Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2010/05/20/M109.100149.DC1.html
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 32, pp. 24575–24583, August 6, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Plant O-Hydroxyproline Arabinogalactans Are Composed of
Repeating Trigalactosyl Subunits with Short Bifurcated Side
Chains*□
S
Received for publication, January 1, 2010, and in revised form, April 14, 2010 Published, JBC Papers in Press, May 20, 2010, DOI 10.1074/jbc.M109.100149
Li Tan‡1, Peter Varnai§2, Derek T. A. Lamport¶2, Chunhua Yuan储2, Jianfeng Xu‡3, Feng Qiu**4, and Marcia J. Kieliszewski‡5
From the ‡Department of Chemistry and Biochemistry, Biochemistry Research Facility, Ohio University, Athens, Ohio 45701, the
§
Department of Chemistry and Biochemistry and the ¶School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QG,
United Kingdom, the 储Campus Chemical Instrument Center, Ohio State University, Columbus, Ohio 43210, and the **Department
of Chemistry, Temple University, Philadelphia, Pennsylvaina 19122
* This work was supported, in whole or in part, by National Institutes of Health
Grant 2-P41-RR05351-06 (to the Complex Carbohydrate Research Center).
This work was also funded by United States Department of Agriculture Grants
2004-34490-14579), Herman Frasch Foundation Grant 526-HF02, The Ohio
University Biomimetic, Nanoscience, and Nanotechnology Program Grant
GC0013845), and by the Ohio University Molecular and Cellular Biology
Program.
□
S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Tables I–V and Figs. 1–10.
1
Present address: 315 Riverbend Rd., CCRC, University of Georgia, Athens, GA
30602.
2
These authors contributed equally to this manuscript.
3
Present address: Arkansas Biosciences Institute, Arkansas State University,
Jonesboro, AR 7240.
4
Present address: R&D, Bristol-Myers Squibb Co., 311 Pennington-Rocky Hill
Rd., Pennington, NJ 08534.
5
To whom correspondence should be addressed: 350 West State St., Biochemistry Research Facility, Ohio University, Athens, OH 45701. Tel.: 740-593-9466;
Fax: 740-597-1772; E-mail: [email protected].
AUGUST 6, 2010 • VOLUME 285 • NUMBER 32
Hydroxyproline-rich glycoproteins of the cell surface comprise groups of related structural proteins, including the
extensins that form cell wall scaffolding networks essential for
cytokinesis (1) and the classical arabinogalactan proteins (2)
that are largely at the membrane wall interface (3) and have
diverse functions (4). O-Hyp6 glycosylation characterizes the
hydroxyproline-rich glycoproteins and is of much interest as it
defines molecular properties and, hence, biological function.
Arabinogalactan proteins are highly glycosylated mainly with
O-Hyp-arabinogalactan polysaccharides (5, 6). Extensins are
less highly glycosylated mainly with small O-Hyp arabinooligosaccharides (7, 8), whereas the related proline-rich proteins are
minimally glycosylated also with arabinooligosaccharides (9).
Peptide sequence directs O-Hyp glycosylation by the addition
of small oligosaccharides to contiguous Hyp residues and larger
acidic arabinogalactan polysaccharides to clustered noncontiguous Hyp (10, 11). For example, clustered Ala-Hyp and Ser-Hyp
are typical AGP glycosylation motifs (12), whereas the Hyp residues in repetitive blocks of Ser-Hyp2 orSer-Hyp4 are arabinosylated. The “hyperglycosylation” of closely related AGPs complicates their purification, a problem that can be overcome by
expressing single individual AGPs as GFP fusion glycoproteins,
the hydrophobic GFP tag enabling chromatographic purification (13, 14). This approach also allows purification of neoAGPs containing single repeating AGP glycosylation motifs, for
example (Ala-Hyp)n or (Ser-Hyp)n, for base-catalyzed peptide
bond hydrolysis. Base hydrolysis releases alkali-stable Hyp-arabinogalactan glycoamino acids, designated Hyp-AGs (5, 6),
that can be further purified by size-exclusion chromatography.
Here we report the complete structural elucidation of three
such Hyp-AGs ranging in size from 14 to 22 sugar residues. The
structures were determined using multidimensional homonuclear and heteronuclear NMR spectroscopy in conjunction
with molecular simulations in the presence of water.
Ala-Hyp-polysaccharide-1 isolated earlier from (Ala-Hyp)51GFP expressed in tobacco cells represented the first complete
structure of a small Hyp-AG (15) characteristic of type II arabi6
The abbreviations used are: Hyp, hydroxyproline; AGP, arabinogalactanprotein; AG, arabinogalactan; GFP, green fluorescent protein; COSY, homonuclear correlation spectroscopy; TOCSY, total correlation spectroscopy;
NOE, nuclear Overhauser effect; NOESY, NOE spectroscopy; HSQC, heteronuclear single quantum coherence; HMBC, heteronuclear multiple bond
coherence.
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Classical arabinogalactan proteins partially defined by type
II O-Hyp-linked arabinogalactans (Hyp-AGs) are structural
components of the plant extracellular matrix. Recently we
described the structure of a small Hyp-AG putatively based
on repetitive trigalactosyl subunits and suggested that AGs
are less complex and varied than generally supposed. Here we
describe three additional AGs with similar subunits. The
Hyp-AGs were isolated from two different arabinogalactan
protein fusion glycoproteins expressed in tobacco cells; that
is, a 22-residue Hyp-AG and a 20-residue Hyp-AG, both isolated from interferon ␣2b-(Ser-Hyp)20, and a 14-residue
Hyp-AG isolated from (Ala-Hyp)51-green fluorescent protein. We used NMR spectroscopy to establish the molecular
structure of these Hyp-AGs, which share common features:
(i) a galactan main chain composed of two 133 ␤-linked trigalactosyl blocks linked by a ␤-136 bond; (ii) bifurcated side
chains with Ara, Rha, GlcUA, and a Gal 6-linked to Gal-1 and
Gal-2 of the main-chain trigalactosyl repeats; (iii) a common
side chain structure composed of up to six residues, the
largest consisting of an ␣-L-Araf-(135)-␣-L-Araf-(133)-␣L-Araf-(133- unit and an ␣-L-Rhap-(134)-␤-D-GlcUAp(136)-unit, both linked to Gal. The conformational ensemble
obtained by using nuclear Overhauser effect data in structure
calculations revealed a galactan main chain with a reverse
turn involving the ␤-136 link between the trigalactosyl
blocks, yielding a moderately compact structure stabilized by
H-bonds.
Repetitive Structure of Hyp-arabinogalactans
EXPERIMENTAL PROCEDURES
Gene Construction and Expression in Tobacco Cells—Genes
encoding Interferon ␣2-(Ser-Hyp)20 and (Ala-Hyp)51-GFP
were constructed and expressed as described in detail earlier
(12, 18). Briefly, proteins were targeted for secretion using a
tobacco extensin signal sequence, and gene expression was
under control of the 35 S cauliflower mosaic virus promoter.
The genes were subcloned into the plant transformation vector
pBI121 and expressed in tobacco Bright Yellow-2 cells selected
and maintained as described earlier (12, 18).
Isolation of the Interferon ␣2-(Ser-Hyp)20 and (Ala-Hyp)51GFP Fusion Glycoproteins—Interferon ␣2-(Ser-Hyp)20 and
(Ala-Hyp)51-GFP were isolated from the medium of suspension-cultured tobacco Bright Yellow-2 cells by hydrophobic
interaction chromatography on a phenyl-Sepharose column as
described earlier (18).
Isolation of Hyp-arabinogalactans—Two hundred mg of
Interferon ␣2-(Ser-Hyp)20 were hydrolyzed in 20 ml of 0.44 N
NaOH solution at 108 °C for 18 h. The cooled solution was
titrated to pH 7.8 with cold 1 N HCl and then freeze-dried.
24576 JOURNAL OF BIOLOGICAL CHEMISTRY
Hydrolysates were fractionated on an analytical Superdex-Peptide column (HR 10/30, GE Healthcare) equilibrated in 20%
acetonitrile (aqueous) and eluted at a flow rate of 0.3 ml/min
(15).
Fractions (0.6 ml total volume each) were freeze-dried and
analyzed for Hyp and monosaccharides colorimetrically or by
gas chromatography using methods described earlier (18). The
fraction containing the most Hyp (fraction 16 described in Ref.
18, interferon Hyp-polysaccharide-1) and a fraction containing
later-eluting Hyp-glycans (fraction 18, Ref 18, interferon Hyppolysaccharide-2) were rerun on the Superdex column, freezedried, and then used for NMR analyses. The Hyp-glycan AlaHyp-polysaccharide-2 from (Ala-Hyp)51-GFP was isolated by a
combination of cation exchange and gel filtration chromatography as described earlier (15, 20).
NMR Spectroscopy—A 1-mg sample of each Hyp-AG was
dissolved in 0.5 ml of 99.996% D2O (Cambridge Isotope Laboratories, Andover, MA). NMR experiments were carried out
either at 55 °C on a Bruker DMX-800 equipped with a cryoprobe or at 25 °C on a Bruker DMX-600 spectrometer equipped
with a triple-resonance probe and three-axis gradient coils. The
parallel data sets include one-dimensional 1H, two-dimensional 1H-homonuclear correlation spectroscopy (COSY), total
correlation spectroscopy (TOCSY) (mixing time 60 and 90 ms),
rotating frame NOE spectroscopy (ROESY) (200 ms), and
nuclear Overhauser effect spectroscopy (NOESY) (mixing time
150, 300 and 500 ms), and two-dimensional 13C,1H heteronuclear single quantum coherence (HSQC) and heteronuclear
multiple bond coherence (HMBC) NMR spectra. In addition,
several more interferon Hyp-polysaccharide-1 experiments
were recorded in an effort to resolve assignment ambiguities
such as magnitude COSY with one-, two-, and three-step relay
transfer and two-dimensional 13C,1H heteronuclear HSQCTOCSY and HSQC-NOESY together with diffusion-ordered
spectroscopy for measuring the diffusion constant. Water suppression was achieved by either presaturation or WATERGATE
techniques. Data were processed with NMRPipe (21) and
visualized using NMRView (22) Chemical shifts were referenced to an external standard: 4,4-dimethyl-4-silapentane1-sulfonic acid.
NMR Structure Calculations—Interferon Hyp-polysaccharide-1 was constructed in an arbitrary extended conformation
using the LEaP module of Amber 10 (23). The starting model
was subjected to a restrained simulated annealing conformational search protocol to obtain an ensemble of structures consistent with the NMR data. All assigned NOESY cross-peaks
were classified as strong (1.8 –2.7 Å), medium (1.8 –3.7 Å), weak
(1.8 –5.0 Å), and very weak (1.8 – 6.0 Å) interproton distance
restraints according to their intensities. Beyond these bounds, a
quadratic penalty potential was applied with a force constant of
20 kcal mol⫺1 Å⫺2. A total of 49 distance restraints were used
for interferon Hyp-polysaccharide-1 of which 34 were assigned
non-ambiguously to protons in sequential residues. Crosspeaks that correspond to non-sequential assignments gave rise
to ambiguous restraints where either more than one proton
pair contributes to the NOESY volume or unambiguous assignment was not possible. Ambiguous peaks were interpreted as
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nogalactans, which are defined by ␤-1,3-linked galactose residues.
Ala-Hyp-polysaccharide-1 consisted of a 5-residue 1,3-␤-D-Gal
backbone interrupted by a single ␤-1,6 linkage connecting Gal-3
and Gal-4 (numbered from the reducing end). Galactose residues
1 and 2 had small-bifurcated side-chain substituents at C-6. The
side chains possessed a single ␤-D-Gal di-substituted at C-3 with
␣-L-arabinose di- or trisaccharides and at C-6 with ␤-D-glucuronic
acid or ␣-L-rhamnosylglucuronic acid (12, 15). We proposed that
⬃15-residue repetitive blocks of decorated ␤-(1–3) trigalactosyl
subunits connected by ␤-1,6 linkages create the larger arabinogalactan polysaccharides of AGPs, consistent with the
small blocks separated by periodate-sensitive residues suggested earlier (16, 17).
As type II arabinogalactans are often considered intractably
complex, and because Ala-Hyp-polysaccharide-1 was only a
single example of a Hyp-AG subunit, we determined the structure of three additional Hyp-AGs derived from two different
AGP motifs, repetitive Ala-Hyp and Ser-Hyp. Two of the HypAGs designated interferon-polysaccharide-1 (interferon Hyppolysaccharide 1) and interferon-polysaccharide-2 were isolated from a fusion glycoprotein of human interferon ␣2b fused
to a (Ser-Hyp)20 AGP glycomodule (18). The third Hyp-AG,
designated Ala-Hyp polysaccharide-2, was isolated from (AlaHyp)51-GFP similar to Ala-Hyp-polysaccharide-1 described
earlier (15). Here we identified the fundamental similarities
between these Hyp-arabinogalactans and determined if the
non-glycosylated domains (interferon ␣2 versus GFP) or AGP
motifs (Ser-Hyp versus Ala-Hyp repeats) influenced the glycan
structure. Significantly, the six-residue galactan backbone of
these new Hyp-AGs consisted of two ␤-1,3-linked galactosyl
trisaccharides connected by a ␤-1,6 linkage. Such “decorated”
⬃15-residue trisaccharide subunits likely constitute the fundamental building blocks of type II arabinogalactan polysaccharides; hence, they are far less complex than commonly supposed
(4, 19). Finally, the NMR analyses and molecular modeling of
the glycans revealed major conformers that include a moderately compact folded structure.
Repetitive Structure of Hyp-arabinogalactans
RESULTS
The Structures of Interferon Hyp-polysaccharide-1, interferon Hyp-polysaccharide-2, and Ala-Hyp-polysaccharide-2
were determined based on earlier composition analyses and on
the chemical shifts (15, 26) observed in one-dimensional 1H
NMR spectra and two-dimensional COSY, TOCSY, HSQC,
and HMBC NMR spectra as follows:
Primary Structure of Interferon Hyp-polysaccharide-1—Sizefractionated base hydrolysates of Interferon␣2-(Ser-Hyp)20
yielded a single peak containing sugar and Hyp residues,
described earlier (26). Two subfractions were chosen for structural analyses; one contained the major Hyp-AG species, designated Interferon Hyp-polysaccharide-1, with 22 glycosyl residues estimated by the Hyp to monosaccharide molar ratios,
and a second fraction contained a smaller, less abundant
Hyp-arabinogalactan of 20 glycosyl residues, designated
interferon Hyp-polysaccharide-2.
Sugar Composition and Configuration—The integrated areas
in the anomeric region in a one-dimensional 1H NMR spectrum (Fig. 1) corroborated the molar ratios Hyp Gal10 Ara8
GlcU2 Rha2 of interferon Hyp-polysaccharide-1 (26), confirming interferon Hyp-polysaccharide-1 as a 22-residue arabinogalactan attached to Hyp. The anomeric configurations were conAUGUST 6, 2010 • VOLUME 285 • NUMBER 32
FIGURE 1. 1H NMR spectrum of Hyp-AG interferon Hyp-polysaccharide-1
at 55 °C. Signals A and B were assigned respectively to H-1 of six and two
␣-L-Ara residues, signal C to H-1 of two ␣-L-Rha residues, signal D (shoulder
peak of C) to H-4 of Hyp, and signal E to the five ␤-D-Gal residues of the
galactan backbone. Signal F was assigned to H-1 of ␤-D-Gal linked to Hyp and
signal G to H-1 of the four side chain ␤-D-Gal residues and two ␤-D-GlcUA
residues.
firmed by the corresponding anomeric carbon signals in the
HSQC spectrum (Fig. 2, supplemental Table I). Interferon Hyppolysaccharide-1 contained a total of eight ␣-L-Araf residues; 6
of the 8 ␣-Araf residues were either terminal, 1,5-linked, or
1,3-linked (⬃5.25 ppm), and 2 gave signals that were consistent
with being both terminal and 13 5-linked to L-Araf residues
(5.087 ppm). Interferon Hyp-polysaccharide-1 also contained 2
␣-L-Rhap residues (4.797 ppm), 2 ␤-D-GlcUAp residues (⬃4.5
ppm), and a total of 10 ␤-D-Galp residues; 1 was O-linked to
Hyp (4.57 ppm), 5 were consistent with a galactan backbone
(⬃ 4.70 ppm) (15), and 4 others that produced signals typical of
side-chain ␤-D-Galp residues (4.40 – 4.54 ppm). The chemical
shift at 4.786 ppm identified 4-H of Hyp.
The Interferon Hyp-polysaccharide-1 Hyp-galactose Linkage—
Signals arising from the Hyp residue were also identified in the
TOCSY (supplemental Fig. 1) and HSQC spectra (Fig. 2). The
chemical shifts are shown in supplemental Table I. The H-4 and
C-4 resonances characteristic of non-glycosylated Hyp shifted
downfield from 4.62 and 70.6 to 4.786 and 78.02 ppm,
respectively, judging from the HSQC spectrum. This indicated that the hydroxyl group of Hyp was galactosylated (15,
18). The HMBC spectrum (Fig. 3) confirmed this in crosspeak G, which arose from C-4 of Hyp (78.02 ppm) and H-1 of
a ␤-D-Galp residue (4.57) ppm, designated G1 in Fig. 4 and
supplemental Table I.
Galactan Backbone and Side Chain Gal Residues—In addition to G1 linked to Hyp, there were another five ␤-D-Galp
residues in the interferon Hyp-polysaccharide-1 galactan backbone (designated G1– 6 in supplemental Table I and Figs. 4 and
5) judging by H-1 resonances at ⬃4.70 ppm in the one-dimensional 1H spectrum. Four of the five backbone Gal residues were
3-linked to each other and to G1, as deduced from cross-peaks E in
the HMBC spectrum (Fig. 3), which correlated backbone Gal H-1
signals with backbone Gal 3-C signals. A fifth backbone Gal participated in a 13 6 linkage to another backbone Gal residue
deduced from cross-peak F in the HMBC spectrum. Thus, based
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an ⬍ r⫺6 ⬎⫺1/6 averaged value of the contributing interproton
distances.
For a restrained molecular dynamics conformational search,
initial models were energy-minimized without restraints and
subjected to 200 simulated annealing cycles. A cycle started
from the structure obtained in the previous cycle and included
10-ps heating from 300 to 1000 K followed by an equilibration
of 10 ps at 1000 K without any restraints applied. The force
constants for all restraints were then scaled gradually from 0 to
the final values during 20 ps at 1000 K followed by cooling the
system to 300 K over 30 ps. Atomic interactions within the
system were calculated using the Glycam06 parameter set for
sugars (24) and Generalized Born solvation (igb ⫽ 2) with
monovalent salt concentration corresponding to 0.1 M. The last
structures in each cycle were energy-minimized using the same
parameters and restraints as described above. Ten best models
for an average structure were selected based on NMR restraint
violations and the potential energy of the molecule to undergo
further refinement in explicit water and counterion environment. Each of these model structures was placed in a truncated
octahedral box of about 5000 TIP3P water molecules and two
K⫹ counterions to neutralize the total charge. In one case we
used Ca2⫹ to neutralize the charge of the uronic acids. Parameters related to water and counterions were taken from the
standard Amber libraries. The system was energy-minimized
and then heated to 300 K at constant volume during 50 ps,
whereas the solute was kept under positional restraints with a
force constant of 25 kcal mol⫺1 Å⫺2. The positional restraints
were gradually removed over 300 ps at constant pressure (1
atm) and temperature (300 K), and a production phase was
initiated for 2 ns with the full set of restraints applied. The final
structures were energy-minimized and used for subsequent
analysis. The hydrodynamic radius was calculated for the
model structures using HYDROPRO Version 7c2 (25).
Repetitive Structure of Hyp-arabinogalactans
FIGURE 3. HMBC spectrum of Hyp-AG interferon Hyp-polysaccharide-1 at
55 °C. This helped identify the interferon Hyp-polysaccharide-1 monosaccharide sequence. Cross-peak A correlated Ara H-1 with Ara C-5, cross-peak
B correlated Ara H-1 with Ara C-3, cross-peak C correlated Ara H-1 with sidechain Gal C-3, cross-peak D correlated Rha H-1 with GlcUA C-4, cross-peak E
correlated backbone Gal H-1 with backbone Gal C-3, cross-peak F correlated
backbone Gal H-1 with backbone Gal C-6, cross-peak G correlated G1 H-1 with
Hyp C-4, cross-peak H correlated GlcUA H-1 with side-chain Gal C-6, and crosspeaks I and J correlated side-chain Gal H-1 to backbone Gal C-6.
on our earlier work (15) and the NMR spectra presented here, the
interferon Hyp-polysaccharide-1 galactan backbone had the
structure ␤-D-Galp-(133)-␤-D-Galp-(133)-␤-D-Galp-(136)-␤D-Galp-(133)-␤-D-Galp-(133)-␤-D-Galp-(134)-O-Hyp.
The final 4 of the 10 Gal residues of interferon Hyp-polysaccharide-1 occurred in side chains (designated Ga, Gb, Gc, Gd in
supplemental Table I and Figs. 4 and 5) linked 136 to the backbone Gal residues. This was deduced from one-dimensional 1H
and two-dimensional 13C,1H HMBC spectra. Resonances at
4.40 – 4.54 ppm in the one-dimensional 1H NMR spectrum
were consistent with four Gal side chains attached to the galactan backbone (15), and cross-peaks I and J in the HMBC spec-
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FIGURE 2. HSQC spectrum of Hyp-AG Interferon Hyp-polysaccharide-1 at
55 °C. Cross-peaks A and B were assigned to H-1/C-1 of ␣-L-Ara, C to H-1/C-1
of ␣-L-Rha, D to the H-1/C-1 of ␤-D-Gal residues of the galactan backbone, and
E to H-1/C-1 of ␤-D-Gal linked to Hyp. Cross-peak F was assigned to H-1/C-1 of
␤-D-GlcUA residues, G to H-1/C-1 of side-chain ␤-D-Gal, H to H-4/C-4 of Hyp, I
to H-2/C-2 of Hyp, and J to H-5/C-5 of Hyp.
trum indicated that the four side-chain Gal residues were 136
linked to backbone Gal residues.
Interferon Hyp-polysaccharide-1 Side-chain Composition
and Linkages—The two ␣-L-Rha residues, designated R1 and
R2, were terminal, deduced by a comparison of their assigned
chemical shifts (supplemental Table I) obtained from TOCSY
(supplemental Fig. 1) and HSQC spectra (Fig. 2) with those of
earlier characterized Ala-Hyp-polysaccharide-1 (15). Rhamnose residues R1 and R2 were linked to O-4 of ␤-D-GlcUAp
(UA1 and UA2 of supplemental Table I), deduced from crosspeak D in the HMBC spectrum (Fig. 3). Cross-peak H in the
HMBC spectrum indicated side-chain Gal residues were substituted at O-6 with glucuronic acid residues UA1 and UA2. The
same chemical shifts arising from ␣-L-Rhap, ␤-D-GlcUAp, and
side-chain ␤-D-Galp residues were identified earlier on sidechain Gal residues in Ala-Hyp-polysaccharide-1 (15). This indicated the side chains were attached to backbone Gal residues
nearest Hyp; therefore, we assigned the two Rha-(134)-GlcUA
subunits to side-chain Gal residues closest to Hyp, Ga, and Gb
(supplemental Table I and Figs. 4 and 5) to give two side chains:
␣-L-Rhap-(134)-␤-D-GlcUAp-(136)-␤-D-Ga and -Gb.
The Ara residues occurred in small side chains that were
3-linked to the side-chain Gal residues. The HMBC spectrum
cross-peak A identified Ara 5-linked to another Ara (Fig. 3). It
arose from H-1 of ␣-L-Araf residues (5.087 ppm, A1 and A4 in
Fig. 4a) and 5-C of other Ara residues (67.1 ppm, A2 and A5 in
Fig. 4a) This was consistent with the one-dimensional 1H NMR
spectrum that indicated there were two Ara-(135)-Ara linkages in interferon Hyp-polysaccharide-1. HMBC signals
arising from the ring carbon atoms of A1 and A4 (C-2, C-3,
and C-4) showed that they were terminal residues (15);
hence, two diarabinosyl structures occurred having the
structure ␣-L-Araf-(135)-␣-L-Araf-(13).
The HSQC C-1/H-1 signals at 109.2 ppm and ⬃5.25 ppm
arose from six Ara residues (A2, A3, A5, A6, A7, A8 in
supplemental Table I, Figs. 4 and 5) and indicated two other
types of linkages in the arabinose side chains. Cross-peak B in
the HMBC spectrum indicated linkage of ␣-L-Araf to O-3 of
another ␣-L-Araf residue (3-C signal at 83.88 ppm) as in the
structure ␣-L-Araf-(133)-␣-L-Araf (i.e. A2 to A3 and A5 to A6),
whereas cross-peak C indicated ␣-L-Araf-(133)-␤-D-Gal linkages (side-chain Gal residues Ga-d in supplemental Table I). The
side-chain Gal H-3 chemical shifts (⬃3.73 ppm) (15) present in
the TOCSY spectrum (supplemental Fig. 1) indicated each of
the four side-chain Gal residues was O-3 substituted. As four
Ara residues were attached to Gal, and the other four occurred
in two ␣-L-Araf-(135)-␣-L-Araf-(13 units, we concluded that
two of the arabinosyl side chains had the structure ␣-L-Araf(135)-␣-L-Araf-(133)-␣-L-Araf-(133)-Galsc, and two had
the structure ␣-L-Araf-(133)-Galsc.
A comparison of the interferon Hyp-polysaccharide-1
HSQC and HMBC spectra with those of Ala-Hyp-polysaccharide-1 (15) showed that the interferon Hyp-polysaccharide-1
anomeric proton/carbon chemical shifts arising from Gc and
Gd (⬃103.6/4.447 and 103.2/4.41 ppm), the side-chain Gal
residues furthest from Hyp in Fig. 4, differed from the other
side-chain Gal residues, Ga and Gb (both ⬃103.4/4.50). Furthermore, the signals from Ga and Gb in Interferon Hyp-
Repetitive Structure of Hyp-arabinogalactans
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calculated value for model structures of 13.9 ⫾ 0.3 Å (see below).
The intensity of NOEs arising
from protons between sequential
O-linked sugar residues, for example H-1 of R1 and H-4 of glucuronic
acid residue UA1 (supplemental
Table III and Fig. 3) suggested a
somewhat restricted conformation
around glycosidic links rather than
free rotation. Furthermore, the
single set of chemical shifts rules
out the existence of several stable
conformations.
Although the major spectral
region was not amenable to unequivocal analysis due to considerable resonance overlap, a region
possessing unique chemical shifts
showed two NOE clusters at ⬃5.25/
4.50 and ⬃5.25/4.70 ppm. The first
cluster included three NOEs indicating long-range interactions; (i) ␦
5.241/␦ 4.526 attributed to H-1 of
Ara residue A7 or A8 and H-1 of Gal
residue Ga; (ii) ␦ 5.233/␦ 4.500
attributed to H-1 of A7 or A8 and
H-1 of Gb, UA1, or UA2; (iii) ␦
5.262/␦ 4.511 attributed to H-1 of
Ara A3 or A6 and H-1 of UA1 or
UA2. NOEs (i) and (ii) indicate a side
chain Ara of the second trigalactosyl
unit is close to the side-chain
Gal/UA residues of the first trigalactosyl unit; this suggests that the
␤-1– 6 linkage between two triFIGURE 4. Interferon Hyp-polysaccharide-1 and its canonical side chain. a, the primary structure of
the 22-residue Hyp-arabinogalactan Interferon Hyp-polysaccharide-1 is shown. Residue labeling includes galactosyl units allows the main
the linkage of each residue and corresponds to that featured in supplemental Table I. The Hyp residue is chain to fold. The second cluster
positioned to the right, beneath rhamnose residue R1. b, the canonical bifurcated AG side chain is shown.
also included diagnostic NOEs; (i)
5.237/4.694 ppm attributed to H-1
polysaccharide-1 were identical to those of Ala-Hyp-polysaccha- of Ara A7 or A8 and H-1of Gal G2; (ii) 5.265/4.700 ppm attribride-1 characterized earlier (15). Therefore, we designated the uted to H-1 of A3 or A6 and H-1 of G4. The intensity of these
specific side-chain Gal residues in the two ␣-L-Araf-(135)-␣- NOEs indicates a distance of 6 Å between the respective protons that suggests the molecule is folded. The possible effect of
L-Araf-(133)-␣-L-Araf-(133)-Gal units as Ga and Gb. They
were part of the two bifurcated six-residue side chains of Inter- spin diffusion can be excluded because these NOEs could be
feron Hyp-polysaccharide-1. The Gal residues in the two ␣-L- observed with a short mixing time of 150 ms.
Primary Structure of Interferon Hyp-polysaccharide-2—NeuAraf-(133)-side-chain Gal units were designated Gc and Gd
tral sugar, uronic acid, and Hyp analyses of interferon Hyp(supplemental Table I, Figs. 4 and 5).
Interferon Hyp-polysaccharide-1 Long-range Interactions— polysaccharide-2 gave a molar ratio of Hyp Gal10 Ara5 GlcUA4
Lowering the temperature of NMR analyses from 55 to 25 °C Rha. The interferon Hyp-polysaccharide-2 1H NMR spectrum
(see the chemical shift assignments in supplemental Table II) (supplemental Fig. 4) gave a very similar signal pattern in the
provided the following lines of evidence for a folded Interferon anomeric proton region as interferon Hyp-polysaccharide-1,
Hyp-polysaccharide-1 conformer (Fig. 6).
except the peak area ratios differed, as interferon Hyp-polysacA diffusion-ordered spectroscopy spectrum (supple- charide-2 contained two more GlcUA and one less Rha and
mental Fig. 2) gave a diffusion coefficient of (1.58 ⫾ 0.1) only five Ara residues. Of the five ␣-Araf residues, only one
⫻10⫺10 m2/s. Using the Stokes-Einstein equation, this value (anomeric proton signal at ⬃5.08 ppm) was 5-linked to another
corresponds to a hydrodynamic radius of 15.6 ⫾ 1.0 Å, which Ara; the other four (anomeric proton signal at ⬃5.24 ppm) were
is consistent with a globular folded structure and close to the terminal, 1,3-linked, or 1,5-linked. The ␣-Rhap residue was ter-
Repetitive Structure of Hyp-arabinogalactans
FIGURE 6. Molecular model of Interferon Hyp-polysaccharide-1. A range
of galactan backbone conformations (in gray) is consistent with the experimental NOE data. A representative conformation is shown in color with the
terminal Hyp residue upper right.
minal, and of the 10 ␤-Galp residues, 6 were backbone Gal
residues. Five of the backbone Gal residues gave H-1 signals at
4.68 – 4.71 ppm, and the sixth, G1 in supplemental Table IV,
was linked to Hyp. The other four Gal residues were part of the
side chains (4.39 – 4.49 ppm). The H-1 signals of ␤-GlcUAp
were not resolved from those of the side-chain Gal; however,
the signal peak integral combined with chemical analyses of
24580 JOURNAL OF BIOLOGICAL CHEMISTRY
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FIGURE 5. Comparison of the primary structures of Hyp-AGs. a, Ala-Hyppolysaccharide-1 (15). b, interferon Hyp-polysaccharide-1. c, interferon Hyppolysaccharide-2. d, Ala-Hyp-polysaccharide-2. The yellow circles indicate ␤-DGalp residues, the green squares indicate ␣-L-Araf, the purple diamonds
indicate ␣-L-Rhap, the red triangles indicate ␤-D-GlcUAp, and the blue blocks
indicate Hyp. The residue labels inside the shapes (A3, G1, etc.) correspond to
the residues described under “Results,” Fig. 4, and the supplemental Tables I–V.
interferon Hyp-polysaccharide-2 indicated it had four GlcUA
residues. Together, the 1H NMR spectrum and the sugar analyses indicated interferon Hyp-polysaccharide-2 was a 20-sugar
residue Hyp-arabinogalactan (Fig. 5c). We assigned the chemical shifts of interferon Hyp-polysaccharide-2 (supplemental Table IV) using two-dimensional TOCSY (supplemental
Fig. 5), HSQC (supplemental Fig. 6), and HMBC (supplemental Fig. 7) spectra discussed below.
Hyp-Gal Linkage and Galactan Backbone—Interferon Hyppolysaccharide-2 had the same galactan backbone structure as
interferon Hyp-polysaccharide-1. The Hyp-Gal linkage was
established by cross-peak G in the HMBC spectrum (supplemental Fig. 7). Like interferon Hyp-polysaccharide-1, the
galactan backbone of interferon Hyp-polysaccharide-2 was
composed of five ␤-D-Galp residues (G2-G6 in supplemental Table IV) and six Gal linked to Hyp (G1). Cross-peak E
indicated the backbone Gal residues were mainly 133-linked,
although a 136 link occurred between G3 and G4 (cross-peak F
in supplemental Fig. 7 and Fig. 5c).
Interferon Hyp-polysaccharide-2 Side Chains—The sidechain structures were similar to those of interferon Hyp-polysaccharide-1, but generally smaller. The four side-chain Gal
residues evident in the one-dimensional 1H NMR spectrum
were attached to backbone Gal residues through the O-6 position, deduced from cross-peaks I and J in the HMBC spectrum
(supplemental Fig. 7) (side-chain Gal H-1 at 4.39 – 4.49 ppm to
backbone Gal 6-C at ⬃70.0 ppm). Interferon Hyp-polysaccharide-2 had only one terminal ␣-L-Rhap but four ␤-D-GlcUAp
residues. Cross-peak D in the HMBC spectrum indicated an
␣-L-Rhap-(134)-␤-D-GlcUAp unit, and the chemical shifts of
the other GlcUA residues (supplemental Table IV) indicated
they were unsubstituted and, therefore, terminal. Cross-peak H
showed that all four GlcUA residues were ␤-13 6-linked to
side-chain Gal residues. In Fig. 5c, we assigned the Rha-(134)GlcUA unit to one of the two side-chain Gal residues closest to
Hyp, Gb, based on earlier work with Ala-Hyp-polysaccharide-1
(15); however, we had no direct evidence for this assignment,
and any one of the four side-chain Gal residues were
candidates.
HMBC cross-peaks A, B, and C (supplemental Fig. 7) corresponded to the following side chain arabinosyl units: ␣-L-Ara(135)-␣-L-Ara, ␣-L-Araf-(133)-␣-L-Araf, and ␣-L-Araf(133)-Gal (15). These units were part of two arabinosyl side
chains, ␣-L-Ara-(135)-␣-L-Ara-(133)-␣-L-Araf-(133 and
␣-L-Araf-(133)-␣-L-Araf-(133, that we assigned to side-chain
Gal residues Ga and Gb rather than Gc and Gd because the H-3
resonances (supplemental Table IV, Fig. 5c) in the TOCSY
spectrum (supplemental Fig. 5) indicated residues Gc and Gd
were unsubstituted at O-3. However, we could not discern the
precise distribution of the diarabinosyl and triarabinosyl units
between Ga and Gb.
Primary Structure of Ala-Hyp-polysaccharide-2—Ala-Hyppolysaccharide-2 derived from base-catalyzed hydrolysates of
(Ala-Hyp)51-enhanced green fluorescence protein was a
14-sugar residue Hyp-AG determined from the peak areas of its
one-dimensional 1H NMR spectrum (not shown) and sugar/
Hyp analyses corresponding to Hyp1 Gal8 Ara2 GlcUA2 Rha2,
one residue less than Hyp1 Gal7 Ara5 GlcUA2 Rha of Ala-Hyp-
Repetitive Structure of Hyp-arabinogalactans
DISCUSSION
Complete elucidation of type II arabinogalactan structure
has been a major goal since the isolation of Hyp-AGs more than
30 years ago (5, 6). Churms et al. (16, 27, 28) notably suggested
AUGUST 6, 2010 • VOLUME 285 • NUMBER 32
a repetitive structure with “AG substituents showing blocks of
1,3–1inked galactan backbone interrupted by periodate susceptible residues (kinked region).” The structure of Ala-Hyppolysaccharide-1 supported the repetitive subunit hypothesis
and also suggests that the arabinogalactan structure is highly
conserved in both classical AGPs and the less abundant chimeric glycoproteins of the cell surface (29). As Ala-Hyp-polysaccharide-1 was only the first Hyp-AG (15), we characterized
additional Hyp-AGs to test the possibility that AGs are highly
varied structures dictated by regional peptide sequence and too
complex for structural elucidation. We expressed the two most
frequently occurring AGP motifs Ser-Hyp and Ala-Hyp in
tobacco Bright Yellow-2 cells as chimeric fusion glycoproteins
with non-glycosylated partners interferon ␣2b and green
fluorescence protein, respectively. This allowed comparison
of Hyp-AGs isolated from Ser-Hyp and Ala-Hyp repeats in
the fusion proteins interferon ␣2-(Ser-Hyp)20 (15, 30) and
(Ala-Hyp)51-GFP.
Interferon ␣2-(Ser-Hyp)20 yielded Hyp-AGs that ranged
from 18 to 26 residues. Interferon Hyp-polysaccharide-1 was
the largest characterized and contained 22 residues corresponding to the composition Gal10 Ara8 GlcUA2 Rha2. HypAGs isolated from (Ala-Hyp)51-GFP were marginally smaller
and somewhat more disperse (14 –25 residues). Significantly,
all the newly characterized Hyp-AGs (Fig. 5) had a six-residue
galactan main chain consisting of two ␤-1,3-linked trigalactosyl
blocks linked by a ␤-1,6 linkage. Ala-Hyp-polysaccharide-1 also
shared this main-chain structure except that it lacked the sixth
(terminal) Gal residue (15).
The side-chain substituents linked to the galactan main
chain were similar in position and composition. The small HypAGs, Ala-Hyp-polysaccharide-1, and Ala-Hyp-polysaccharide-2 contained only two side chains attached to main-chain
Gal residues G1 and G2 (15) and differed from each other only in
their Rha and Ara content (Fig. 5). In contrast, the larger HypAGs, interferon Hyp-polysaccharide-1 and interferon Hyp-polysaccharide-2 each had four side chains ranging from two to six
glycosyl residues linked to G1, G2, G4, and G5 (Figs. 4 and 5).
These three new Hyp-AGs and Ala-Hyp-polysaccharide-1
shared an ⬃15-residue subunit consisting of a repetitive trisaccharide with two bifurcated acidic side chains (Figs. 4 – 6), each
with a maximum of six residues; these side chains are attached
to C-6 of G1 and G2, i.e. the first and second Gal residues, numbered from the reducing end of the galactan backbone.
This relatively invariant Hyp-AGs structure with bifurcated
side chains is apparently widespread; it is consistent with compositional data from diverse species (16, 27, 28) and with the
fact that AGPs from diverse species selectively co-precipitate
with the ␤-Yariv reagent. The six-residue side chain of interferon Hyp-polysaccharide-1 is identical with the gum arabic
side chain in the legume Acacia senegal (31) except for the
addition of terminal 5-linked Araf in interferon Hyp-polysaccharide-1. Furthermore, neither the type of Hyp-AGs peptide
motif (Ser-Hyp versus Ala-Hyp) nor the attached non-glycosylated domain (interferon versus GFP) affected the composition
or structure of this 15-residue glycan subunit, again consistent
with a highly conserved structure. The “type II” (i.e. ␤-1,3linked) Hyp-AGs represented here by interferon Hyp-polysacJOURNAL OF BIOLOGICAL CHEMISTRY
24581
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polysaccharide-1 (15). The chemical shifts of Ala-Hyp-polysaccharide-2 (supplemental Table V) were assigned based on
HSQC (supplemental Fig. 8) and HMBC (supplemental Fig. 9)
spectra compared with those of Ala-Hyp-polysaccharide-1.
Ala-Hyp-polysaccharide-2 Hyp-Gal Linkage and Galactan
Backbone—Cross-peak G of Ala-Hyp-polysaccharide-2 in the
HMBC spectrum established the linkage of ␤-D-Galp (G1) to
position 4 of Hyp (Fig. 5c). G1 with five other backbone ␤-DGalp residues comprised the galactan backbone, confirmed by
the four ␤-D-Galp-(133)-Gal linkages evident in cross-peak E
and by a single ␤-D-Galp-(136)-Gal linkage (cross-peak F).
Thus, Ala-Hyp-polysaccharide-2 had one more backbone Gal
residue than Ala-Hyp-polysaccharide-1.
Ala-Hyp-polysaccharide-2 Side Chains—Like Ala-Hyppolysaccharide-1, interferon Hyp-polysaccharide-1, and interferon Hyp-polysaccharide-2 side chain Gal residues (Ga and
Gb) of Ala-Hyp-polysaccharide-2 were attached to the galactan
backbone through 1– 6 linkages, deduced from cross-peak I.
These two Gal side chains were assigned to G1 and G2 (Fig. 5c,
supplemental Table V), as the chemical shifts were like those
already characterized in Ala-Hyp-polysaccharide-1 (15).
Like the other Hyp-AGs, the ␤-D-GlcUAp residues of Ala-Hyppolysaccharide-2 O-4 substituted with terminal ␣-L-Rhap residues
(cross-peak D in supplemental Fig. 9 and Table V). Furthermore,
the GlcUA residues were O-6-linked to Galsc deduced from crosspeak H. Therefore, Ala-Hyp-polysaccharide-2 contained two sidechain units of ␣-L-Rhap-(134)-␤-D-GlcUAp-(136)-␤-D-Gal.
Judging by the C/H chemical shifts at ␦ 82.3/4.21 ppm in the
HSQC spectrum (supplemental Fig. 8 and Table V), the two
␣-L-Araf residues were terminal residues. Cross-peak C in the
HMBC spectrum indicated two side-chain units of ␣-L-Araf(133)-␤-D-Gal.
NMR Structure Calculations of the Hyp-AGs—Model structures of interferon Hyp-polysaccharide-1 were generated by
simulated annealing, and the 10 best models were further
refined in explicit water and ion environment. These models,
consistent with NOE distance information obtained at 25 °C
(supplemental Table III), depict an overall folded structure with
backbone residues Gal-6 and Gal-1 in proximity forming a
sharp bend or “reverse turn” formed by the ␤-1,6-link between
repetitive ␤-1,3-linked trigalactosyl subunits. We note that
some conformational flexibility was seen in the NMR ensemble
without violating the experimental distance information (Fig.
6). Significantly, ⬃10 intramolecular H-bonds stabilized interactions in which uronic acid carboxyls appeared close enough
to chelate Ca2⫹, a possibility supported by NOE-restrained
molecular dynamics simulation of interferon Hyp-polysaccharide-1 in explicit water and Ca2⫹ (supplemental Fig. 10). The
chelated ion remained strongly bound to the uronic acids without violating the available NOE distance data throughout the
simulation. As an additional test of the global properties of the
structural models, we calculated the hydrodynamic radius of
the structures as 13.9 ⫾ 0.3 Å.
Repetitive Structure of Hyp-arabinogalactans
24582 JOURNAL OF BIOLOGICAL CHEMISTRY
role of ⬃10 intramolecular H-bonds and shrinkage of conformational space (43) by the bulky bifurcated AG side chains;
these limit the conformational AG landscape particularly when
restrained even further in vivo by their attachment to the
polypeptide backbone.
A conserved core structure for type II arabinogalactans suggests a conserved function and some speculations about the
precise roles played by the Hyp-arabinogalactans that decorate
naturally occurring AGPs and the numerous AGP chimeras
that populate the plasma membrane-cell wall interface. Indeed,
in tobacco cells complete coverage of the plasma membrane by
classical AGPs is likely (3).
Although the sheer abundance of classical AGPs at the cell
surface suggests they (and their glycans) are structural molecules, roles for AGPs in signal transduction might arise from
the composition and presentation of residues at the periphery
of the glycans, the regions that also exhibit microheterogeneity.
The compact folded structure deduced here for interferon
Hyp-polysaccharide-1 indicates that the side chains are readily
available for homophilic and heterophilic interactions and the
galactan backbone somewhat less so, especially near the reducing end of the polysaccharide where the larger side chains shield
the galactan backbone.
For example, although all Hyp-arabinogalactans possess a
galactan backbone with arabinosyl side chains, some lack the
abundant uronic acid residues prevalent in the Hyp-arabinogalactans of tobacco (44, 45) where charge repulsions of the
uronic acid residues may form a compression buffer at the cell
surface analogous to animal proteoglycan compression buffers
(15). On the other hand, the close proximity of glucuronic acid
residues may favor Ca2⫹ chelation (supplemental Fig. 10),
which may be relevant to processes involving calcium signaling
(45). Finally, wall AGPs may play a role in regulating cell extension, perhaps by acting as pectic plasticizers (3). The structures
described here may help test these hypotheses.
Acknowledgments—We gratefully acknowledge the School of Life Sciences and the University of Sussex. We are also grateful to Charles
Cottrell of the Ohio State University Campus Chemical Instrument
Center for contributions to the NMR analyses.
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charide-1, interferon Hyp-polysaccharide-2, Ala-Hyp-polysaccharide-2 (Figs. 4 and 5), and Ala-Hyp-polysaccharide-1 (15)
confirm that tobacco Bright Yellow-2 cell Hyp-AGs consist of
small ⬃15-residue subunit repeats with a common composition and linkage pattern differing mainly in the number of trigalactosyl subunits decorated with two side chains each composed of up to 6 residues. Thus, larger Hyp-AGs of up to 150
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example, with the complex rhamnogalacturonan-II pectic
polysaccharide with 12 different sugar residues linked by more
than 20 different glycosidic linkages (35).
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dominate current AGP discussion. However, the location of
classical AGPs at the cell surface and their sheer physical abundance (3) predict primarily structural functions. Structural conservation implies that Hyp-AGs play similar conserved roles in
both classical AGPs and AGP chimeras (3, 29). Nevertheless,
despite considerable speculation (4, 36), specific biological
roles of classical AGPs and their Hyp-AGs remain to be elucidated. The ubiquity of AGPs at the cell surface and involvement
of classical AGPs in numerous fundamental developmental
processes is clear (3, 37– 41), yet AGP function at molecular
levels is relatively unexplored. The current structural elucidation including computer simulations support structural roles
for classical AGPs as follows.
Exclusively ␤-1–3-linked galactans belong to the compact
hollow helix polysaccharide family (42). However, interspersed
␤-1– 6-linkages form “kinks” (17) that may be analogous to the
classical reverse ␤-turns of polypeptides. A molecular model
(Fig. 6) depicts interferon Hyp-polysaccharide-1 as a folded
polysaccharide that forms a moderately compact spheroid consistent with both the NOE data and the hydrodynamic radius
determined experimentally.
Modeling also revealed other conserved features of interferon Hyp-polysaccharide-1 that may contribute to its stability
and molecular function. They include the general stabilizing
Repetitive Structure of Hyp-arabinogalactans
AUGUST 6, 2010 • VOLUME 285 • NUMBER 32
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Supplemental Tables
Supplemental Table I. Chemical shifts arising from Interferon-Hyp-polysaccharide-1 glycosyl and
Hyp residues at 55°C. Two sets of chemical shifts for Hyp residues resulted from base hydrolysis
during preparation of Hyp-AGs. Residue designations correspond to those in Figures 4 and 5
C-1/H-1
C-2/H2
Residues
C-3/H-3
C-4/H-4
C-5/H-5
C-6/H-6
ppm
t-Ara
107.542/5.087
81.047/4.135
76.665/3.960
84.171/4.099
61.440/3.833,3.723
A2
5-Ara
109.179/5.259
81.353/4.228
76.951/4.019
82.473/4.260
67.076/3.891,3.816
A3
3-Ara
109.179/5.261
81.200/4.220
83.877/3.955
84.098/4.147
61.440/3.833,3.723
A4
t-Ara
107.542/5.087
81.047/4.135
76.665/3.960
84.171/4.099
61.440/3.833,3.723
A5
5-Ara
109.179/5.259
81.353/4.228
76.951/4.019
82.473/4.260
67.076/3.891,3.816
A6
3-Ara
109.179/5.261
81.200/4.220
83.877/3.955
84.098/4.147
61.440/3.833,3.723
A7
t-Ara
109.179/5.264
81.352/4.228
76.665/3.960
84.171/4.099
61.440/3.833,3.723
A8
t-Ara
109.179/5.264
81.352/4.228
76.665/3.960
84.171/4.099
61.440/3.833,3.723
R1
t-Rha
100.746/4.797
70.451/3.948
70.582/3.750
72.223/3.538
69.178/4.021
16.628/1.267
16.628/1.267
R2
t-Rha
100.746/4.797
70.451/3.948
70.582/3.750
72.223/3.538
69.178/4.021
UA1
4-GlcUA
103.135/4.511
73.431/3.391
74.791/3.728
79.477/3.625
74.484/3.585
UA2
4-GlcUA
103.135/4.511
73.090/3.361
75.858/3.716
79.477/3.625
74.484/3.585
Ga
3,6-Gal(s)
103.310/4.536
70.009/3.664
80.378/3.728
68.524/4.146
73.567/3.923
69.169/4.041,3.921
Gb
3,6-Gal(s)
103.546/4.487
70.009/3.664
80.378/3.728
68.524/4.146
73.562/3.923
69.169/4.041,3.921
Gc
3-Gal(s)
103.556/4.447
70.838/3.607
80.378/3.728
68.590/4.112
72.497/3.691
61.068/3.784
Gd
3-Gal(s)
103.201/4.406
70.947/3.557
80.378/3.728
68.590/4.112
72.497/3.691
61.068/3.784
G1
3,6-Gal(b)
101.439/4.571
70.030/3.689
82.129/3.854
68.546/4.232
72.869/3.662
69.465/4.041,3.921
G2
3,6-Gal(b)
103.920/4.691
70.030/3.689
81.939/3.812
68.546/4.232
72.869/3.662
70.098/4.041,3.921
G3
6-Gal(b)
104.363/4.644
71.920/3.539
72.869/3.662
69.092/3.903
74.943/3.730
70.435/4.041,3.921
G4
3,6-Gal(b)
103.800/4.703
70.030/3.689
82.135/3.868
68.542/4.232
72.869/3.662
69.185/4.041,3.921
G5
3,6-Gal(b)
103.800/4.703
70.030/3.689
81.821/3.893
68.542/4.232
72.869/3.662
69.986/4.041,3.921
G6
t-Gal(b)
103.695/4.725
71.920/3.539
72.869/3.662
69.092/3.903
75.357/3.712
61.068/3.784
set 1
59.978/4.356
35.130/2.650,2.214
78.020/4.786
51.889/3.683,3.569
set 2
59.978/4.244
34.895/2.564,2.452
77.303/4.708
51.999/3.774,3.483
Hyp
1
Downloaded from www.jbc.org at OHIO UNIV, on October 29, 2010
A1
Supplemental Table II. Chemical shifts arising from Interferon-Hyp-polysaccharide-1 glycosyl and
Hyp residues at 25°C. Two sets of chemical shifts for Hyp residues and G1 resulted from base
hydrolysis during preparation of Hyp-AGs. Residue designations correspond to those shown in
Figures 4 and 5
C-1/H-1
C-2/H2
C-3/H-3
Residues
C-4/H-4
C-5/H-5
C-6/H-6
ppm
t-Ara
107.411/5.075
80.899/4.128
76.559//3.946
83.941/4.089
61.232/3.824,3.718
A2
5-Ara
109.250/5.252
81.316/4.224
76.721/4.009
82.330/4.243
66.747/3.875,3.795
A3
3-Ara
109.250/5.264
81.018/4.223
83.781/3.938
83.880/4.053
61.213/3.825,3.695
A4
t-Ara
107.411/5.075
80.899/4.128
76.559//3.946
83.941/4.089
61.232/3.824,3.718
A5
5-Ara
109.250/5.252
81.316/4.224
76.721/4.009
82.330/4.243
66.747/3.875,3.795
A6
3-Ara
109.250/5.264
81.018/4.223
83.781/3.938
83.880/4.053
61.213/3.825,3.695
A7
t-Ara
109.250/~5.241
81.298/4.207
76.547/3.939
83.788/4.119
61.189/3.827,3.696
A8
t-Ara
109.250/~5.241
81.298/4.207
76.547/3.939
83.788/4.119
61.189/3.827,3.696
R1
t-Rha
100.801/4.732
70.203/3.934
70.115/3.756
71.950/3.430
69.137/4.020
16.442/1.255
R2
t-Rha
100.801/4.732
70.203/3.934
70.115/3.756
71.950/3.430
69.137/4.020
16.442/1.255
UA1
4-GlcUA
102.685/4.505
73.283/3.367
74.286/3.570
79.245/3.577
76.191/3.741
UA2
4-GlcUA
102.685/4.505
72.855/3.337
75.483/3.516
79.175/3.589
75.476/3.712
Ga
3,6-Gal(s)
103.149/4.532
69.879/3.658
80.185/3.758
68.468/4.141
73.139/3.948
69.394/4.026,3.913
Gb
3,6-Gal(s)
103.149/4.492
69.879/3.663
80.185/3.724
68.505/4.106
73.139/3.948
69.394/4.026,3.913
Gc
3-Gal(s)
103.615/4.432
70.652/3.527
80.196/3.646
69.278/3.925
75.030/3.692
60.970/3.768
Gd
3-Gal(s)
103.251/4.388
70.738/3.581
80.196/3.662
69.278/3.944
75.030/3.692
60.970/3.768
Set 1
101.257/4.565
69.870/3.681
81.853/3.859
68.488/4.239
75.097/3.680
69.201/4.026,3.913
Set 2
101.89/4.485
3,6-Gal(b)
G1
G2
3,6-Gal(b)
103.868/4.686
70.115/3.763
81.801/3.869
68.462/4.248
75.097/3.680
69.171/4.026,3.913
G3
6-Gal(b)
104.305/4.622
69.857/3.615
72.456/3.663
69.171/3.924
74.885/3.689
69.433/4.031,3.913
G4
3,6-Gal(b)
103.851/4.708
70.135/3.774
81.689/3.882
68.478/4.238
76.758/3.994
69.171/4.026,3.913
G5
3,6-Gal(b)
103.772/4.723
70.235/3.777
81.668/3.879
68.478/4.252
76.758/3.994
69.171/4.026,3.913
G6
t-Gal(b)
103.887/4.660
70.333/3.774
71.734/3.714
68.940/3.955
75.032/3.772
60.968/3.778
Hyp
set 1
173.98
59.800/4.336
35.186/2.635,2.173
77.970/4.780
51.980/3.675,3.540
59.800/4.236
34.952/2.524,2.438
77.364/4.693
52.010/3.765,3.450
set 2
2
Downloaded from www.jbc.org at OHIO UNIV, on October 29, 2010
A1
Supplemental Table III. NOE assignments of Interferon-Hyp-polysaccharide-1 at 25°C. Italicized
sections highlight the NOEs between Hyp and Gal residue G1, bolded sections highlight nonsequential NOEs and corresponding residues, sequential NOEs are neither bolded nor italicized.
When two distinct proton pair sets produced the same NOEs both pairs are presented.
NOEs
Assignments
4.790/4.579
Hyp-H4/G1-H1
4.791/4.240
Hyp-H4/G1-H4
4.576/3.532
G1-H1/Hyp-H5
Hyp-H4/G1-H3
A7-H1 or A8-H1/Ga-H1
5.233/4.500
A7-H1 or A8-H1/Gb-H1 or UA1-H1 or UA2-H1
5.262/4.511
A6-H1, or A3-H1/UA1-H1, or UA2-H1
4.709/4.146
G4-H1/Ga-H4, or A1-H2, or A4-H2
4.518/4.245
UA1-H1 or UA2-H1 or Ga-H1/A6-H4 or A2-H4 or G2-H4
5.237/4.694
A7-H1 or A8-H1/G2-H1
5.265/4.700
A3-H1or A6-H1 /G4-H1
4.690/4.245
G2-H1/G1-H4
4.677/4.217
G6-H1/G5-H4
4.710/4.038
G4-H1/G3-H61
4.706/3.913
G4-H1/G3-H62
4.392/4.251
Gd-H1/G5-H4
4.436/4.237
Gc-H1/G4-H4
4.620/3.870
G3-H1/G2-H3
4.616/3.767
G3-H1/G2-H2
4.013/5.085
A2-H3/A1-H1, or A5-H3/A4-H1
5.265/3.645
A3-H1/Ga-H2, or A6-H1/Gb-H2
4.220/3.647
A3-H2/Ga-H2, or A6-H2/Gb-H2
5.241/3.653
A7-H1/Gc-H3 or A8-H1/Gd-H3
3.726/4.683
Gb-H3/G2-H1
3.520/4.045
UA2-H3/Gb-H62
4.436/4.702
Gc-H1/G4-H1
4.574/4.687
G1-H1/G2-H1
4.724/3.568
R1-H1/UA1-H4 or R2-H1/UA2-H4
4.724/3.373
R1-H1/UA1-H2
4.029/3.566
R1-H5/UA1-H3
3.373/4.020
UA1-H2/Ga-H61
3.372/3.879
UA1-H2/Ga-H62
3.371/3.921
UA1-H2/Ga-H5
3.374/4.137
UA1-H2/Ga-H4
4.505/4.032
UA1-H1/Ga-H61
4.505/3,888
UA1-H1/Ga-H62
3.351/3.891
UA2-H2/Gb-H61
3.348/4.045
UA2-H2/Gb-H62
4.506/3.912
UA2-H1/Gb-H61
4.247/5.086
A2-H4/A1-H1 or A5-H4/A4-H1
3.886/5.086
A2-H51/A1-H1 or A5-H51/A4-H1
3.798/5.086
A2-H52/A1-H1 or A5-H52/A4-H1
4.540/4.039
Ga-H1/G1-H61
3
Downloaded from www.jbc.org at OHIO UNIV, on October 29, 2010
4.789/3.863
5.241/4.526
4.490/4.245
Gb-H1/G2-H4
4.394/3.902
Gd-H1/G5-H61
4.395/4.028
Gd-H1/G5-H62
4.440/4.037
Gc-H1/G4-H61
4.440/3.891
Gc-H1/G4-H62
4.618/4.241
G3-H1/G2-H4
5.084/4.245
A1-H1/A2-H4, or A4-H1/A5-H4
5.084/3.801
A1-H1/A2-H51, or A4-H1/A5-H51
5.085/3.887
A1-H1/A2-H52, or A4-H1/A5-H52
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4
Supplemental Table IV. Chemical shifts (collected at 55 °C) arising from Interferon-Hyppolysaccharide-2 glycosyl and Hyp residues. Residue designations correspond to those shown in
Figure 5
C-1/H-1
C-2/H2
C-3/H-3
t-Ara
108.0/5.08
81.4/4.21
77.1/3.94
84.4/4.13
61.6/3.71,3.82
A2
5-Ara
109.4/5.24
81.4/4.21
77.5/4.00
84.4/4.13
67.1/3.79,3.88
A3
3-Ara
109.4/5.24
81.1/4.12
82.9/4.24
84.0/4.09
61.6/3.71,3.82
A4
t-Ara
109.4/5.24
81.4/4.21
77.1/3.94
84.4/4.13
61.6/3.71,3.82
A5
3-Ara
109.4/5.24
81.1/4.12
82.9/4.24
84.0/4.09
61.6/3.71,3.82
R1
t-Rha
101.1/4.79
71.0/3.93
70.7/3.69
72.5/3.53
68.2/4.02
Residues
A1
C-4/H-4
C-5/H-5
C-6/H-6
Ppm
t-GlcUA
102.9/4.52
73.8/3.39
76.2/3.78
71.3/3.53
75.8/3.52
UA2
4-GlcUA
102.9/4.52
73.1/3.34
77.0/3.75
79.5/3.62
74.8/3.58
UA3
t-GlcUA
102.9/4.52
73.8/3.39
76.2/3.78
71.3/3.53
75.8/3.52
UA4
t-GlcUA
102.9/4.52
73.8/3.39
76.2/3.78
71.3/3.53
75.8/3.52
Ga
3,6-Gal(s)
103.5/4.49
70.4/3.66
80.5/3.72
68.5/4.13
74.0/3.92
Gb
3,6-Gal(s)
103.5/4.49
70.4/3.66
80.5/3.72
68.5/4.13
74.0/3.92
69.5/4.03,3.92
Gc
6-Gal(s)
104.0/4.43
71.9/3.53
73.1/3.60
68.2/3.93
75.1/3.73
69.5/4.03,3.92
Gd
6-Gal(s)
103.7/4.39
71.9/3.53
73.1/3.60
68.2/3.93
75.1/3.73
69.5/4.03,3.92
G1
3,6-Gal(b)
101.8/4.56
70.4/3.66
82.2/3.83
68.5/4.22
73.0/3.68
70.3/4.03,3.92
G2
3,6-Gal(b)
104.2/4.68
70.4/3.66
82.2/3.85
68.5/4.22
73.0/3.68
70.3/4.03,3.92
G3
6-Gal(b)
104.2/4.68
71.9/3.53
73.1/3.68
68.2/3.93
75.1/3.73
71.0/4.03,3.92
G4
3,6-Gal(b)
104.2/4.68
70.4/3.66
82.2/3.86
68.5/4.22
73.0/3.68
69.5/4.03,3.92
G5
3,6-Gal(b)
104.2/4.71
70.4/3.66
82.2/3.88
68.5/4.22
73.0/3.68
69.5/4.03,3.92
G6
t-Gal(b)
104.2/4.71
61.2/3.77
Hyp
71.9/3.53
73.1/3.64
68.2/3.93
76.0/3.71
set 1
60.5/4.34
35.2/2.62,2.20
78.4/4.78
52.0/3.56,3.67
set 2
60.2/4.23
35.0/2.44,2.55
77.6/4.69
52.3/3.47,3.77
5
69.5/4.03,3.92
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UA1
17.0/1.25
Supplemental Table V. Chemical shifts (collected at 60 °C) arising from Ala-Hyp-polysaccharide-2
glycosyl and Hyp residues. The residue labeling corresponds to the labeling in Figure 5.
C-1/H-1
C-2/H2
C-3/H-3
t-Ara
110.3/5.25
82.3/4.21
77.7/3.95
A2
t-Ara
110.3/5.25
82.3/4.21
R1
t-Rha
101.7/4.81
71.4/3.95
Residues
A1
C-4/H-4
C-5/H-5
C-6/H-6
ppm
85.1/4.13
62.4/3.71,3.82
77.7/3.95
85.1/4.13
62.4/3.71,3.82
71.4/3.78
73.2/3.42
70.1/4.02
17.6/1.25
17.6/1.25
t-Rha
101.7/4.81
71.4/3.95
71.4/3.78
73.2/3.42
70.1/4.02
4-GlcUA
103.7/4.51
74.4/3.45
77.4/3.73
80.2/3.63
75.5/3.56
UA2
4-GlcUA
103.7/4.51
74.4/3.45
77.4/3.73
80.2/3.63
75.5/3.56
Ga
3,6-Galsc
104.5/4.47
71.7/3.65
81.7 /3.71
69.6/4.11
74.7/3.91
70.5/4.04,3.94
Gb
3,6-Galsc
104.6/4.48
71.7/3.65
81.7/3.71
69.6/4.11
74.7/3.91
70.5/4.04,3.94
G1
3,6-Galbb
102.5/4.57
71.6/3.65
83.0/3.87
69.7/4.23
74.8/3.77
70.5/4.04,3.94
G2
3,6-Galbb
105.0/4.64
71.6/3.65
83.0/3.85
69.7/4.23
74.8/3.77
70.5/4.04,3.94
G3
6-Galbb
105.0/4.67
72.7/3.54
74.0/3.65
70.0/3.92
74.8/3.77
70.5/4.04,3.94
G4
3-Galbb
105.0/4.68
71.6/3.65
83.0/3.87
69.7/4.12
76.0/3.70
62.3/3.78
G5
3-Galbb
105.0/4.69
71.6/3.65
83.0/3.87
69.7/4.12
76.0/3.70
62.3/3.78
G6
t-Galbb
105.0/4.72
72.7/3.54
74.0/3.65
70.5/3.93
76.2/3.70
62.3/3.78
set 1
61.0/4.32
36.5/2.62,2.19
79.1/4.77
52.9/3.54,3.64
Hyp
set 2
61.3/4.21
36.1/2.40,2.55
78.4/4.68
52.9/3.47,3.77
6
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R2
UA1
Supplemental Figure Legends
Supplemental Figure 1. A TOSCY spectrum of Interferon-Hyp-polysaccharide-1 collected at 55oC
aided in the assignment of 1H chemical shifts and Cross-peaks A indicated side-chain Gal residues Gc
and Gd were substituted at position 3.
Supplemental Figure 2. DOSY spectrum of Interferon-Hyp-polysaccharide-1 collected at 25oC used to
calculate the diffusion coefficient.
Supplemental Figure 3. NOESY spectrum of Interferon-Hyp-polysaccharide-1 (25 °C).
Supplemental Figure 4. 1H NMR Spectrum of Hyp-AG Interferon-Hyp-polysaccharide-2 collected at
55 °C. Signals A and B were assigned to H-1 of α-L-Ara, signal C to H-1 α-L-Rha, signal D (shoulder
of C) to H-4 of Hyp, and signal E to the β-D-Gal residues of the galactan backbone. Signal F was
assigned to H-1 of β-D-Gal linked to Hyp, and signal G to H-1 of side-chain β-D-Gal and β-D-GlcUA
residues.
Supplemental Figure 6. HSQC Spectrum of Hyp-AG Interferon-Hyp-polysaccharide-2 collected at 55
°C. All labelling corresponds to those featured in Figure 2.
Supplemental Figure 7. HMBC Spectrum of Hyp-AG Interferon-Hyp-polysaccharide-2 collected at
55°C. All labelling corresponds to those featured in Figure 3.
Supplemental Figure 8. HSQC spectrum of Ala-Hyp-polysaccharide-2 collected at 60 °C. All
labelling corresponds to those featured in Figure 2.
Supplemental Figure 9. HMBC spectrum of Ala-Hyp-polysaccharide-2 collected at 60 °C. All
labelling corresponds to those featured in Figure 3.
Supplemental Figure 10. A stable folded conformer of Interferon Hyp-polysaccharide-1 depicts a
‘reverse-turn’ galactan backbone (dark blue) with carboxylate chelation of a calcium ion (yellow) by
sidechain glucuronic acid residues (purple). Peripheral -L-arabinosyl trisaccharides are depicted by
light blue 5-membered rings and sidechain Gal residues by light blue 6-membered rings. Not all
residues are visible in this presentation.
7
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Supplemental Figure 5. 2D TOCSY spectrum of Interferon-Hyp-polysaccharide-2 collected at 55 °C.
The spectrum was used for 1H chemical shift assignments. Based on our earlier work (15) Signal A
indicated the side-chain Gal residues Gc and Gd were unsubstituted at position 3.
Supplemental Figure 1
Downloaded from www.jbc.org at OHIO UNIV, on October 29, 2010
8
Supplemental Figure 2
Downloaded from www.jbc.org at OHIO UNIV, on October 29, 2010
9
Supplemental Figure 3
Downloaded from www.jbc.org at OHIO UNIV, on October 29, 2010
10
Supplemental Figure 4
Downloaded from www.jbc.org at OHIO UNIV, on October 29, 2010
11
Supplemental Figure 5
Downloaded from www.jbc.org at OHIO UNIV, on October 29, 2010
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Supplemental Figure 6
Downloaded from www.jbc.org at OHIO UNIV, on October 29, 2010
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Supplemental Figure 7
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Supplemental Figure 8
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Supplemental Figure 9
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Supplemental Figure 10
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