Vol. 269, No. 5, Issue of February 4, pp. 3331-3338, 1994
Printed in U.S.A.
THEJOURNAL
OF BIOIKCIC~.
CHEMI~Y
0 1994 by The American Society for Bioehemietry and Molecular Biology, Inc
'H N M R Studies on an Asn-linked Glycopeptide
GlcNAc-1 C2-N2 BOND IS RIGID I N HZO*
(Received for publication, May 27, 1993)
Jeffery T.Davis$$, Shirish Hiraninll, Catherine Bartlettn, and BrianR. Reid**
From the $Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742, the
YGenzyme Corporation, Cambridge, Massachusetts 02139, and the **Departments of Chemistry and Biochemistry,
University of Washington, Seattle, Washington 98195
33131
Downloaded from www.jbc.org at UNIV OF MARYLAND on June 12, 2008
tide conformation signals N-glycosylation; predominant peptide conformations have been proposed for the N-glycosylation
site (Bause and Legler, 1981; Bause, 1983;Abbadi et al., 1986,
Beintema, 1986;Imperiali and Shannon,1991; Imperiali et al.,
1992). Whatever the peptide's conformational preference, N glycosylation might be expected to alter this structure. However, little is known about intramolecular sugar-peptide interactions and theirrole in controlling glycoprotein structure and
function.
The crystal structure of a human IgG F, domain, with carbohydrate linked to Asn2", shows hydrogen bonds and hydrophobic interactions between polypeptide and oligosaccharide
domains (Deisenhofer, 19811, and energycalculations for these
specific glycan-peptide interactions have been done (StuikePrill and Meyer, 1990). Solution studies have alsobeen undertaken on a variety of Asn-linked carbohydrates. While much
NMR work has utilized asparaginyl oligosaccharides, with a
single Asn attached to the glycan (Bush et al., 1982; Brisson
and Carver, 1983a,
1983b; Sawidou etal., 1984; Homans et al.,
1987a, 1987b; Berman etal., 19881, these studies havefocused
on determining saccharide and
glycoside conformation, and not
on peptide-carbohydrate interactions. Other studies have addressed thetopic of peptide-carbohydrate interactions.Solution
NMR of the model compounds GlcNAc@l-NAsn (Bush et al.,
1980) and t-butoxycarbonyl-Asn (G1cNAc)-Gly-Ser-OMe(Ishii
et al., 1985) did not show any peptide-sugar interactions.Also
'H NMR showed that glycosylation of a cyclic hexapeptide with
GlcNAc attached to a Gln y-carboxamide did not change the
peptide backbone conformation (Kessler etal., 1991).
Largersystemshave also been examined.
NMR studies
on RNAse B indicated that theoligosaccharides have little effect on protein conformation and dynamics (Berman et al.,
1981). Brockbank and Vogel(1990) used 'H NMR to study the
The attachmentof oligosaccharide to theAsn y-carboxamide glycoprotein phosvitin, which has complex-type oligosacchariin generic Asn-Xaa-Serfl'hr N-glycosylation sites is a cotrans- des linked toa single N-glycosylation site. They concluded that
lational protein modification (Kornfeld and Kornfeld, 1985). oligosaccharide conformation is unperturbed by the protein.
Eukaryotic oligosaccharyltransferasesrequire an Asn-Xaa-Serl Wormald et al. (1991) studied the conformational effects of
"hr tripeptide for N-glycosylation (Marshall, 1972). Modifica- oligosaccharide on an IgM 22-amino acid tailpiece fragment.
has been statistically evaluated They also found no changes in the oligosaccharide or peptide
tion at the consensus sequence
for a number of proteins; Pro neveroccupies the Xaa position, backbone structureandsuggested
that the oligosaccharide
or position 4 following the SerPThr residue (Gavel and von causes a decrease in the
peptide's conformational mobility near
Heijne, 1990). This strict sequenceselectivity implies that pep- the glycosylation site. Joao et al. (1992) also concluded that
N-glycosylation has little effect on RNAse protein structure,
* This work was supported in part by National Institutes of Health but may affect the local dynamics and stability of the protein.
Small Business Innovative Research Grant Ft44GM39656-02 (to the Similarly, NMR investigations of 0-linked glycopeptides sugGenzyme Co.). The costs of publication of this article were defrayed in
part by the payment of page charges. This article must therefore be gest that the majorconsequences of 0-glycosylation is to
hereby marked "advertisement" in accordance with 18 U.S.C. Section change protein dynamics and to limit thelocal conformational
1734 solelyto indicate this fact.
space available to the peptide
(Dill and Carter,1986; Dill et al.,
0 Supported by NationalInstitutes of Health National Research Ser- 1990; Andreotti and Kahne,1993).
vice Award Postdoctoral FellowshipGrant F32 GM14340-01. To whom
The general consensusof the studies reported to date
is that,
correspondence should be addressed.
11 Current address: Immulogic Pharmaceutical Co., Waltham, hlA despite the size and complexity of the carbohydrate, N-glyco02154.
sylation causes little structural change in thepeptide. There-
The conformation of an Am-linked glycopeptide in
HzO was studied by two-dimensional 'H NMR. Nonexchangeable proton and exchangeable amide (NH)proton resonances were assigned for the hen ovomucoid
glycopeptideI , SerIle-Glu-Phe-Gly-Thr"ne-Ser-Lys,
with pentasaccharide Manu13 (Manu14)Manpl4GlcNAcpl4GlcNAc~1-NH
attached to the Asn7 ycarboxamide. The pentasaccharide increases the local correlation times of amino
acid
residues near the
N-glycosylation site. Nuclear Overhauser effect (NOE)
measurements on I and the corresponding Man3GlcNAcz pentasaccharide 3 show that theattached p e p
tide doesnot perturb 0-glycosideconformation.Sequential dm (i, i+l) NOES in the Thrs-SePregion
indicate populations of folded structure near the N-glycosylation site of both glycopeptide I and aglycosyl
peptide 2. However, the Man,GlcNAcz pentasaccharide
does not dramatically affect the average conformation
of either the peptide backbone or the Asn' side chain.
GlcNAc NH protons were studied at pH 3.0; and NOE
and %JNHdata were used to constrain the glycopeptide's GlcNAc-1 side chain dihedral angle (7)(Cl-C2-N2C7(Ac)). The glycopeptide's core GlcNAc-1 C2-N2 side
chain bond is not flexible in H,O. A strong GlcNAc-1
NH2-H3 NOE, a medium strength NH2-HI NOE, and a
weak NH2-H2 interaction suggest that GlcNAc-1 has a
rigid C2-N2 bond, with T between 95 and 115".No evidence was found
for intramolecular hydrogen bondsrestricting this C2 side chain torsion. It may be that
GlcNAc-1's rigid planar N-glycosidic linkage limits the
conformational space availableto the adjacent C2 acetamido side chain.
3332
Glycopeptide NMR Studies
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fore, we wanted to determineif the peptide influences the core peptide NH chemical shift temperature dependence was donein aqueGlcNAc's conformation. In 0-linked glycopeptides, where Gal- ous solution over a temperature range of 1 0 3 0 "C. Increments of 3 "C
NAc is attached to a Ser or Thr y0, the a-linked sugar may were used, and the probe temperature was calibrated with a MeOH
standard. For the temperaturedependence experiments a spectral winstabilize structure by forming anintramolecular hydrogen dow of 4386 Hz was collected into 16,000 complexpoints. Temperature
bond between GalNAc's C2 acetamidocarbonyl and the Thr NH coefficients are reported in partdbillioddegree Kelvin.
(Maeji et al., 1987). Similar sugar-peptide interactions may
Zbo-dimensional NMR Experiments-NOESY experiments in DzO
exist inN-linked glycopeptides. To gain information about local and Me2SO-d,utilized the standardpulse sequence (Kumar et al., 1981)
peptide-sugar interactions, we used lH NMR in H20to exam- using the phase-sensitive method (States et al., 1982).In each NOESY
ine exchangeable GlcNAc NH protons in a homogenous experiment the mixing time was varied by 10%to eliminate zero-quantum coherence. Forall NOESY experiments, 2048 complexpoints in t2
N-linked glycopeptide. Only recently have sugar NH-sugar lH and 800 points in t l were collected with relaxation delays of 2.5 s and
correlations been usedto studyN-linked glycoprotein structure a spectral width of 4386 Hz. For each tl experiment 32 scans were
(Brockbank and Vogel, 1990) or to determine hexosamine side obtained. The NOESY data was zero-filled to 2048 points in each dichain conformation in GlcNAc-containing oligosaccharides mension and apodized with a sine-squared 90% phase-shifted function
(Dabrowski and Poppe, 1989; Poppe et al., 1990; Cagas et al., to 800 points in t2 and 400 points in t l . NOESY experiments in 90%
HzO, 10%DzO were doneusing the 1-1 echo water-suppression method
1991).
(Sklenar and Bax, 1987). Delays
of T~ = 140 ps and 7, = 280 ps were used
We report an NMR study of the glycopeptide 1 , Ser-Ile-Glu- to maximize NH excitation. A 10-ms homospoilpulse was applied durPhe-Gly-Thr-A-sn'Ile-Ser-Lys, with
the
pentasaccharide
ing the mixingperiodtoremove
transverse water magnetization.
Manal-3(Mana1-6)Manp1-4GlcNAc~1-4GlcNAcpl-NH at- COSY-45 (Bax and Freeman, 1981)and TOCSY experiments (Davis and
tached to the Asn7 y-carboxamide. We provide a comparison of Bax,1985) facilitated resonance assignments. TOCSY experiments
NMR data in aqueous solution for glycopeptide, 1, aglycosyl were performed in the TPPI mode (Drobny et al., 1979).
and Man,peptide 2, Ser-Ile-Glu-Phe-Gly-Thr-Am-Ile-Ser-Lys,
RESULTS AND DISCUSSION
GlcNAc2 pentasaccharide 3. Although it appears that the oligosaccharide and protein do not affect each other's solution
Glycopeptide NMR-TheB,-2
glycopeptide, containing the
structure, the glycopeptide's GlcNAc-1 Cl-N2 side chain bond Asn53N-glycosylation site, was obtained by proteolysis of hen
has a rigid orientation in HzO. This leads to the question of ovomucoid (Yet et al., 1988). A single Asn-Xaa-Serrnr protein
whether the peptide, attached to GlcNAc-1 C1, has a role in N-glycosylation site may have different oligosaccharide serestricting the sugar's C2 side chain conformation.
quences associated with it. For instance, B,-2 has at least 10
different complex-type oligosaccharides attachedto
(BeeEXPERIMENTALPROCEDURES
ley, 1976; Yet et al., 1988), withnumerous p-Gal and p-GlcNAc
Materials and Sample Preparation-The glycopeptide 1, Ser-Ile-Glu- sugars joined to the a1,3 and u1,6 antennae of an invariant
Phe-Gly-Thr-Asn'Ile-Ser-Lys,with Mano!l-3(Mana1-6)Man~l-4Glc- ManaGlcNAcz core. One of our goals is to determine if the
NAcpl-4GlcNAcpl-NH attached to A
m
' (M, 1908)was prepared from attached peptide influences sugar C2 side chain conformation
a hen ovomucoid B,-2 mixture (Yet et al., 1988).Terminal sugars on the
in the internal GlcNAcpl-4 GlcNAc disaccharide. In order to
B,-2 oligosaccharide's Manal,3 and Manal,6 antennae were removed
by sequential treatment with jack bean P-galactosidase (Sigma) and achieve this goal we needed to be able to correlate theGlcNAc
jack bean P-N-acetylglucosaminidase(Sigma). B,-2 glycopeptide (30 side chain NHwith the nonexchangeable GlcNAc ring protons.
mg) was incubated with P-galactosidase (4 units) in 0.1 M sodium cit- Brockbank and Vogel (19901, in their study of complex-type
rate, pH 4.0, at 37"C for 48 h. This material was then treated with oligosaccharides attached to thephosvitin glycoprotein, found
P-N-acetylglucosaminidase(40 units) in 0.1 M sodium citrate, pH 5.0,at that the antennary GlcNAc NH and GlcNAc-1 NH chemical
37 "C for 72 h.
shifts were severelyoverlapped. We simplified NMR analysis of
Glycopeptide 1 was purified by reverse-phase chromatography on a
Sep-Pak Cls cartridge (Waters). Free sugars were eluted with MeOH, GlcNAc NH protons by removing @Gal and P-GlcNAc residues
and the glycopeptide was eluted with HzO. Purity was assayed by from the a1,3 and a1,6 antennae.Glycopeptide 1 , Ser-Ile-Glureverse-phase HPLC,' carbohydrate analysis, amino acidanalysis (Wa- Phe-Gly-Thr-Asn*Ile-Ser-Lys,
withManal-B(Manal-6)Manters Pico-Tag),and 500-MHz 'H NMR. Aglycosylpeptide 2 , Ser-Ile-Glu- /31-4GlcNAc/31-4GlcNAc~l-NH
connected to A m 7 , was prePhe-Gly-Thr-Am-Ile-Ser-Lys,
was prepared by solid-phase synthesis. pared from B,-2 as described under "Experimental Procedures"
Peptide purity was established by HPLC and one-dimensional NMR
and summarized inFig. 1.
analysis. NMR samples were typically between2 and 10 m~ in peptide
NMR in D,O: Nonexchangeable Protons and 0-Glycoside
in 50 m~ sodium phosphate (pH or pD 3.0-4.0).
The Man,GlcNAc, pentasaccharide 3 was prepared from glycopep- Conformation-The 500-MHz 'H NMR spectra of glycopeptide
shown in Fig. 2. Glycopeptide 1 by digestion with recombinant peptide-N4-(N-acetyl-p-~-glu-1 and pentasaccharide3 in D20 are
cosaminy1)asparagineamidase F (N-GlycanaseTM)
(Hirani et al., 1987). tide resonances were assigned
from two-dimensional COSY,
A solution containing glycopeptide 1 (9 mg) and 75 units of N-Gly- TOCSY, and NOESY experiments and are listed in
Table I.
canaseTM(Genzyme Co.) was incubated in 0.1 sodium phosphate, pH
Sugar proton assignments werefacilitated by comparison with
8.6, a t 37 "C for 24 h. Pentasaccharide 3 was separated from deglyco1983;
sylated peptide on a Sep-Pak CIS cartridge by elution with MeOH. data on related oligosaccharides (Vliegenthartetal.,
Pentasaccharide 3 purity was assayed by HF'LC, carbohydrate analysis, Brisson and Carver, 1983a, 1983b; Homans etal., l986,1987a,
1987b). Fig. 2 shows bothcompounds to be analytically pure; all
and 500-MHz 'H NMR.
One-dimensionalNMR Experiments-NMR spectra were acquired at the antennary p-Gal and p-GlcNAc residues were removed by
500 MHz on either a Bruker AM-500 spectrometer or on a home-built the exoglycosidase treatment. Characteristic sugarresonances
spectrometer.2 One-dimensional spectra in DzO and MezSOd6 were
belong to the H1
anomeric protons,located between 5.2 and 4.6
collected using 8192 complex points with an acquisition time of 0.66 s
and a spectral width of 4386 Hz. The field homogeneity was adjusted ppm, and thetwo GlcNAc methyl group protons near 2.0 ppm.
until the HDO resonance line width was no greater than 2-3 Hz. For The rest of the glycopeptide's nonexchangeable sugar protons,
samples in 90% H,O, 10%D,O solvent suppression was achieved using with various aminoacid C" and CP protons, are located between
a 1-1 echo method(Sklenar andBax, 1987).NMR data were transferred
4.6 and 3.5 ppm.
to an IRIS 4D/20 workstation and processed using the Felix NMR soRMany of the nonexchangeableprotons of the individual
ware program (Hare Research Inc., Woodinville,WA). Determination of monosaccharide spin systems in
glycopeptide 1 were identified
from the combined TOCSY and NOESY experiments (Fig. 3).
The abbreviations used are: HPLC, high performance liquid chro- The strong35coupling between p-GlcNAc H1 and H2(3J1,
2 =
matography; Me2S0, dimethyl sulfoxide; NOE,nuclear Overhauser ef8.0
Hz)
distinguished
hexosamine
protons
from
those
of
manfect.
nose ( 3 J l , 2 = 1-2 Hz) inthe TOCSY spectrum (Fig. 3A).
J. Gladden and G. P. Drobny, unpublished data.
Glycopeptide NMR Studies
f'
G~,,,-GICNAC,-M=~~
\
/
1
A
3333
I
GLYCOPEPTIDE1
2 1
6~an~1~GlcNAc~l4GlcNAc~l-N~-~7
3
G~,,,-GICNAG-M-~
I
3
LyslO
1) p-Gakctosidase
2) 0-N-Acetyl-
&
C
O
d
~
~
7
6 ManOl4GlcNAc~1-1GlcNAc~l-NH-Aw~
1
/3
3
2
ManPl
1
1
I
Maw1
3
B
I
PENTASACCHARIDEB
2
,I'
3) N-GlycanaseTM
Ma@4GlcNAcpl4GlcNAc
a
Scheme showing enzymatic preparation of glycopep3 from hen ovomucoid
tide I and MansGlcNAcp pentasaccharide
B,-2. The heterogenous glycopeptide B,-2 mixture was prepared
by
proteolysis of hen ovomucoid according to Yetet al. (1988). The peptide
sequence of B,-2 was NH2-SIEFGTN'ISK-COOH, andthe starting glycopeptide had a mixture of oligosaccharides attachedto Asn' (for composition see Yet et al., 1988). Procedures for the exoglycosidase and
N-GlycanaseTMdigestions aredescribedunder"ExperimentalProcedures." The numbering of the pentasaccharideprimarystructure is
according to Homans et al. (1987a).
FIG. 1.
GlcNAc-1 ring protons, H1, H2, H3, H4, and H5 are all clearly
resolved (Table I), with GlcNAc-1 H2 and H3 being separated
by almost 0.1 ppm. This chemical shift resolution is crucial
when using side chain NH2-sugar 'H NOEs to determine the
GlcNAc C2-N2 torsion angle (see below). The NOESY experiment in DzO,besides showing intraresidue sugar NOEs, identified sequential sugar connectivities in glycopeptide 1 (Fig.
3B). Interresidue NOEs, due to dipolar interactions across glycosidic linkages, can be used to determine 0-glycosidic bond
angles (Homans et al., 1987a).
With O,T, close to 1.0 at 500 MHz and 25 "C, negative NOEs
are not seen in DzO for the aglycosyl peptide 2. In order to
observe NOEs for
2, it isnecessary to either lower the temperature to 1-5 "C or perform the experiment in the more viscous
MezSO. In contrast, intraresidue amino acidNOEs are observed in glycopeptide 1 at 25 "C in DzO.Apparently, the rigid
pentasaccharide causes a significant decrease in the peptide's
conformational mobility near the N-glycosylation site. In addition to the sugar correlations, relatively strong NOE crosspeaks are observed between the main chain C"H and side chain
C@Hof Thr6, A m 7 , and Ile8, and between T h r 6 C@H-%H,and
Iles CPH-OH. Very weak C"H-C@HNOEs are observedfor
Phe4, and no connectivities are seen for the N-terminal residues, Ser1-Glu3,or the C-terminal Selg-Lys'O residues. These
observations are similar to those of Wormald et al. (1991) in
their study of N-glycosylation effects on an IgM peptide fragment; they also found that thelocal correlation times of amino
acid side chains were noticeably increased by N-glycosylation.
No dramatic changes in 0-glycoside conformationoccur
when the decapeptide is attached to the MansGlcNAcz pentasaccharide. NOESY experiments with 7, = 800 ms, where longer range distances would be observable, revealed no sugarpeptide NOE cross-peaks. Also, time-dependent NOESY
experiments in DzO ( T =~200-800 ms) showed that interresi-
6
4:8
4:O
3:2
2:4
1:s
PPm
FIG.2. A, one-dimensional 'HNMR spectrum of glycopeptide 1 in
D20 (50 m~ sodium phosphate, pD 3.0)at 25 "C. Thepentasaccharide's
H1 anomeric protons and GlcNAcmethyl groups are indicated. B , onedimensional 'H NMR spectrum of MansGlcNAcz pentasaccharide3 in
D20at 5 "C.
due and intraresidue sugar NOE intensities were similar for
glycopeptide 1 and pentasaccharide 3 (Table 11).Due to spectral
overlap intersaccharide NOEs intheManpl-tGlcNAcpl4GlcNAccore (NOEs between Man3 H1-GlcNAc2H4 and
GlcNAc2 H1-GlcNAcl H4) were not measured. However the
cross-peak volumes of these overlapped peaks were similar in
the two compounds.Relative NOE intensities, using the intraresidue Man Hl-H2 NOE as a reference, could be compared for
both the Mancul-3 linkage (NOEs between Man4 H1-Man3 H3
and Man4 H1-Man3 H2) and Mancul-6 linkage (NOEs between
Man4' H1-Man3 H6, H6') in1 and 3 (see Fig. 3B). There are no
long-range sugar-peptide interactions that perturb the NOEs
across these two 0-glycosidic linkages.
NMR Studies in HzO: Exchangeable Amide NH ProtonsGlycopeptide 1's NMR spectrum in HzO has a single set of 12
NH resonances between 8.0 and 9.0 ppm. Nine of the signals
are due to peptide main chain NH protons and the other three
resonances belong to the pentasaccharide, namely the N-glycosidic NH (GlcNAc-1 NH1) and the two GlcNAc C2 side chain
NH protons (GlcNAc-1 NH2and GlcNAc-2 NH2). NH chemical
shifts, ,JNH values and NH temperature coefficients (-AWAT)
in HzO for glycopeptide1 and peptide 2 are presented in Table
111. The NH chemical shifts and 3 J m coupling constants are
consistent with values for a random coil peptide (Wuthrich,
1986). Importantly, GlcNAc-1 NH2at 8.28 ppm is well resolved
from the other NH resonances.
Peptide Structure-For aglycosyl peptide 2 in HzO at 5 "C,
sequential daN (i, i+l) NOES are observed for Phe4-Lys10 (Fig.
41, consistent with an extended conformation in this region.
Sequential d m (i, i+l) NOEs, present only betweenThr6-Asn7,
Asn7-Ile8,and Iles-SeP, indicate transient secondary structure
near the peptide's N-glycosylationsite. Sequential d m (i, i+l)
NOEs in flexible peptides reflect populations of turn or helical
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/
3
LyslO
Glycopeptide NMR Studies
3334
TABLEI
H chemical shifis of nonexchangeable protons for glycopeptide 1, in D20a t 25 "C (in ppm)
A. Pentasaccharide
Residue
H1
GN1
3.85
GN2
3.75
Man3
4.28
Man4
Man4'
.76
.56
.80
5.03
3.78
4.61
4.79
3.90
5.12
4.93
H2
H3
H4
H5
ND
NDND
ND
ND
ND
ND
ND
3.78
4.09
4.00
ND
3.91
H6
H6'
ND
ND
3.83
ND
CH3
2.01
2.09
3.94
ND
B. Decapeptide
Residue
CPH
Ser'
Ile2
~ 1
Phe4
Gly6
1.90
C"H
0.87
~
3
4.01
4.23
The
2.77
1.89
3.88
1.82
Asn7 2.89,
Ile8
SeP
Lys'O
4.49
4.40
4.38
4.62
3.92,
4.35
4.78
4.22
4.49
4.36
CaH
3.87
0.90,
1.89
C'H
CH3
1.48, 1.20
2.20, 2.12
3.23, 3.17
1.19
1.41
1.71
3.00
TOCSY
3 H2
3 H1
0
3
4' H1
FIG.3. Two-dimensional TOCSY
and NOESY spectra of glycopeptide
1. Glycopeptide 1 (5 m)was dissolved in
400 pl of D20 150 m sodium phosphate,
pD 3.0, 25 "C). A, expanded Hl-H2, -3, -4,
-5, -6 region of the two-dimensional
TOCSY ( T = 100 ms),showing nonexchangeable sugar 'H sugar 'H correlations for individual saccharide spin systems. Theanomeric H1 resonances for the
five sugars arelabeled. GlcNAc-1 H1, H2,
H3, H4, and H5 resonances are all clearly
resolved. E , the same expanded region of
the two-dimensional NOESY experiment
showing interresidueandintraresidue
sugar 'HJH NOE cross-peaks. Interresidue NOE cross-peaks indicating dipolar
interactions across glycosidic linkages are
boxed.
4 H1
4'
H2
4
H3
H4
OflD &
Q
1 H2
0
-
\
Iri
~~
2H1-1H4
B
2 HI '<
'
.Iri
4 H2
-.
~
3 H1
H5
NOESY
j
3H1-2H4
o
0
4'H1-3H6'
4'H1-3H6
4' H1
-_
,_""
i"""
#
"
4.'1
4.'0
G3
\
3:9
I"
""
3.6
3.'?
316
3.
PPm
structures, with dm = 2.3-3.4 (Wuthrich, 1986; Dyson et al.,
1988). In an extended conformation the average d m is 4.5 A,
too long of a distance to show an NOE for a molecule of M, 1900
and W T ~= 1.1.
Because the NOESY cross-peaks are weak in HzO, due to O T ~
= 1.0,experiments were repeated in the more viscous Me2S0 at
18 "C. Time-dependent NOESYs ( T =~ 80, 120, 200, and 400
ms) in MezSOrevealed that thesequential NH-NH cross-peaks
Downloaded from www.jbc.org at UNIV OF MARYLAND on June 12, 2008
A
Glycopeptide NMR Studies
TABLE
I1
Relative nuclear Overhauser enhancements for the Manal3Man
and Manal-6Man units in glycopeptide 1 and inMan3GlcNAc2
pentasaccharide 3 in D20 a t 25 "C
The intrasaccharide Man H1-H2 NOE in each linkage was used as a
reference. Cross-peak volumes were measured above and below the
diagonal in NOESY experiments with 7, = 400 ms.
Relative cross-peak volume
NOE Cross-peak
Glycopeptide 1
Pentasaccharide 3
1.0
2.3
0.27
1.0
2.1
1.0
0.28
0.56
1.0
0.24
0.61
Man4 1-3 Man3 linkage
Man4 Hl-H2
Man4 H1-Man3 H3
0.37
Man4
H1-Man3 H2
Man4' 1-6 Man 3 linkage
Man4' Hl-H2
Man4' H1-Man3 H6
Man4' H1-Man3 H6'
~
8.70 (ND)
8.60 (8.59)
8.60 (8.71)
8.49 (8.54)
8.08 (8.23)
(8.70)8.64
8.07 (8.27)
8.46 (8.57)
8.10-8.50'
8.68
8.28
4.68.51
(7.66)
(6.95)
GN1 HZ
i
3
P
E
a
I_
P
Chemical
shifts"
3
GN1 H 3
AWAP
6.0
ND (7.5)
ND (7.2)
ND (7.0)
ND
7.0 (7.3)
7.3 (7.2)
7.3 (7.9)
6.5 (ND)
7.3 (7.7)
9.0
9.1
8.2
(6.0)
5.8 (5.8)
6.6 (4.8)
6.6 (5.9)
5.5 (5.7)
4.6 (5.0)
5.5 (4.3)
5.2 (4.5)
5.2 (5.4)
5.1
6.3
P
Am7
8.4
8.0
PPm
F'Ic. 5. Expanded region of the two-dimensionalNOESY spectrum (7, = 400 ms) for glycopeptide 1 in 90% H20, 10%D20(50
m~ sodium phosphate, pH 3.0,5 "C) showing d, (i, i+l)connectivities. Intraresidue doN(i, i+l) NOEs from Gly" to Serg are labeled.
to Lys'O are connected by a line.
Sequential daN (i, i+l)NOES from Gly"
The intraresidue sugar NOEs, GlcNAc-1 NHl-H2, GlcNAc-1 NH2-H2,
and GlcNAc-1 NH2-H3 are boxed.
other regions of the peptide backbone.
Glycopeptide 1,compared with peptide 2, has a similar NOE
Ilea
pattern in HzO (Fig. 4). Fig. 5 shows the fingerprint NH-C"H
SeP
region for glycopeptide 1. Sequential daN (i, i+l) connectivities
Lys'O
are observed only for GlyS-LyslO. Glycopeptide 1 also had seGNl NH1
quential dm (i, i+l) NOEs between Thr6 NH and A m 7 NH,
GN1 NH2
GN2 NH2
Asn7 NH and Ile* NH, and between Ile8 NH and S e P NH,
(6.0)
hn7E
confirming the presence of nonrandom secondary structure in
Asn7z
(7.1)
the N-glycosylation site. However, these d m (i, i+l) crossa Chemical shifts in ppm were measured at 5 "C and 3Jm coupling
peaks remained weak, just barely out of the noise at 7, = 400
constants (Hz) were measured at 10 "C.
ms, indicating thatglycosylation does not significantly change
NH temperature coefficients (AUAT) are presented in -ppbiK.
The chemical shift of Lys'O NH is sensitive to pH, at pH 4 . 0 : =
~ 8.10, the average peptide backbone structure. Also, N-glycosylation
while at pH 3.0: u = 8.50.
at A m 7 did not cause anynew medium range peptide NOES to
appear in the
glycopeptide's spectrum. Theseexperiments show
5
10
1
that both glycopeptide 1 and aglycosyl peptide 2 sample nonS I E F G T N ' I S K
random secondary structure in H20.
While the N-glycosylation
site, Am7-Iles-Sere, can adopt folded backbone conformations
in both compounds, the NMR data suggest that the pentasaccharide does not alter the peptide backbone structure.
N-Glycosylation has been proposed to limit the conformational space around theAsn side chain (Wormald et al., 1991).
The restrictionof conformational space due to N-glycosylation
might be indicated by a change in either theAsn7 C"-C@bond
angle (xl) andor the C@-Cvbond angle (xz).There are three
major rotamers for xl:one trans ( t = 180") and two gauche (g+
= 60" andg- = 60') (Ishii et al., 1985) whose populations can be
FIG.4. Sequential intraresidue and interresidue
NOEs involv- estimated from 3J coupling between Asn C"H and C@protons
ing backboneNH protons in glycopeptide1 and aglycosyl pep- (Cung and Marruad, 1982). The Asn7 C"H-C@H,H' coupling
tide 2. The solid bars above the lines represent NOE cross-peaks pre- constants, JAx and JBX, were similar for both glycopeptide 1
sent in glycopeptide 1,whereas the hatched bars represent cross-peaks
and aglycosyl peptide 2:JM = 6.0 and JBx = 6.8 in glycopeptide
present in aglycosyl peptide 2.
1 , and JAx = 6.3 and JBx= 7.4 in aglycosyl peptide 2. For these
in the Asn-Ile-Ser glycosylation site are due to direct dipolar measurements a spectral width of 4386 H z was collected into
interactions and are not
products of spin diffusion. At 7, = 400 16,000 complex points giving a resolution of 0.5 Hdpoint. These
ms in Me2S0, NOE cross-peaks of varying intensities were similar coupling constants indicate that g+ = 60" is the most
evident between all sequential (i, i+l) NHs, butonly the A m 7 - populated conformer for the A m 7 Ca-C@bond in both comIle8 and the Iles-SePNH-NH NOES had developed at 7, = 80 pounds and thatN-glycosylation has littleeffect on the Asn7 x1
ms. Given the slow cross-relaxation rate for such a relatively rotamer population.
small molecule, the specific time-dependent d m (i, i+l)NOEs
Qualitative comparison of NOESY data in H20
for glycopepprovide evidence that theN-glycosylation site in theaglycosyl tide 1 and peptide 2 showed that glycosylation also has little
peptide 2 samples folded structures that are not present in
effect on the Asn7 C W @bond (xz).NOESY experiments inH 2 0
The
A m 7
Downloaded from www.jbc.org at UNIV OF MARYLAND on June 12, 2008
Ser'
Ilez
~ 1
Phe4
Gly"
GN1 N H l
I
TABLE111
NH chemical shifls (ppm), 3JNHvicinal coupling constants (Hz) and
NH temperature coefficients (-ppb /K)for NH protons in glycopeptide
1 and peptide 2
Values for the aglycosyl peptide 2 are shown in parentheses.
Residue
-
3335
Glycopeptide NMR Studies
3336
TABLEIV
Relative nuclear Overhauser enhancementsfor the exchangeable sugar
NH protons in glycopeptide 1 in H,O at 5 "C
The measuremepts were made froma NOESY experiment with T, =
400 ms at 5 "C at p H 3.0.
NOE cross-peak
GN1
H1NHl-GN1
GN1 NH1-GN1 H2
GN1 NH1-hn7
58 CBHb
GN1 NH1-hn' CBHb
Measured
volume"
Relative
volume
12
1.0
75
6.3
43
4.8
3.6
"*
GlcNAcl NH
0
64
GN1 NH2-GN1 H1
GN1 NH2-GN1 H2
GN1 NH2-GN1 H3
GN1
CH3
NH2-GN1
25
12
62
2.0
1.0
5.'0
4.5
4.0
3:5
3.'0
2.'0
215
*I
5.2
5.2
GN2
H1 NH2-GN2
GN2 NH2-GN2 H m 3 '
H3
hc. 6. A NOESY contour plot stripand one-dimensional slice
through the GlcNAc-1NH2 region of 1 in fW% H,O, 10%DpO(60
m~ sodium phosphate, pH 3.0, at 5 "C). The figure shows crosspeaks for GlcNAc-1 NH2 to GlcNAc-1 sugar protons H1, H2, H3, and
CH3. The correspondingNOE cross-peak volumes are presented in
Table IV.A GlcNAc-1 C2-N2 dihedral angle of T near 100" is schematically depicted. The strong GlcNAc-1 NH2-H3 NOE is indicated by the
arrow.
provide some information about the orientationof xz by come- Glycopeptide 1's trans glycosidic amide contrasts with the cis
latingAsn7 C@H, H' with adjacent
y-carboxamide NH protons: y-amide seen at the glycosylated Amzs7 in the IgG F, crystal
Asn7 NYHE in aglycosyl peptide 2, and GlcNAc-1 NH1 in gly- structure (Deisenhofer, 1981). Trans-cis amide isomerization
copeptide 1. The diastereotopic Asn7 CS protons were not as- has an activation barrier of20-25kcaVmol
(Jorgensen and
signed, but we assumed that the relative
chemical shifts of the Gao, 1988). Presumably during glycoprotein biosynthesis, and
Asn7 CS pro-R and pro-S protonsdo not change uponglycosyla- before protein folding, glycosidic amide bonds are trans. Local
tion. In peptide 2 the cross-peaks between Asn7 NVHE and the and long-range peptide-oligosaccharide interactionsinthe
with the folded IgG F, may provide the energy neededfor cis amide bond
upfield Asn7 CSH (u= 2.77 ppm) was larger than that
downfield CSH proton (u = 2.89 ppm). Glycosylation did not formation at AsnZs7.
GlcNAc Side Chain Conformation-In 0-linked glycopepchange this NOE pattern. In glycopeptide 1, GlcNAc-1 NH1
again has a stronger NOE with the upfield Asn7 CSH (Table tides, where GalNAc is attachedto a Ser or Thr $3, the
IV).Just as N-glycosylation had littleinfluence on the average a-linked sugar may stabilize structure by forming an intramopeptide backbone structure, it also appears notto dramatically lecular hydrogen bond between GalNAc's C2 acetamidocarbony1 and the Thr NH (Meaji et al., 1987). Similar sugar-pepaffect the Asn7 side chain conformation.
N-Glycosidic Linkage-Information about the conformation tide interactions could exist in N-linked glycopeptides, as the
at the N-glycosidic linkage inglycopeptide 1 was obtained from core GlcNAc-1 acetamido group could hydrogen bond with dothe water NOESY experiment (90% HzO, 10% DzO, 7, = 400 nors in the nearbypeptide. To gain information about C2 side
ms, pH 3.0, 5 "C). The N-glycosidic bond, defined by the dihe- chain conformation and local peptide-sugar interactions, we
dral angle 4 (05-C1-N1-Asn7$), has GlcNAc-1 H1 and NH1 examined exchangeable GlcNAc NH protons in HzO at pH 3.0
trans in various
Asn-linked glycopeptides: the crystal structure for both the glycopeptide 1 and theMan3GlcNAc2pentasaccharide 3. Cagas et al. (1991) recently showed that Lewis blood
of 2-acetamido-l-N-(~-aspart-4-oyl)-2-deoxy-~-~-glucop~anosyl amine (Ohanessian et al., 1980), CD, and 35coupling data group oligosaccharides have rigid hexosamineC2 side chains in
for asparaginyl oligosaccharides (Bush et al., 19821, and NOE aqueous solution. Specifically, they found thatthe LND-1
and 35 measurements on a 22-amino acid IgM glycopeptide GlcNAc has 7 = 90 f 20°, and LNF-1 GlcNAc has T = 60 5 30".
(Wormald et al., 1991) all indicate trans N-glycosidic bonds. Following the approach of Cagas et al., we used 3 J m 2 , 2and
GlcNAc-1 Cl-C2-N2Glycopeptide 1 is no different. The NOESY experiment in90%/ NOE measurementstoconstrainthe
10% HzO/DzO showed that theglycosidic NH (GlcNAc-1 NH1) C7(Ac) dihedral angle (7)in glycopeptide 1.
Since 3Jm2,H2
= 9.1 Hz for GlcNAc-1 (Table 1111, its C2-N2
has a strong dipolar interaction with GlcNAc-1 H2 and a very
weak NOE with the anomeric GlcNAc-1 H1 (Table IV).Assum- bond is near a trans conformation with regard to H2 on the
10.2 Hz is
ring andNH2 on the side chain. If 3Jm2,H2=
1
ing a 4C1 ring conformation, this NOE pattern and 3 J ~ ~ 1= , ~sugar
9.0 Hz (Table 111) indicate that the ovomucoid glycopeptide's taken as the maximum for a trans orientationof NH2 and H2
N-glycosidic bond is near trans in HzO (although these mea- (Bush, 1982; Cagas et al., 1991), the reduced 3J value for the
glycopeptide's GlcNAc-1 NH2 indicates two possible staggered
surements do not accurately define +).
The single GlcNAc-1 NH1 resonance, the strong NOES be- orientations about the C2-N2 bond: NH2 syn to H 1 (7 near
tween GlcNAc-l NH1 andAsn7CSH, H' (Table IV), and thelack 100") or NH2 syn to H3 (7 near 140"). Fig. 6 shows a NOESY
of a GlcNAc-1 Hl-Asn7 CPH, H' NOE suggest that the Asn7 contour plot, andthe correspondingone-dimensional slice
y-amide bond, connecting carbohydrate to peptide, is in the Z through the NOE cross-peaks, for the glycopeptide's GlcNAc-1
conformation. Wormald et al. (1991) also found the glycosidic NH2 proton. The NH sugar 'H NOE cross-peak volume meaamide bond to be planar and trans in the IgM glycopeptide. surements from a n experiment with 7, = 400 ms are listed in
Downloaded from www.jbc.org at UNIV OF MARYLAND on June 12, 2008
41
3.4
70
5.8
35
GN2 NH2-GN1 H4
2.9
GN2 NH2-GN2 CH3
55
4.7
Cross-peak volumes were measured usingthe integration routine
in the Felix N M R software (Hare Research). The estimated error is
2 0 4 0 % for cross-peaks with volumes of 10-20, while for cross-peaks
with volumes of 30-70 the error is 10-20%. Volume measurements of
the base-line noise typically gave values from -5
to 5.
*The diastereotopic Asn7 CS protons were not assigned. For this
tableAsn7CSH is the upfield resonanceat 2.77 ppm andAsn CBH' is the
downfield resonance at 2.89 ppm. Wormald et al. (1991) assigned the
downfield resonance to be the pro-S proton in a similar glycopeptide.
e Due to the chemical shift redundanceof GN2 H2 and GN2 H3 this
volume represents an overlapped NOE cross-peak.
Glycopeptide NMR Studies
3337
Incontrast to glycopeptide 1's relatively rigid GlcNAc-1
C2-N2 bond, Kessleret al. (1991) found that theGlcNAc C2side
I
I
chain in thehexapeptide cyclo(-~-Pro-Phe-Ala-[N-2-acetamido2-deoxy-~-~-glucopyranosyl]Gln-Phe-Phe)
is mobile in MezSO
solution. Specifically, their NOE data were inconsistent with
the acetamido side chain adopting a single conformation, as
GlcNAc NH2 showed strong NOEs of similar intensity toprotons (H2 and H1)on both faces of the GlcNAc sugar ring. It is
interesting that the
GlcNAc C2-N2bond behaves so differently
for ovomucoid glycopeptide 1 and Kessler's synthetic cyclic glycopeptide. In the cyclic peptide GlcNAc is attached to a Gln
y-carboxamide, instead of to an Asn residue. The Gln side
chain, with an extra methylene group, has greater conformational mobility than an Asn side chain. It may be that when
GlcNAc is in the context of a natural N-glycosylation site its
I
I
I,
I
2 '
acetamido side chain conformation is restricted. The relatively
-- 11 02 00
-60
0
60
118200
rigid N-glycosidic linkage in glycopeptide 1, extending from
TAU (Degrees)
GlcNAc-1 C1 to Asn7 CY, may restrict theconformational space
FIG.7. Variation of GlcNAc-1 NH2-H1, -H2,and -H3interproton available to the sugar's C2 acetamido group.
distances as a function of the GlcNAc Cl-C2-N2-C7(Ac)dihedral
We wanted to determine if GlcNAc-1 C2 side chain conforangle ( 7 ) . The distances were calculated after 10" increments of 7 using
the PDB crystal structure coordinates for GlcNAc-1 from the IgM F, mation is influenced by the P-linked peptide. Treatment of
fragment (Deisenhofer,1981). The molecularmodelingprogram In- glycopeptide 1 with N-GlycanaseTMcleaved the peptide and
sight11 (Biosym) was usedto measure these interproton distances. The generated ananomeric mixture of the MansGlcNAcz pentasacdotted l i n e represents the minimum energy conformation calculated for charide, 3 ( d p = 0.63/0.37 in 0.1mM sodium phosphate, pH 3.0).
7 in GlcNAc (Pincus et al., 1976).The boxed region between T = 95-115"
chemical shift redundance of
represents the conformation for the glycopeptide 1's GlcNAc-1 C2-N2 Unfortunately, duetothe
GlcNAc-1 H2 and H3 inboth anomers, determination of 7 was
bond, as determined from NMR data in H,O (Fig. 6 and Table IV).
not possible using side chain NH sugar ringNOEs.
With 7 = 95-115", the GlcNAc-1 N-acetamidocarbonyl would
Table IV. At 7, = 400 ms, spin diffusion will not dominate ina
pentasaccharide (Homas et al., 1987a1, and a two-spin analysis be pointed away from the P-linked GlcNAc-2 and pointed tomay be used to approximate GlcNAc-1 NH2-'H distances. Fig. ward the glycopeptide linkage (see diagram in Fig. 6). We rea7 shows calculated GlcNAc NH2-GlcNAc H1, H2, H3 distances soned that peptide residues may help stabilize this GlcNAc-l
as a function of the C2-N2 dihedral angle 7.
C2 side chain conformation. One possibility is that thesugar's
Our experimental observations (Fig. 6, Table IV)of a strong C2 acetamidocarbonyl can form an intramolecular hydrogen
NH2-H3 NOE, a weak NH2-H2 NOE, and a medium-strength bond with a hydrogen bond donor in theN-glycosylation site, or
NH2-H1 cross-peak, are consistent with GlcNAc-1 NH2 being alternatively, with GlcNAc-1 NH1. However we have yet t o
syn toH3. Even consideringthe 20-30% error in measuring theobserve evidence for an intramolecularhydrogen bond. None of
weaker NH2-'H cross-peak volumes, itisclearthatthe
the glycopeptide's NH protons had reduced NH temperature
GlcNAc-1C2 side chainadopts a predominant orientation coefficients in HzO (Table 111). NH temperature coefficients
which places NH2 closer to the sugarring's H3 thanto H1. In may indicate hydrogen bonds in smallflexible peptides (Dyson
fact, the NH2-H3 NOE cross-peak volumes are more than twice et al., 1988; Perczelet al., 1991). In polar solvents, AWAT coefthose of NH2-H1, indicating a difference in average distance of ficients of 0 to -4 ppb/K are characteristic of NH protons in0.14.2 A.From the measured NOE volumes, and the torsion volved in weak, but significant, intramolecular hydrogen
map inFig. 7, GlcNAc-1 T was conservatively constrained to be bonds. Coefficients between -4 and -9 ppbK are typical for
between 95 and 115".
solvent-exposed NH protons. Moreover, glycosylation did not
The cis conformation (7 = -60") was calculated to be the cause any pronounced change in the NHH/D exchange rates.
minimum energy conformer for GlcNAc mono- and disacchari- Dissolution of glycopeptide 1 (10 mM) in DzO (50 mM sodium
des (Pincus et al., 1976), being more stable than other confor- phosphate, pD 3.0, 5 "C) showed that Ile* NH ( t , = 20 min)
mations by 2 kcal/mol. But the cis C2 conformation is appar- and another NHproton, either SergNH or Lys'O NH (tllz= 10
ently not very populated in HzO for glycopeptide 1. The NOE min), were relatively long-lived when compared with the other
measurements indicate that the glycopeptide's GlcNAc-1C2
were also
10 NH protons (tllz = 1-2 min). Iles NH and Sers NH
side chain must berelatively rigid with limited torsional oscil- the slowest to exchange in the aglycosyl peptide 2. These relation about C2-N2. As depicted in Fig. 7, if the C2-N2 bond duced exchange rates for Ile8 NH and S e 9 NH were not related
were mobile it would sample conformations that, on average, to their location in anN-glycosylation site; transposition of Gly
than to either H1or H3 and Ile resulted in Ile5 NH (tllz = 20 min) and Thr6NH (tllz=
place NH2 closer to H2 (dav= 2.4
(dav= 2.8 A). Because of the r6 dependence of the NOE, con- 6 min) having the longest lived NH protons in the related
formational averaging caused by free rotation about the
C2-N2 peptide, Ser-Ile-Glu-Phe-Ile-Thr-Asn-Gly-Ser-Lys,
whereas
bond would result in theNH2-H2 NOE cross-peak volume be- G l p and Serg NHswere fastto exchan&tllz = 1-2 min). The
ing at least two to three times larger than the NH2-H1 and reduced exchange rates for Iles NH and Sers NH in glycopepNH2-H3 volumes ((2.8/2.4)6= 2.57). This is clearly not thecase tide 1 and peptide 2 are probably due to the stericbulk of the
for the glycopeptide's GlcNAc-1 NH2 (Fig. 6, Table IV). Defini- Ile' isopropyl side chain (Robertson and Baldwin, 1991)and not
chemical shift to any hydrogen bond interaction inHzO.
tion of 7 for GlcNAc-2 was less certain due to the
overlap of H2 and H3resonances. However, the intrasaccharide
In summary, NMR studies have shown that N-glycosylation
GlcNAc-1, and there of a flexible decapeptide does not perturb either thepeptide or
GlcNAc-2 NH2-H1 NOE is greater than in
is also a relatively strongintersaccharide
NOE between glycoside conformation. However, the glycopeptide's core
GlcNAc-2 NH2 and GlcNAc-1 H4 (Table IV). These two NOEs GlcNAc-1 has a relatively rigid C2 side chain conformation in
indicate that theGlcNAc-2 side chain is able to sample confor- HzO. No evidence for intramolecular hydrogen bonds contribmations where 7 = 120-160", with NH2 syn to H1.
uting to thistorsional rigidity was found. It may be that, when
GN1
Downloaded from www.jbc.org at UNIV OF MARYLAND on June 12, 2008
A)
Glycopeptide NMR Studies
3338
in the context of an N-glycosylation site, the rigid N-glycosidic
linkage at GlcNAc-1 limits the conformational space of the
adjacent C2-N2 acetamido side chain.
Acknowledgments-We thank Dr. Jya-Wei (David) Cheng, Russell
Bigelow, and Krystyna Haptas for assistance.
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