Key words: conformation/glycopeptide/molecular modeling/NMR/proteoglycan
Proteoglycans (PGs) are ubiquitous components of mammalian cells and tissues. They contain a protein core to which side chains of glycosaminoglycans (GAGs) including heparin/heparan sulfate (HS), chondroitin sulfate, and dermatan sulfate are covalently attached. These macromolecules participate in a variety of biological activities such as inhibition of blood coagulation, modulation of cellular proliferation, and cell adhesion. They have been also implicated in the pathogenesis of atherosclerosis and interactions with a variety of ligands such as growth factors, hormones, and neurotransmitters (Roden, 1980; Poole, 1986; Kjellen and Lindahl, 1991; Silbert and Sugamaran, 1995). The GAGs are covalently attached to a serine in the protein core through a linkage tetrasaccharide GlcpA[beta](1->3)Galp[beta](1->3) Galp[beta](1->4)Xylp[beta]. Based upon the nature of the repeating disaccharides, GAGs have been subgrouped into two types, galactosaminoglycans and glucosaminoglycans. The galactosaminoglycans chondroitin sulfate (CS) and dermatan sulfate (DS) contain GalpNAc and uronic acid, while the glucosaminoglycans heparin and HS contain GlcpNAc and uronic acid.
The glycosyl transferases which catalyze the transfer of hexosamine ([beta]-GalpNAc or [alpha]-GlcpNAc) to the linkage tetrasaccharide core, and hence determine the type of GAG to be synthesized thereafter, are thought to be different from those involved in the subsequent chain elongation steps (Esko and Zhong, 1996). Sugahara and his coworkers identified an [alpha]-N-acetylgalactosaminyl transferase in fetal bovine serum which catalyzes the transfer of an [alpha]-GalpNAc to the linkage tetrasaccharide and hexasaccharide-serines (Kitagawa et al., 1995). Recently, we reported the partial purification of [alpha]-N-acetylglucosaminyltransferase I from the wild-type CHO cells and from rat liver that transfers GlcpNAc to linkage tetrasaccharides having benzyl and naphthalenemethanol as an aglycone and observed significant dependence concerning its specificity (Fritz et al., 1997). These studies provided in vivo basis for the possible role of core protein in the biosynthesis of specific GAGs (Fritz et al., 1997).
To better understand the structural basis for the biosynthesis of proteoglycans, we have investigated the solution structure of oligosaccharide fragments from the carbohydrate-protein linkage region, such as, Galp[beta](1->4)Xylp[beta](1->O)-Serine (GXS), and Galp[beta](1->3) Galp[beta](1->4)Xylp[beta](1->O)-Serine (G[prime]GXS) by a combination of 2D NMR spectroscopy and molecular modeling methodology, and identified a single family of conformations that are compatible with the solution-phase NMR data (Krishna et al., 1990; Choe et al., 1991). Recently, we reported structural studies for bis-glycosylated-hexapeptide (BGH) [Ser(Galp[beta](1->4) Xylp[beta])-Gly-Ser-Gly-Ser(Galp[beta](1->4) Xylp[beta])-Gly] and tris-glycosylated hexapeptide (TGH) [Ser(Galp[beta] (1->4)Xylp[beta])-Gly-Ser-(Galp[beta](1->4) Xylp[beta])-Gly-Ser (Galp[beta](1->4)Xylp[beta])-Gly] (Curto et al., 1996). In the present study, we have extended these structural investigations to two glycopeptides, Ser(GlcpA[beta](1->3)Galp[beta] (1->3)Galp[beta](1->4) Xylp[beta])-Gly-Ser-Gly-Ser(GlcpA[beta](1->3) Galp[beta] (1->3)Galp[beta](1->4)Xylp[beta])-Gly (1) and Ser(GlcpA[beta](1->3)Galp[beta] (1->3)Galp[beta](1->4)Xylp[beta])-Gly (2), designated as OSHP (octasaccharide hexapeptide) and TSDP (tetrasaccharide dipeptide), respectively. A hexapeptide, Ser-Gly-Ser-Gly-Ser-Gly (3) was also synthesized as the unglycosylated control peptide. It is worth mentioning here that using a combination of mass spectrometry and NMR spectroscopy, several groups have characterized A-GlcpA[beta](1->3) Galp[beta](1->3)Galp[beta] (1->4)Xylp[beta]-B type linkage oligosaccharides (where A is the disaccharide [Delta]4,5 GlcpA[beta](1->4) GalpNAc-4 or 6-O-sulfate and B is serine), but complete 1H NMR assignments are not always reported (Van Halbeek et al., 1982; Goto and Ogawa, 1993; Sugahara et al., 1994, 1995; Liu et al., 1995; Manzi et al., 1995; Neumann et al., 1996).
A variety of glycopeptides have been studied by NMR to elucidate the impact of O-linked sugar chains on the conformation and flexibility of peptides (Gerken et al., 1989; Kessler et al., 1992; Andreotti and Kahne, 1993; Liang et al., 1995; Mer et al., 1996; Gervais et al., 1997). The 13C NMR relaxation measurements undertaken on the bovine maxillary mucin have shown that O-linked carbohydrates alter the peptide-core conformation and decrease the mobility of the glycosylated residues (Gerken et al., 1989). Kessler et al. have concluded from NMR studies on glycosylated and unglycosylated cyclic hexapeptides that there is no change in the backbone conformation of peptide upon glycosylation. From the measurements of amide-exchange-rates, a decrease of conformational mobility has been observed for fucosylated proteinase inhibitor, and this result has been correlated with an enhancement of the protein thermal stability upon fucosylation (Mer et al., 1996). Based upon sequential amide proton NOE, Kahne and his coworkers were able to show that peptide backbone conformation is radically different depending on whether a mono- or a disaccharide is attached (Andreotti and Kahne, 1995; Liang et al., 1995). NMR studies on glycosylated recombinant human granulocyte-colony-stimulating factor, suggest that carbohydrate moiety reduces the local mobility around the glycosylation site, which could be responsible for the stabilizing effect observed in glycoprotein (Gervais et al., 1997).
A fundamental problem in the homonuclear NMR analysis of a glycopeptide is the difficulty in completely assigning each resonance in the proton spectra either to carbohydrate or peptide moiety because of severe overlap in the 3.5-4.0 p.p.m. region. Thus, the first step in the assignment procedure is to achieve distinction between the spin-systems belonging to carbohydrate chain and the peptide backbone. This was accomplished primarily by homonuclear 2D NMR experiments (COSY, TOCSY, and NOESY) in combination with 1H-13C HSQC spectra, following standard procedures (Wüthrich, 1986; Dabrowski, 1994). Our goal in this study is to investigate 1H and 13C NMR chemical shifts and coupling constants, and to identify the most dominant conformation about the glycosidic linkages present in these glycopeptides in aqueous solution using a combination of NOE spectroscopy and molecular modeling calculations, and to compare peptide backbone conformation of 1 with that of unglycosylated hexapeptide. 1H and 13C chemical shift assignments
The primary structures of OSHP (1) and TSDP (2) are shown in Figure
Figure 1. Schematic representation of the octasaccharide hexapeptide (OSHP) 1 and tetrasaccharide dipeptide (TSDP) 2 with the labeling of individual pyranose units as used in text. Sequential connectivities were determined by locating NOE cross-peaks between anomeric-H and aglycone-H across the glycosidic bond in 2D NOESY (Figure Glucopyranuronoic acid-galactose-galactose-xylose conformation
The 400 ms NOESY spectrum of 1 (Figure
Table I.
Figure 2. Two-dimensional NOESY spectrum of 1 in D2O at 278 K at 600 MHz with a mixing time 400 ms. Interresidue NOE contacts are indicated along with residue positions along the diagonal. Serines 1, 3, and 5 are denoted by S1, S3, and S5. The xylose residues attached to S1 and S5 are denoted as X1 and X5, respectively. Because of chemical shift degeneracy, G is used to denote G1 and G5, G[prime] to denote G1[prime] and G5[prime], and U to denote U1 and U5.
Figure 3. Fingerprint region of a 400 ms NOESY spectrum of 1, indicating resonance assignments. Serines are denoted as S1, S3, and S5, while G2, G4, and G6 denote the glycines.
In the present studies for each interglycosidic linkage, NOE was observed from H1[prime] to Hn and H1[prime] to He (n+1) proton (where He (n+1) indicates the equatorial proton attached to higher adjacent carbon with respect to Cn (n = 3 or 4)). The ratios of the NOEs between H1[prime] to Hn, and H1[prime] to He (n+1) ranges between 3-4.5:1 and the corresponding derived average distances were 2.4-2.5 Å and 2.9-3.2 Å respectively. These observations suggest greater proximity between H1[prime] and Hn, and a syn-periplanar-like arrangement of protons across the C1[prime]-Cn axes in all the three interglycosidic linkages. The NOEs observed between UH1 and G[prime] H3 and G[prime] H4, G[prime] H1 and G H3 and G H4, G H1 and XH4 and XH5e, were principally used to define the orientation of U-G[prime] [beta](1->3), G[prime]-G [beta](1->3), and G-X [beta](1->4) linkages. Conformation of glycopeptide bond
The orientation of the peptide chain with respect to xylopyranose is determined by a combination of three torsional angles affecting bonds between the xylopyranosyl anomeric carbon and the serine C[alpha] ([phis], [psi] and [chi]). Present studies reveal that the X-S glycopeptide linkage representing both N- and C-terminals adopts a well defined conformation (i.e., [phis]1/[psi]1 torsional angles) as indicated by two sugar-peptide NOEs (XH1/S H[beta] and XH1/S H[beta][prime]). The more intense XH1 - S H[beta][prime] cross peak (compared to XH1-S H[beta]) in the NOESY spectra of both 1 and 2 clearly indicated a slightly smaller distance between the XH1 and the S H[beta][prime], than the S H[beta] proton. Similar kind of NOE observations have been reported for related Xyl-Ser containing glycopeptides (Paulsen, 1990; Paulsen et al., 1991). However, the two Xyl-Ser glycopeptide linkages (X1-S1 and X5-S5) are slightly different in 1 as inferred from a weak NOE between X5 H1-S5 H[alpha]. This NOE is absent between X1 H1-S1 H[alpha]. The DG/SA calculations suggest -sc and +ac conformations for the [phis]1 and [psi]1 torsional angles, respectively. The preference of [phis]1 for the -sc range is, in accordance with the concept of exo-anomeric effect (Lemieux et al., 1979) and with the studies on glycoceramides incorporating [beta]-linked glucose or galactose moiety (Howard and Prestegard, 1995; Bruzik and Nyholm, 1997). Peptide backbone conformation of 1
The fingerprint region of the NOESY (Figure
Figure 4. Amide proton region of the 200 ms ROESY (A), and 400 ms NOESY spectrum (B) of 1. Unglycosylated peptide
The 1H NMR spectrum of (ser-gly)3 hexapeptide (3) (Table I) was assigned as discussed earlier for 1. There were a number of strong intra-residue d[alpha]N (i, i) NOEs for glycines, d[beta]N (i, i) for S3 and S5. The presence of strong d[alpha]N (i, i+1) ROEs together with the absence of dNN (i, i+1) and nonsequential ROEs are consistent with an extended flexible structure (Dyson and Wright, 1991). Influence of glycosylation on backbone conformation
To determine the influence of tetrasaccharide substitution at S1 and S5 of (Ser-Gly)3 hexapeptide, comparison of ROESY data for 1 and 3 was performed. Under identical experimental conditions, both of these compounds exhibit strong d[alpha]N (i, i+1) NOEs. The backbone dNN ROEs (sequential and/or nonsequential) were absent in case of 3. The dNN (i, i+1) ROEs were also absent in case of 1 except a weak S5/G6 dNN peak. The presence of a weak dNN (i, i+1) ROE for S5 suggests that glycosylation modifies the peptide bond torsional angle of this position to some minor extent. Distance geometry/simulated annealing refinement
Figure 5. Family of 13 best conformers obtained from distance geometry/simulated annealing calculations on TSDP (2). Protons are not shown.
In order to interpret the NMR parameters in terms of conformational models, DG/SA calculations were performed for 2 and 1. For example, a total 20 NOESY constraints were used in the calculations of 2 including 12 inter-residue constraints. Tight dihedral angle and distance constraints within each sugar were used to fix its 4C1 chair conformation. From 32 initial DG/SA structures, a total of 13 acceptable SA structures were obtained with the lowest averaged constraints violations (NOE distance violations <0.3 Å, dihedral violations <10°). The average values of [phis] and [psi] for U-G[prime], G[prime]-G, G-X, and X-S linkage for these structures are reported in Table III, along with deviations. The deviations from the average values (Table III) in the glycosidic torsional angles [phis] and [psi] for the tetrasaccharide moiety ranged from ±3° to ±14°. The family of these structures (Figure
Table II.
We have presented the complete 1H and 13C assignments for two glycopeptides representing the carbohydrate-protein linkage region of connective tissue proteoglycans. The 1H and 13C chemical shift assignments observed for the tetrasaccharide moiety of 1 and 2 were quite similar to those partly reported by other authors (Neumann et al., 1996; Sugahara et al., 1994, 1995), but differ in some respects from those reported earlier by for methyl analog of the tetrasaccharide, i.e., GlcA[beta](1->3)Gal[beta](1->3)Gal[beta](1->4) Xyl[beta]-OMe (Nielsson et al., 1993). Specifically, the values we report for G[prime] H2 and G[prime] H3 and U H2 differ significantly (by 0.30, 0.08, and 0.35 p.p.m., respectively). Our 1H NMR chemical shift values were consistently 0.15-0.16 p.p.m. lower field than those reported by Neumann et al. (1995) for the tetrasaccharide moiety of Gly(GlcA[beta](1->3)Gal[beta](1->3) Gal[beta](1->4) Xyl[beta])-Ser-Gly-Glu, but the order of the chemical shift assignments for anomeric-H is quite similar. The reported chemical shift [delta] 4.96 for XH5e (Neumann et al.,1995) also seems to be unusual as it has been generally reported to appear at [delta] 4.11 ± 0.2 p.p.m. (Van Halbeek et al., 1982; Krishna et al., 1990; Choe et al., 1991; De Waard et al., 1992; Sugahara et al., 1992; Curto et al., 1996; Fritz et al., 1997).
In order to establish the role of glucuronopyranosyl substitution, the 1H and 13C chemical shifts for 2 were compared with those reported for G[prime]GXS (Choe et al., 1991) which reflect major alteration in the chemical shifts of G[prime] residue due to its involvement in interglycosidic linkage with GlcA but without any significant changes in the chemical shifts of inner G and X residues. The 1H NMR chemical shifts of the xylose in 2 were also comparable ([le]0.02 p.p.m.) with those reported for BGH in D2O (Curto et al., 1996) reflecting that GlcA[beta](1->3)Gal substitution to the C-3 of Gal[beta](1->4)Xyl of BGH does not modify significantly the conformational behavior of G-X linkage in case of 1. Previous studies of BGH in 60:40 (by volume) H2O:TFE suggested a conformational manifold that included some folded structures, including some containing a weak salt bridge between the N- and C-termini (Curto et al., 1996), and reflect the role of the solvent (TFE) in stabilizing some structures.
For all the three interglycosidic linkages (GlcA-Gal[prime], Gal[prime]-Gal, and Gal-Xyl), NOE was not only observed for proton linked to aglycone carbon but also to equatorial proton occupying adjacent higher position. A comparison of NOE cross-peak volume integral in the NOESY spectra of 1 and 2 revealed that H1-He (n+1) cross-peak was weak as compared with H1-Hn cross-peak. Both of these NOEs across each interglycosidic linkage were taken into consideration to define the geometry of a glycosidic bond. The average [phis] and [psi] values generated a model for 2 which is in good agreement with experimental NOE derived inter-residual hydrogen distances (Table III). These studies suggest that the [phis] torsion angle, irrespective of monosaccharide residues (GlcA/Gal/Xyl) as well as site of glycosidic linkage ([beta](1->3),[beta](1->4) /[beta](1->O)) shows strict preference for the -sc range. This feature is in accordance with the concept of exo-anomeric effect (Lemieux et al., 1979). The torsion angle [psi], on the other hand, exhibits -ac conformation for [beta](1->3) interglycosidic linkages but +ac for [beta](1->4) interglycosidic linkage and for glycopeptide bond. The observed [phis] and [psi] dihedral angles for the average tetrasaccharide structure (A) were found to be with in 2° for [phis] and within 20° for [psi] when compared with published model disaccharide fragments such as [beta]-d-Glcp(1->3)-[alpha]-d-Galp-OMe (Baumann et al., 1989), [beta]-d-GlcpA(1->3)-[beta]-d-Galp-O-benzyl (Zsiska and Meyer, 1993), and [beta]-d-Xylp(1->4)-[beta]-d-Xylp-OMe (Hricovini et al., 1990).
Table III. Introduction
Results
Position
1
2
Position
1
2
3
1H
13C
1H
13C
1Hb
13C
1H
13C
1H
X1
Ser 1
1
4.44
104.14
4.47
104.19
[alpha]
4.38
54.05
4.35
53.72
4.10
2
3.34
73.66
3.37
73.69
[beta][prime]
4.07
68.62
4.09
68.19
3.87
3
3.58
74.83
3.62
74.87
[beta]
4.26
4.22
3.87
4
3.84
77.46
3.87
77.51
5e
4.08
64.05
4.12
64.09
Gly 2
5a
3.40
3.41
NH
8.79
8.77
[alpha]
4.13
43.49
3.90
43.35
3.99
X5
[alpha][prime]
4.00
3.70
3.99
1
4.40
104.22
2
3.30
73.69
Ser 3
3
3.57
74.87
NH
8.46
8.52
4
3.83
77.46
[alpha]
4.51
56.50
4.38
5e
4.08
64.05
[beta][prime]
3.86
62.15
3.79
5a
3.36
[beta]
3.90
3.79
G1,G5
Gly 4
1
4.50
102.67
4.53
102.68
NH
8.55
8.56
2
3.64
70.90
3.67
70.93
[alpha]
4.00
43.53
3.94
3
3.82
83.17
3.82
83.30
[alpha][prime]
3.98
3.89
4
4.17
69.28
4.19
69.35
5
3.70
76.12
3.72
76.10
Ser 5
6[prime]
3.75
62.06
3.79
62.04
NH
8.44
8.28
6[prime][prime]
3.71
3.75
[alpha]
4.69
54.39
4.40
[beta][prime]
3.88
70.0
3.77
G[prime]1,G[prime]5
[beta]
4.17
3.79
1
4.63
105.19
4.66
105.19
2
3.70
71.23
3.76
71.25
Gly 6
3
3.78
83.61
3.82
83.46
NH
8.17
8.23
4
4.17
69.34
4.19
69.35
[alpha]
3.80
44.29
3.73
5
3.66
75.94
3.68
75.93
[alpha][prime]
3.71
3.78
6[prime]
3.75
62.06
3.79
62.03
6[prime][prime]
3.71
3.75
U1,U5
1
4.67
105.08
4.67
105.03
2
3.40
74.26
3.41
74.25
3
3.48
76.44
3.51
76.43
4
3.48
72.89
3.51
72.91
5
3.75
77.39
3.72
77.34
Residue
1
2
Residue
1
2
3
X1
Ser 1
J12
7.6
7.7
J[alpha][beta]
5.0
5.9
4.8
J23
9.3
9.4
J[alpha][beta][prime]
4.0
4.0
4.8
J34
9.1
9.2
J[beta][beta][prime]
-11.6
-11.4
-11.8
J45e
4.9
5.3
J45a
9.8
9.4
Gly 2
J5e5a
-12.2
-12.0
JNH[alpha]
6.0
5.7
JNH[alpha][prime]
6.0
5.7
X5
J[alpha][alpha][prime]
-17.2
-17.0
-17.1
J12
7.6
J23
9.3
Ser 3
J34
9.1
JNH[alpha]
7.9
7.3
J45e
4.9
J[alpha][beta]
4.9
4.9
J5e5a
-11.5
J[alpha][beta][prime]
5.1
5.0
J[beta][beta][prime]
-11.4
-11.5
G1,G5
J12
7.6
7.9
Gly 4
J23
9.3
9.8
JNH[alpha]
6.0
6.0
J34
3.2
3.2
JNH[alpha][prime]
6.0
6.0
J45
<1
<1
J[alpha][alpha][prime]
-16.9
-17.0
G[prime]1,G[prime]5
Ser 5
J12
7.4
7.9
JNH[alpha]
7.9
7.1
J23
9.3
9.5
J[alpha][beta]
4.9
4.8
J34
3.5
3.2
J[alpha][beta][prime]
5.0
4.9
J45
<1
<1
J[beta][beta][prime]
-11.9
-11.8
U1,U5
Gly 6
J12
7.7
7.8
JNH[alpha]
5.7
6.1
J23
9.3
9.6
JNH[alpha][prime]
5.7
6.1
J34
9.1
8.8
J[alpha][alpha][prime]
-17.1
-17.0
J45
9.4
9.0
Discussion
Residues
Angles in degrees
X-S
[phis]1
Xyl O5-Xyl C1- O-Ser C[beta]
-84(3)
[psi]1
Xyl C1-O-Ser C[beta] - Ser C[alpha]
+103(8)
G-X
[phis]2
Gal O5-Gal C1- O-Xyl C4
-59(14)
[psi]2
Gal C1-O-Xyl C4-Xyl C3
+115(5)
G[prime]-G
[phis]3
Gal[prime] O5-Gal[prime] C1-O-Gal C3
-57(6)
[psi]3
Gal[prime] C1-O-Gal C3-Gal C2
-149(6)
U-G[prime]
[phis]4
GlcA O5-GlcA C1-O-Gal[prime] C3
-61(5)
[psi]4
GlcA C1-O-Gal[prime] C3-Gal[prime] C2
-148(7)
Table IV.
Proton pair | Experimental | Calculated |
UH1-G[prime]H3 | 2.3 | 2.27 |
UH1-G[prime]H4 | 3.3 | 3.52 |
G[prime]H1-GH3 | 2.3 | 2.28 |
G[prime]H1-GH4 | 3.3 | 3.47 |
GH1-XH4 | 2.3 | 2.26 |
GH1-XH5e | 3.1 | 3.20 |
XH1-SH[beta] | 2.5 | 2.22 |
XH1-SH[beta][prime] | 3.1 | 3.01 |
An extensive similarity between the 1H NMR multiplicity pattern and 1H and 13C NMR chemical shifts for the tetrasaccharide moiety in case of 2 with those of 1 was indicative of (1) similar conformation of tetrasaccharide chain in both glycopeptides, and (2) elongation of the peptide chain does not exert any pronounced effect on the conformation of G-X, G[prime]-G, and U-G[prime] linkages. The absence of long-range NOEs between tetrasaccharide and peptide backbone resonances in the 2D NOESY data of 1 and 2 indicate that the tetrasaccharide chain extends into solution, away from the peptide in both glycopeptides. The two tetrasaccharide moieties do not interact with each other in the case of 1 as indicated by the absence of long-range NOE between them.
Although, DG/SA calculations indicated that the hexapeptide backbone is somewhat extended but otherwise not well defined in case of 1, the coincidence of the 1H and 13C NMR chemical shift of UG[prime]G portion of both tetrasaccharide arms in 1 suggests that these are solvated and do not interact with each other. A comparison of the 1H NMR chemical shifts of 1 with those noted for 3 reflects that the two [beta]-methylene protons of S1 and S5 residues almost overlap in 3 but get well resolved in 1 (Table I). Strong deshielding effects (0.16-0.40 p.p.m.) are observed for H2[beta] and H[alpha] resonances of the S1 and S5 residues. Among [beta]-methylene protons, glycosylation causes greater downfield shift (about three times) for [beta]-H as compared with [beta][prime]-H. The proximity between pyranosidic oxygen (O5) of Xyl and S H[beta] and 1,3-syn diaxial interactions between XH1 and S H[beta][prime] seems to be responsible for the observed differential deshielding effect caused by glycosylation (Baumann et al., 1989, 1990).
In order to determine the effects of peptide elongation on the conformation of the glycopeptide (Xyl-Ser) bond, we compared NOESY data (400 ms) for 1 and G[prime]GXS which revealed that XH1-SH[beta]' NOE is predominant in both cases but XH1-SH[beta] NOE present in the case of former was absent in case of later. The contrast between the NOESY spectra of 1 and G[prime]GXS suggests that peptide backbone influences conformation of the Xyl-Ser linkage to some minor extent. In an analogous manner, a comparison of the NOESY data (400 ms) for 1 and 3 had been made to determine the effects of glycosylation on the peptide component of 1. These studies revealed that the number and intensity of the observable d[alpha]N NOEs were comparable within experimental limits and the only difference lies in the presence of the weak dNN (i, i+1) NOE for S5/G6 in case of 1 which is absent in case of 3. In both cases, other amino acid residues do not exhibit the dNN (i, i+1) NOEs. These results indicate that O-glycosylation does not exhibit any pronounced effect on the hexapeptide backbone conformation. This observation is in line with the recent structural studies of O-glycopeptides (Huang et al., 1997; Saha et al., 1997). Huang et al. (1997) have reported NMR studies for glycosylated analogues of the principal neutralizing determinant of gp120 having O-glycosylated [alpha]-d-GalNAc at Thr and Ser residues and observed stronger NOEs around Thr19 glycosylation site indicative of more local rigidity as compared with Ser6 site. In O-glycopeptides having [beta]-d-GlcNAc substituted to serine, NMR studies suggested only minor variation of the rotamer distribution about Ser [chi] torsions, and no evidence for any long-range contact between carbohydrate and amino acid residues (Saha et al., 1997).
In conclusion, the combined use of NMR spectroscopy, and distance geometry/dynamical simulated annealing protocol allowed us to characterize the three-dimensional structures of the carbohydrate-protein linkage tetrasaccharide sequence. The results described in this report are significant in several aspects. (1) Complete 1H and 13C assignments and vicinal coupling data (3JHH) for two glycopeptides, representative of the carbohydrate-protein linkage regions of connective tissue proteoglycans have been presented (Table I). (2) Table II indicates that [phis], which is independent of the position of the interglycosidic bond (1->3)/(1->4) and type of monosaccharide residue (GlcA, Gal, Xyl), acquires -sc orientation. (3) As seen in Table III, reasonable agreement is obtainable between theoretical and experimental parameters when a single rigid geometry is assumed. (4) The effects of O- glycosylation on peptide conformation are limited to Xyl-Ser linkage, with only a very minor perturbation on the peptide backbone at Ser5. And (5) elongation of the core peptide chain from a dipeptide (2) to a hexapeptide (1), does not alter the conformation of the tetrasaccharide moiety, in the glycopeptides studied in the current investigation.
Synthesis of compounds 1 and 2 was described previously (Rio et al., 1993). NMR experiments were performed on 1 mM solutions (500 µl) either in 100% D2O or 90% H2O/10% D2O. Proton chemical shifts were referenced relative to external acetone at [delta] = 2.225 p.p.m.. NMR spectra were recorded on a Bruker AM 600 spectrometer equipped with an Aspect 3000 computer. All 2D experiments were performed in the phase sensitive mode (TPPI) at 293 K. Time domain data sets normally consisted of 1024 complex data points in t2 dimension and 256 to 512 t1 increments with spectral widths of 2404 Hz in 1H and 10,417 Hz in 13C. The following experiments were performed in D2O for 1 and 2: NOESY (400 ms and 800 ms), TOCSY (69 ms spin lock time), ROESY (150 ms), PS-COSY, 1H-13C HSQC, and 2D 1H-13C NOESY-HSQC (400 ms). NOE buildup curves for 1 in D2O (10, 50, 100, 150, 200, 300, 400, 600, and 800 ms mixing times) were also recorded. All data were zero filled to 1K × 1K real points. For the assignments of the exchangeable NH resonances of the 1, the pH was adjusted to 4.1 using 0.1 M DCl or HCl solutions. A combination glass electrode was used to measure the pH directly in the 5 mm NMR tube. TOCSY (70 ms spin-lock time) and NOESY (800 and 400 ms mixing time) spectra were recorded with a spectral width of 6024 Hz in both dimensions. For the NOESY experiments, the time domain data were multiplied by 90° shifted sinebell and, baseline corrected with a third-order polynomial fit in both dimensions. Data were processed on a Silicon Graphics Indigo work station using Felix (Biosym Technologies, San Diego, CA).
A 3D NOESY-TOCSY spectrum of 1 in D2O was collected according to phase cycling method of Vuister et al. (Vuister et al., 1988). Incrementation in the second evolution period was accomplished with the use of an external timing accessory (Tschundin Associates). Mixing times of 70 ms and 800 ms were used, respectively, for the TOCSY and NOESY segments of the 3D experiment each with 64 increments. The data were zero-filled to 256 (real) points and baseline corrected using a third order polynomial function in all three dimensions.
Molecular modeling and structure calculations
Molecular modeling calculations were performed using X-PLOR/QUANTA/CHARMM package (version 4.1, Molecular Simulations, Inc., Burlington, MA). A hybrid distance geometry/simulated annealing protocol as described before (Lee et al., 1994) was used to generate structures compatible with experimental restraints. The protocol consisted of four stages: initial regulation (I), regulation and refinement (II), refinement (III), and release of experimental constraints (IV). The first two stages consist of 200 cycles of Powell minimization, restrained MD at 1000 K followed by cooling to 300 K in steps of 25 K, and 200 cycles of Powell minimization. These two stages differ in the manner in which the force constants are set. The third stage consists of restrained MD with cooling in steps of 20 K followed by minimization. The final stage consists of 200 cycles of Powell minimization without NOE and torsion angle constraints. NOESY/ROESY peak volumes were calculated from the spectrum collected with 400 ms mixing time and were divided into three groups of strong (1.8-2.7 Å), medium (1.8-3.3 Å), and weak (1.8-5.0 Å), respectively. The ranges in these constraints allow for variations due to conformational flexibility and spin diffusion effects, if any. In the presence of significant freedom for rotation across the interglycosidic bonds, several other inter-residue NOEs would have been expected, in addition to the strong H1-Hn and a weak H1-He(n+1) NOEs that were the only ones observed. Thus, our experimental data are suggestive of a predominant conformation or a family of closely related conformations for each interglycodic linkage ([phis],[psi]). The distances between the 1,3 and 1,5 diaxial protons within Gal ring (with 4C1 chair geometry) were used for the calibration of the volume integral. Torsion angle constraints, based on the J-couplings, were derived from the 1D 1H NMR experiments. Sugar intra-residue NOE distance constraints and fixed dihedral angles were used for retaining the sugars in the 4C1 conformation during refinement. A total of 32 structures were generated using this protocol. Of this family of structures, 13 structures with NOE distance violations less than 0.3 Å and dihedral violation less than 10° were considered acceptable and included in the final analysis. The average structure was obtained by taking averages of the atomic coordinates of these acceptable structures. This structure was subjected to further energy minimization without constraints in order to remove bond length and bond angle distortions to obtain the final energy minimized average structure. All calculations were carried out on a Silicone Graphics Indigo workstation.
We thank Dr. Mike Jablonsky for assistance with the NMR measurements and Dr. Ted Sakai for assistance with the modeling calculations. This work was supported in part by grants from the NCI (CA 13148) and the Arthritis Foundation.
GAG, glycosaminoglycan; PGs, proteoglycans; CS, chondroitin sulfate; DS, dermatan sulfate; HS, heparan sulfate; CD2, cluster of differentiation 2;
TSDP, serine-O-([beta]-d-glucopyrauronosyl)-(1->3)-O-([beta]-d-galactopyranosyl)-
(1->3)-O-([beta]-d-galactopyranosyl)-(1->4)-O-[beta]-d-xylopyranosyl)-glycine;
OSHP, serine-O-([beta]-d-glucopyrauronosyl)-(1->3)-O-([beta]-d-galactopyranosyl)-
(1->3)-O-([beta]-d-galactopyranosyl)-(1->4)-O-([beta]-d-xylopyranoside)-glycine-
serine-glycine-serine-O-([beta]-d-glucopyrauronosyl)-(1->3)-O-([beta]-d-galactopyranosyl)-
(1->3)-O-([beta]-d-galactopyranosyl)-(1->4)-O-([beta]-d-xylopyranoside)-glycine;
GXS, [beta]-d-galactopyranosyl-(1->4)-O-[beta]-d-xylopyranosyl-O-serine;
G[prime]GXS, [beta]-d-galactopyranosyl-(1->3)-[beta]-d-galactopyranosyl-(1->4)-O-
[beta]-d-xylopyranosyl-O-serine;
Xyl (X), xylopyranose; Gal (G, G'), galactopyranose; GlcA(U), glucopyrauronoic acid; GalPNAc, N-acetylgalactosaminopyranose; GlcPNAc, N-acetylglucosaminopyranose; Ser (s), serine; gly (g), glycine; s1, s2, and s5 corresponds to serine-1, serine-3, and serine-5 while g2, g4, and g6 corresponds to glycines of the positions 2, 4, and 6 respectively; X1, G1, G[prime]1, and U1, and X5, G5, G[prime]5, and U5 correspond to respective monosaccharide moieties substituted to s1 and to s5, respectively; CHO, Chinese hamster ovary cells; NMR, nuclear magnetic resonance spectroscopy; 2D, two-dimensional; 3D, three-dimensional; COSY, proton correlation spectroscopy; TOCSY, totally correlated spectroscopy; NOESY, nuclear Overhauser enhancement spectroscopy; ROESY, rotating frame nuclear Overhauser enhancement spectroscopy; HSQC, heteronuclear single-quantum correlation spectroscopy; DG, distance geometry; MD, molecular dynamics; SA, simulated annealing; rms, root-mean-square; ap, antiperipalnar (180° ± 30°); ac, anticlinal (120° ± 30°); sc, synclinal (60° ± 30°); -sc, -synclinal -(60° ± 30°); sp, synperiplanar (0°±30°).
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