 µm times 37 cm) at a flow rate of 2 µl/min. CID was performed in the third field free region using helium as the collision gas at a pressure that attenuated the primary ion beam by 75%. The collision cell was floated at 8 kV. Fragment ions were detected by a JEOL MS-ADS11 variable dispersion array detector(37) . A delay time of 100 ms was used between magnetic and electric field steps to allow stabilization of the magnetic field. MS 2 had an approximate resolution of 1000 m/Deltam, and was calibrated with a mixture of LiI, NaI, KI, RbI, and CsI.

Molecular Weight Determinations of Glycosylated and Deglycosylated PHRGP by Matrix-assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS)

We determined the molecular mass of the PHRGP and dPHRGP on a Hewlett Packard LDI 1700XP mass spectrometer operated at 30 kV accelerating voltage and a pressure of 6 times 10 torr. The mass spectrometer was calibrated with a mixture of equine heart cytochrome c (M(r) = 12,360), equine heart myoglobin (M(r) = 16,950) and bovine serum albumin (M(r) = 66,465) to give a mass accuracy of ± 0.2%. Samples were desorbed/ionized from the probe tip with a nitrogen laser ( = 337 nm) having a pulse width of 3 ns and delivering approximately 8 µJ of energy/laser pulse. Aqueous solutions of PHRGP and dPHRGP (10 mg/ml) were diluted 1:8 in aqueous 90% methanol containing 100 mM sinnapinic acid(38) . We vacuum-crystallized 1 µl of the sample/matrix solution onto the probe and then recorded mass spectra over a m/z range of 1-100,000 using a deflector to attenuate the abundance of ions below 10,000 m/z.


RESULTS AND DISCUSSION

Molecular Masses of PHRGP and dPHRGP

Recently, we demonstrated that PHRGP is a hydroxyproline-rich glycoprotein homologous with members of the extensin protein family in that it contains variations of the highly repetitive pentameric motif, Hyp/Pro-Hyp-Val-X-Lys, which characterizes the RPRPs and other extensins(14, 21, 25) . The repetitive PHRGP motifs form a longer 18-residue tandem repeat unit: Pro-Hyp-Val-Hyp-Val-Ile-Pro-Pro-Hyp-Val-Val-Lys-Pro-Hyp-Hyp-Val-Tyr-Lys, which is lightly glycosylated with short chains of arabinofuranosides O-linked to hydroxyproline.

SDS-PAGE gave a molecular weight of 97,400 for PHRGP(21) , while size exclusion chromatography gave a molecular mass of about 669 kDa(^2); however, neither of these methods provide reliable estimates of HRGP size, judging from visualization of HRGP monomers by electron microscopy and polypeptide size as deduced from cDNA clones(9, 13, 39, 40, 41) . Indeed, HRGPs behave as if they are much larger than the equivalent globular protein. Their asymmetric rodlike character arises from a high pyrrolidine ring content and is further reinforced by glycosylation (10, 13-16, 22-25, 42, 43). These conformational constraints maintain the extended conformation, which characterizes all HRGPs examined(10, 22, 39, 43, 44, 45) and probably explains their anomalous behavior on gel filtration(9, 13) . On the other hand, SDS-PAGE probably overestimates PHRGP molecular weight because Lys-rich polycations, such as HRGPs, reduce the overall negative charge due to bound SDS. Glycosylation may further contribute to this effect by sterically restricting the amount of peptide-bound SDS.

In contrast to these conventional methods, which have a mass accuracy of only ± 5-10% for the average globular protein(46) , MALDI-TOF MS is a straightforward, sensitive (to picomolar levels of protein), and accurate (leq0.1-0.2%; (46) and (47) ) method for measuring molecular mass, as the measurements are based on mass and charge. Here, we report that MALDI-TOF mass spectra of glycosylated and HF-deglycosylated PHRGP gave molecular masses of 73,186 ± 146 Da and 53,953 ± 108 Da, respectively (Fig. 1). These estimates were significantly smaller than those obtained by SDS-PAGE or gel filtration and imply that other estimates of HRGP molecular size based exclusively on SDS-PAGE or size exclusion chromatography (48) need to be reevaluated.


Figure 1: MALDI-TOF mass spectrometry of glycosylated (a) and HF-deglycosylated (b) PHRGP. The MALDI-TOF mass spectrum of glycosylated PHRGP (a) contained broad peaks corresponding to the triply (M + ^3H), doubly (M + 2H), and singly charged (M + H) molecular ions at m/z 24538.5, 36415.1, and 73113.2, respectively. b, the spectrum of deglycosylated PHRGP also showed three peaks at m/z 18044.2, 26945.8, and 53834.5, corresponding the the triply, doubly, and singly charged species, respectively.



PHRGP Saccharide Content

The molecular mass of PHRGP was 19,233 Da greater than that of HF-deglycosylated PHRGP (Fig. 1); thus sugar accounts for 26% of the PHRGP mass. Judging from neutral sugar analyses(21) , this corresponds to 127 arabinose residues and 13 galactose residues in the average PHRGP molecule. Combining earlier amino acid and carbohydrate analyses (21) and the MALDI-TOF data, we calculated the following empirical formula for the intact glycoprotein:

Interestingly, the size distribution of the arabinooligosaccharides is skewed and seems non-random, as we estimate from PHRGP Hyp-arabinoside profiles ((21) ; cf.Table 1, column 1) and the empirical formula that PHRGP contains a single Hyp-Ara(4), 40 Hyp-Ara(3), 10 Hyp-Ara(2), and 14 Hyp-Ara(1), with 82 Hyp residues nonarabinosylated. In order to determine if the arabinosylated Hyp residues occurred randomly throughout the protein, or in accordance with the Hyp contiguity hypothesis, we proteolytically degraded the PHRGP into small glycopeptides and characterized the arabinosylation sites biochemically and by MS/MS (see flow chart, Fig. 2).


Figure 2: Experimental flow chart. We digested glycosylated PHRGP with Pronase, then analyzed aliquots of the unfractionated digest by electrospray ionization (ESMS/MS) and fast atom bombardment tandem mass spectrometry (FABMS/MS). To corroborate the results from MS analyses of the Pronase digest and determine the percent arabinosylation of each glycopeptide, we also purified the major peptide and glycopeptide components by a combination of hydrophilic interaction (HILIC) and reverse-phase chromatography. The purified peptide H2P1 and glycopeptides H3P2 and H4P1 were then structurally characterized by Edman degradation, sugar analyses, and mass spectrometry (ESMS and ESMS/MS).



MS Analyses of the PHRGP Unfractionated Pronase Digest

Pronase digestion of PHRGP yielded a relatively few major peptides and glycopeptides as evidenced by analyses of the digests both before and after chromatographic separation. Both ESIMS (Fig. 3) and CF-FABMS mass spectra (not shown) of the unfractionated digest contained only a few molecular ions; we chose those common to both spectra, i.e. the signals at m/z 439, 701, 833, and 965, for further analysis by CID and tandem mass spectrometry.


Figure 3: The PHRGP Pronase digest analyzed by electrospray ionization mass spectrometry. We freeze-dried aliquots of the digest to remove ammonium bicarbonate, dissolved the residue in the appropriate matrix solution (see ``Materials and Methods''), and then analyzed the solutions by both CF-FABMS (not shown) and ESIMS (above). We selected the ions common to both spectra (i.e. ions at m/z 439, 701, 833, and 965) for further analysis by CID and MS/MS.



Glycopeptide m/z 965

The high energy CID spectrum of protonated molecular ion m/z 965 contained fragment ions originating from cleavages of the peptide backbone, O-glycosidic bonds, and the sugar rings ( Fig. 4and Fig. 5, a and c). Fragment ions from the C terminus: y(1) (Val), y(2) (Hyp-Val), y(3)Y(0), (Hyp-Hyp-Val), y(4)Y(0) (Pro-Hyp-Hyp-Val), and Y(0) (Lys-Pro-Hyp-Hyp-Val), and from the N terminus: b(1) (Lys), b(2) (Lys-Pro), and a(4) (Lys-Pro-[Ara(3)]Hyp-Hyp), defined the peptide sequence. Fragment ions Y(1) and Y(2) occurred in both the high and low energy spectra (Fig. 5, a and b) and arose from cleavage of the O-glycosidic bonds of the arabinoside side chain only, corresponding to the mono- and diarabinosylated peptide, respectively. Most importantly, the glycopeptide peptide backbone fragmented yet retained its arabinosyl substituents; thus, we were able to determine which Hyp residue was glycosylated. Although the sequence Lys-Pro-Hyp-Hyp-Val contains two potential glycosylation sites, apparently only Hyp-3 is glycosylated judging by the presence of fragment ions y(2) (Hyp-Val), b(3)Y(1) (Lys-Pro-[Ara]Hyp), y(3)Y(1) ([Ara]Hyp-Hyp-Val), y(3)Y(2) ([Ara(2)]Hyp-Hyp-Val), y(4)Y(1) (Pro-[Ara]Hyp-Hyp-Val), y(3) ([Ara(3)]Hyp-Hyp-Val), and a(4) (Lys-Pro-[Ara(3)]Hyp-Hyp), and from the absence of ions at m/z 627, 495, and 363, that would correspond to the glycosylation of Hyp-4.


Figure 4: Nomenclature of fragment ions produced by high energy CID analysis of molecular ion m/z 965, Lys-Pro-[Ara(3)]Hyp-Hyp-Val. The nomenclature used here to describe the PHRGP peptide and glycopeptide fragment ions combines the Roepstorff and Fohlman system (49) for peptides (as modified by Biemann; (50) ) with that proposed by Costello and Vath (51) for glycoconjugates and oligosaccharides. a, fragment ions designated with lowercase letters (a, b, and y) originate from cleavage of the peptide backbone only, with a and b ions arising from the N terminus, and y ions from the C terminus. Subscript numbers indicate the residue at which cleavage occurred, numbered upward from the respective terminus. a and b, uppercaseletters (X and Y) designate ions arising from oligosaccharide fragmentation only (without cleavage of the peptide backbone), with the charge retained on the ``reducing'' end (i.e. sugar fragments attached to the peptide). Again, subscripts number the sugar residues from the reducing end while the superscripts preceding X ions (e.g.X(2)) define cleavages of carbon-carbon or carbon oxygen bonds within arabinosyl ring(51) . yY ions in a arise from fragmentation of both the peptide backbone and the sugar side chain.




Figure 5: MS/MS analyses and corresponding fragment ion series of PHRGP Pronase glycopeptide, Lys-Pro-[Ara](3)Hyp-Hyp-Val, M + H = 965. Both high energy CID (a) and low energy CID (b) of molecular ion m/z 965 gave similar spectra, indicating that the glycopeptide contained a triarabinosyl chain at Hyp-3. However, only the high energy spectrum contained ions y(1) (Val) and a(4) (Lys-Pro-Hyp-Hyp + 3 Ara), which distinguished Hyp rather than Val at position 4 of the sequence (a and c). High energy CID also produced X fragment ions at m/z 905, 875, 861, 729, and 597, which originated from cleavage within the arabinosyl rings. Because of its intensity, the molecular ion (M + H = 965) was not included in the high energy spectrum. Fragment ions m/z 922 and 551 correspond to the molecular ion minus the valine side-chain (-43 atomic mass units) and the deglycosylated peptide minus water (Y(0) - 18 atomic mass units), respectively. b, the low energy CID spectrum lacked ions defining the sequence of the peptide C terminus, that is, the ions here correspond to either peptide sequence, Lys-Pro-Hyp-Val-Hyp or Lys-Pro-Hyp-Hyp-Val. Fragment ions y(2) and y(3)Y(1) indicate Hyp-3 is the arabinosylation site. c, the fragment ion series arising from high energy CID of molecular ion m/z 965. The masses corresponding to the fragment ion series are listed either above (y, Y, and yY ions), or below (b, a, and bY ions) the glycopeptide. The peptide fragmentation site of ion a(2) (m/z 198) is not shown in c.



Cleavage of the arabinosyl rings occurred only in the FAB mass spectrometer (i.e. high energy CID) giving rise to fragment ions at m/z 905, 875, 861, 729, and 597 which correspond to the X(2), X(2), X(2), X(1), X(0) ion series (Fig. 4b and Fig. 5a).

The peptide sequence itself was established only by the high energy CID spectrum, as the similar, but less complex low energy CID analysis (Fig. 5b) was ambiguous regarding the peptide sequence. However, low energy CID produced fragment ions y(2) (Hyp-Val) and y(3)Y(1) ([Ara]Hyp-Hyp-Val), corroborating Hyp-3 as the glycosylation site.

Glycopeptide m/z 833

The high energy CID spectrum (not shown) of protonated molecular ion m/z 833 (cf.Fig. 3) contained fragment ions originating predominantly from the cleavage of the amide linkage, O-glycosidic bonds, and the sugar rings. The peptide sequence was only partially defined by fragment ions b(1) (Lys), b(2) (Lys-Pro), y(2) (Hyp-Val or Val-Hyp), y(3)Y(0) (Hyp-Hyp-Val or Hyp-Val-Hyp), y(4)Y(0) (Pro-Hyp-Hyp-Val or Pro-Hyp-Val-Hyp), and Y(0) (Lys-Pro-Hyp-Hyp-Val or Lys-Pro-Hyp-Val-Hyp), as we observed no ion that indicated whether Hyp or Val occupied position 4 of the sequence. However, the peptide sequence is probably Lys-Pro-Hyp-Hyp-Val, judging by analyses of the corresponding isolated glycopeptide H3P2 (i.e. Lys-Pro-[Ara] Hyp-Hyp-Val), which is discussed later. Fragment ions Y(1) ([M + H - Ara]) and Y(0), ([M + H - 2Ara]) indicated that the glycopeptide contained 2 glycosyl residues, while fragment ion y(2) (Hyp-Val/Val-Hyp), which contained no sugar, and ions y(3) (i.e. [Ara(2)]Hyp-Hyp-Val or [Ara](2)Hyp-Val-Hyp), y(4) (Pro-[Ara(2)]Hyp-Hyp-Val or Pro-[Ara(2)]Hyp-Val-Hyp), y(4)Y(1)(Pro[Ara]Hyp-Hyp-Val or Pro-[Ara]Hyp-Val-Hyp), and y(3)Y(1) ([Ara] Hyp-Hyp-Val or [Ara]Hyp-Val Hyp), which do contain sugar, suggest that Hyp-3 is the glycosylation site. We also observed fragment ions arising from the cleavage of the sugar rings at m/z 773, 743, 729, and 597, which corresponded to the X(1), X(1), X(1), X(0) series similar to that in Fig. 4. We did not select molecular ion m/z 833 for ESIMS/MS analysis due to the low intensity of molecular ion m/z 833 in the ESI mass spectrum of the Pronase digest (Fig. 3).

Glycopeptide m/z 701

Both high and low energy CID of the Pronase digest protonated molecular ion m/z 701 determined the peptide sequence as Lys-Pro-Hyp-Val-Hyp, as both mass spectra contained series of fragment ions arising from both the C and N terminus (Fig. 6). Both mass spectra also contained the internal fragment at m/z 310 (i.e. Pro-Hyp-Val), which indicated that Val rather than Hyp occurred at position 4, and also the fragment ion y(1) (Hyp-Ara), which points to Hyp-5 rather than Hyp-3 as the arabinosylation site. This was corroborated by ions in the high energy spectrum, b(3), b(4), and a(4), which corresponded to the nonglycosylated sequences: Lys-Pro-Hyp and Lys-Pro-Hyp-Val (Fig. 6a), with no evidence of ions of fragments containing sugar on Hyp-3.



Figure 6: MS/MS analyses of PHRGP Pronase glycopeptide, Lys-Pro-Hyp-Val-[Ara] Hyp, M +H = 701. Both high energy (a) and low energy (b) CID spectra of molecular ion m/z 701 contained fragment ions which determined its peptide sequence, Lys-Pro-Hyp-Val-Hyp, and monoarabinosylation site at Hyp-5. The diagnostic ions for the peptide sequence were b(4) (m/z 438), and internal fragment ion Pro-Hyp-Val at m/z 310, while fragment ion y(1) determined Hyp-5 as the arabinosylation site. Because of its intensity, the molecular ion (M + H = 701) was not included in the high energy spectrum. (c) Fragment ions and masses which belong to the same ion series are listed in rows above (y, yY, and Y ions), or below (a and b ions) the glycopeptide sequence. The peptide fragmentation sites of the a ion series is not shown in c.



Fragment ions corresponding to the cleavage of the sugar ring occurred only in high energy CID (Fig. 6a). That is, the ions at m/z 611 ([M + 1 - 90]) and 597 ([M + 1 - 104]) correspond to the X(0) and X(0) fragments originating from cleavage of carbon-carbon and carbon-oxygen bonds of the glycosyl ring (cf. Fig. 4b).

Peptide m/z 439

Low energy CID (not shown) of protonated molecular ion m/z 439 (cf. Fig. 3) produced fragment ions at m/z 183 (a(2)), 211 (b(2)), 229 (y(2)), and 326 (y(3)), which were consistent with the partial sequence Ile-Pro-Pro-Hyp present in the PHRGP 18-residue repeat reported previously(21) ; however, because Ile and Hyp have identical molecular masses, the MS/MS-derived peptide sequence was ambiguous. As such, we determined the sequence by Edman degradation of the purified peptide, H2P1, described below.

Purification and Characterization of PHRGP Peptides and Glycopeptides Obtained by Pronase Digestion

MS/MS analyses of the PHRGP Pronase digest determined the arabinoside chain lengths and glycosylation site of each glycopeptide and established the complete sequences for two peptides. However, because such MS analyses are not quantitative, we did not know if there was arabinoside chain-length heterogeneity at the arabinosylation sites, nor were all of the deduced peptide sequences completely unambiguous. Therefore, we purified the individual peptides and glycopeptides and determined the percent glycosylation by hydroxyproline arabinoside profile analyses, and we corroborated the peptide sequences by Edman degradation. We also analyzed the purified peptides and glycopeptides by ESIMS and ESIMS/MS to confirm their molecular weights and structures (cf. Fig. 2).

Purification of (Glyco)peptides by HILIC and RPLC Followed by Peptide and Glycopeptide Characterization

HILIC fractionation of the PHRGP Pronase digest (not shown) yielded four major peaks, designated H1-H4, and a few minor peaks. H1 was largely tyrosine (>90 mol %) released as the free amino acid by Pronase. HILIC fractions H2, H3, and H4 eluted as single peaks when fractionated by RPLC, while RPLC fractionation of the minor HILIC peaks each gave several (i.e. 6 or 7) minor components that we did not analyze further. Although direct concentration of the glycopeptides in TEAP/30 mM phosphoric acid buffer led to significant hydrolysis of the acid labile arabinosides, particularly of Hyp-Ara(3), control experiments showed that the arabinosides were stable after removal of the phosphoric acid by Ba(OH)(2) precipitation (Table 1, columns 1-3).

Peptide H2P1 (Ile-Pro-Pro-Hyp) Corresponds to Molecular Ion m/z 439 in the Pronase Digest

Peptide H2P1 (HILIC peak H2, PRP-1 peak 1) was obtained by RPLC fractionation of HILIC peak H2 (not shown; cf. Fig. 2) and sequenced by Edman degradation. H2P1 contained no sugar according to GLC analysis data, while ESIMS confirmed its mass as 439 atomic mass units (not shown). Low energy CID analysis of H2P1 (not shown) was consistent with CID of the molecular ion m/z 439 present in the unfractionated Pronase digest. Thus, molecular ion m/z 439 from the Pronase digest (cf.Fig. 3) and purified H2P1 both originate from the peptide sequence Ile-Pro-Pro-Hyp, which contained a single possible arabinosylation site, yet is never glycosylated: MS analyses of the PHRGP digest (Fig. 3) showed no evidence for molecular ions corresponding to mono-, di-, or triarabinosylated Ile-Pro-Pro-Hyp. This result is consistent with the Hyp contiguity hypothesis, which predicts that single, non-contiguous Hyp residues are rarely arabinosylated, if at all.

Glycopeptide H3P2, Lys-Pro-[Ara]Hyp-Hyp-Val, Corresponds to Molecular Ions m/z 965 and 833 in the Pronase Digest

Reverse-phase purification of HILIC peak H3 (not shown) gave one major glycopeptide, designated H3P2 (HILIC peak H3, PRP-1 peak 2), with the Edman sequence Lys-Pro-Hyp-Hyp-Val, which contains contiguous Hyp residues. A quantitative Hyp-arabinoside profile showed that approximately one-half of the H3P2 Hyp residues are glycosylated, mainly with the triarabinoside, although small amounts of the mono- and diarabinoside also occur (Table 1, column 4).

Consistent with the Hyp-arabinoside profile of H3P2, the ES mass spectrum (not shown) yielded three molecular ions, m/z 965, 833, and 701, corresponding to the tri-, di-, and monoarabinosylated glycoforms, respectively, which apparently cochromatographed on the HILIC and reverse-phase columns. The low energy CID spectrum of H3P2 (not shown) corroborated the structures that had been deduced by CID analyses of the molecular ions m/z 965 and 833 obtained from the unfractionated Pronase digest (cf.Fig. 3and Fig. 5). Thus, Lys-Pro-Hyp-HypVal is always glycosylated at Hyp-3, and usually with a triarabinoside. Such consistent and specific arabinosylation of dipeptidyl-Hyp is also in accordance with the Hyp contiguity hypothesis.

Glycopeptide H4P1, Lys-Pro-Hyp-Val-[Ara]Hyp, Corresponds to Molecular Ion m/z 701 in the Pronase Digest

Fractionation of HILIC peak H4 by RPLC (not shown) yielded the major component, H4P1 (HILIC peak H4, PRP-1 peak 1), which was shown by Edman degradation to have the sequence Lys-Pro-Hyp-Val-Hyp. This peptide is a positional isomer of H3P2 (Lys-Pro-Hyp-Hyp-Val), differing only by the inversion of the Hyp-Val C terminus. This inversion provides a rigorous test of the Hyp contiguity hypothesis, as it permits us to compare the arabinosylation specifics of two peptides of identical composition, but with a crucial distinction between contiguous Hyp residues (H3P2) and non-contiguous Hyp residues (H4P1). Again, data for H4P1 were consistent. A quantitative Hyp-arabinoside analysis of H4P1 showed that 90% of its Hyp residues were not glycosylated, with the remaining 10% monoarabinosylated (Table 1, column 5), while the ES mass spectrum of H4P1 (not shown) contained two molecular ions, m/z 569 and m/z 701, corresponding to the structures, Lys-Pro-Hyp-Val-Hyp and the monoarabinosylated glycoform, Lys-Pro-Hyp-Val-[Ara]Hyp, which co-chromatographed on the HILIC and reversed-phase columns. Low energy CID analysis of H4P1 (not shown) also confirmed the arabinosylation site as Hyp-5 in agreement with the high and low energy CID mass spectra of ion m/z 701 obtained from the Pronase digest (cf.Fig. 3and Fig. 6). Thus, a simple inversion of the Hyp-Val C terminus changes both the site and extent of arabinosylation from consistent arabinosylation predominantly with a triarabinoside on Hyp-3 (H3P2) to an occasional monoarabinosylation of Hyp-5.


CONCLUSIONS

Our recent structural work led us to suggest that Hyp-glycosylation is not random, but follows simple rules, such as Hyp contiguity(21) . To test the Hyp contiguity hypothesis, we needed to sequence a Hyp-rich polypeptide and identify the glycosyl substituents at each position. For reasons already stated, the determination of HRGP arabinosylation site specifics is of great interest, albeit a non-trivial task, as it requires the correct assignment of mono-, di-, tri-, and tetra-arabinosides, or lack thereof, to each Hyp residue in an HRGP sequence. We have virtually achieved this, as the glycopeptides characterized here represent the bulk of the PHRGP glycosylated sequences; we calculate from peptide and sugar recoveries that glycopeptides H3P2 and H4P1 together contained 89% of the PHRGP arabinose residues (i.e. 113 of the 127 residues estimated from the MALDI-TOF mass spectra data). The remaining arabinose, as well as galactose and the amino acids Ser, His, Arg, and Thr, which are minor components of the PHRGP, apparently occurs in the minor HILIC peptides which we did not characterize. Thus, the Douglas fir PHRGP is not only the first HRGP to be weighed by mass spectrometry, but also the first for which it has been possible to define the peptide sequences surrounding the major arabinosylation sites, pinpoint the precise Hyp residues which are arabinosylated, and also determine both the frequency of arabinosylation and arabinoside chain lengths at those sites ( Fig. 7and Fig. 8).


Figure 7: A correlation between Hyp contiguity and Hyp-arabinosylation. The sequences Lys-Pro-Hyp-Val-Hyp (H4P1) and Lys-Pro-Hyp-Hyp-Val (H3P2) are peptide structural, or positional, isomers that differ in the arrangement of their Hyp residues. The Hyp-arabinoside profile of H4P1 (Table 1) indicated that 10% of its total Hyp residues were monoarabinosylated, while CID indicated that arabinosylation occurred only on Hyp-5. Thus, Hyp-5 in the repetitive sequence Lys-Pro-Hyp-Val-Hyp is monoarabinosylated 20% of the time. The Hyp-arabinoside profile of H3P2 (Table 1) indicated that half of the Hyp residues of the sequence Lys-Pro-Hyp-Hyp-Val were arabinosylated predominantly with the triarabinoside, while CID pinpointed Hyp-3 as the only glycosylation site.




Figure 8: Proposed structure of the PHRGP major repetitive glycopeptide. Three peptide sequences, H4P1, H2P1, and H3P2 (underlined) and their glycoforms, comprise the bulk of the PHRGP and occur as part of a larger 18-residue tandem repeat characterized previously(21) . Featured here is the dominant PHRGP glycomotif; however, some variation occurs in the chainlength of the arabinoside (from 1 to 3 residues), and occasionally the single Hyp located between the 2 valine residues (Hyp-5 of H4P1) is monoarabinosylated.



Pronase cleavage of PHRGP yielded three major glycopeptides, which corresponded to glycoforms of the peptide positional isomers, Lys-Pro-Hyp-Val-Hyp and Lys-Pro-Hyp-Hyp-Val, thereby testing the Hyp contiguity hypothesis. The results were consistent with the hypothesis and showed that extensive arabinosylation occurred only for a contiguous Hyp residue, while non-contiguous Hyp is arabinosylated only occasionally, i.e. Hyp-5 of Lys-Pro-Hyp-Val-Hyp, or not at all, as in Ile-Pro-Pro-Hyp. Why Hyp-3 but not Hyp-4 is the inevitable arabinosylation site in H3P2, while Hyp-5 is only an occasional site in H4P1, remains for future work. However, Fig. 8shows that the Hyp-3 triarabinoside of H3P2 is distal rather than proximal to the Val-Tyr-Lys motif, which for dicot extensins, is a putative intermolecular cross-link site (14) and therefore far less likely to sterically hinder cross-link formation than a Hyp-5 triarabinoside. Currently, however, there is no definitive evidence for PHRGP cross-linking, either in vitro or in muro.

Such precise Hyp-arabinosylation suggests a sequence-dependent, rather than a conformation-dependent, enzymic mechanism, as previously suggested for O-Thr/Ser glycosylation (52) and proline hydroxylation(39) . Judging from the number of different arabinosyl linkages in wall proteins (19, 53) and polysaccharides(54) , an arsenal of arabinosyl transferases in plant cells also includes sequence-specific glycosyl transferases.

Remarkably, we captured most of the above structural information in CID spectra of underivatized glycopeptides present in unfractionated PHRGP Pronase digests. This was possible in part because the simple repetitive PHRGP polypeptide backbone produced only a few molecular ions, greatly simplifying MS/MS analyses. However, composition is also a critical factor as the pyrrolidine rings of Hyp and Pro impose conformational constraints which probably lower the dissociation energy of nearby peptide bonds(55) . Thus, HRGPs seem to be uniquely tailored for structural analysis by CID and tandem mass spectrometry. Assuming other HRGPs also fragment readily at Hyp and Pro residues while retaining their saccharide substituents, it may be possible to determine the glycosylation site specifics of any extensin-HRGP family member, including the highly arabinosylated Ser-Hyp(4)-containing extensins, as well as the AGPs and gums which contain both arabinosides and polysaccharides O-linked to Hyp. The Hyp contiguity hypothesis is, therefore, a step toward the elucidation of more precise Hyp glycosylation codes, ultimately leading to a complete description of the HRGP molecular topographies that may be involved in molecular recognition, self-assembly, and morphogenesis of the extracellular matrix.


FOOTNOTES

*
This research was supported by Grant 93-37304-9364 from the United States Department of Agriculture, Grant P41-RR05351 from the National Institutes of Health, and Grant DE-FG09-93ER20097 from the United States Department of Energy Funded Center for Plant and Microbial Complex Carbohydrates. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Complex Carbohydrate Research Center and Biochemistry Dept., 220 Riverbend Rd., University of Georgia, Athens, GA 30602. Tel.: 706-542-4468; Fax: 706-542-4412.

(^1)
The abbreviations used are: HRGP, hydroxyproline-rich glycoprotein; PHRGP, proline-hydroxyproline-rich glycoprotein; RPRP, repetitive proline-rich protein; AGP, arabinogalactan-protein; Hyp-Ara, hydroxyproline arabinoside; RPLC, reverse-phase liquid chromatography; HILIC, hydrophilic interaction chromatography; ESIMS, electrospray ionization mass spectrometry; CF-FABMS, continuous flow fast atom bombardment mass spectrometry; CID, collisionally induced dissociation; MS/MS, tandem mass spectrometry; HF, anhydrous hydrogen fluoride; MALDI-TOF, matrix-assisted laser desorption/ionization time of flight.

(^2)
M. Kieliszewski, unpublished data.


ACKNOWLEDGEMENTS

We thank Drs. Linda Schnabelrauch, Barbara Burgers, and Derek Lamport for helpful comments on the manuscript, and the Suntory Institute (Osaka, Japan) for use of their JEOL HX/HX110A tandem mass spectrometer.


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