NMR and molecular modeling studies on two glycopeptides from the carbohydrate-protein linkage region of connective tissue proteoglycans

Pawan K. Agrawal, Jean-Claude Jacquinet1 and N. Rama Krishna2

Department of Biochemistry and Molecular Genetics, and Comprehensive Cancer Center, The University of Alabama at Birmingham, Birmingham, AL 35294-2041, USA and 1Institut de Chimie Organique et Analytique, UPRES-A CNRS 6005, UFR Sciences, Université d'Orléans, BP 6759, F-45067 Orléans Cedex, France

Received on September 25, 1998; revised on October 2, 1998; accepted on October 5, 1998

Complete 1H and 13C NMR assignments are reported for two glycopeptides representing the carbohydrate-protein linkage region of connective tissue proteoglycans. These glycopeptides are the octasaccharide hexapeptide, 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 the tetrasaccharide dipeptide, Ser(GlcpA[beta](1->3)Galp[beta](1->3)Galp[beta](1->4)Xylp[beta])-Gly (2). The vicinal coupling constant data show that the monosaccharide residues adopt 4C1 chair conformations. Distance geometry/simulated annealing calculations using 2D NOESY derived distance constraints yielded a single family of structures for the tetrasaccharide moiety, with well defined interglycosidic linkage conformations. The [phis] torsion angles of the glycosidic C1[prime]-O1 bonds showed a strict preference for the -sc range whereas the [psi] torsion angles (O1-Cn) exhibited dependence upon the interglycosidic linkage position (-ac for [beta](1->3) linkage, +ac for [beta](1->4) linkage). The predominant conformation about the glycopeptide bond is [phis] = -sc and [psi] = +ac. The presence of strong daN (i, i+1) NOE contacts, and the general absence of dNN (i, i+1) contacts (except for a weak Ser-5/Gly-6 dNN contact) and the dbN (i, i+1) contacts (except for Ser-1/Gly-2) in the ROESY spectrum, suggest that the backbone for 1 is predominantly in an extended conformation. A comparison of the ROESY data for 1 with those obtained from the unglycosylated hexapeptide (3) of the same sequence suggests that glycosylation has only a marginal influence on the backbone conformation of the hexapeptide.

Key words: conformation/glycopeptide/molecular modeling/NMR/proteoglycan

Introduction

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.

Results

1H and 13C chemical shift assignments

The primary structures of OSHP (1) and TSDP (2) are shown in Figure 1. The monosaccharide units of the tetrasaccharide moiety are designated as U, G[prime], G, and X, starting with serine substituted xylose as X, proximal galactose as G, distal galactose as G[prime], and glucopyranuronoic acid as U. Complete 1H and 13C chemical shift assignments for 1 and 2 were derived from TOCSY, PS-COSY, and HSQC spectra. Measured vicinal coupling constants (Table I) confirmed that each sugar ring in 1 and 2 adopts predominantly the expected 4C1 chair conformation (Krishna et al., 1990; Choe et al., 1991). Strong NOE was observed between H4 and H6 protons with small coupling constants, inferring gt conformation of hydroxymethyl group of both Gal residues (Zsiska and Meyer, 1993).


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 2) and in 3D NOESY-TOCSY experiments (data not shown). The chemical shifts of both Gal (G1+G5 and G[prime]1+ G[prime]5) and GlcA (U1+U5) moieties in 1 are completely degenerate for both tetrasaccharide chains substituted to Ser1 and Ser5, respectively. Some of the 1H NMR resonances of X1 residue appear at relatively lower field than the resonances of X5 residue and reflect shift perturbations due to the inductive effects from the ionizable groups. The 1H NMR assignments for 2 were found to be quite similar to that of the tetrasaccharide moiety of 1 which was substituted at s1. Table I gives the complete 1H and 13C chemical shift assignments for 1 and 2. The stereospecific assignment of the serine [beta] proton resonances was achieved by standard analysis of NH/H[beta], H[alpha]/H[beta] NOE's intensities observed in a 400 ms NOESY spectrum (Figures 2, 3) in conjunction with J[alpha][beta] coupling constants (Basus, 1989). The 3Jab and 3Jab' coupling constants for all the three serine residues range between 4 and 5 Hz. A population analysis among the three rotamers (Bystrov, 1976) suggests that the side chains adopt the [chi] = 60° rotamer predominantly.

Glucopyranuronoic acid-galactose-galactose-xylose conformation

The 400 ms NOESY spectrum of 1 (Figure 2) showed inter- as well as intra residue NOESY cross-peaks. The intra-residue NOE (H1/H3 and H1/H5) overlap very often with the interglycosidic NOEs due to chemical shift degeneracy. This requires special effort to accurately calculate interglycosidic NOEs between G H1 and XH4, G[prime] H1 and G H3, and U H1 and G[prime] H3. The volume of the well resolved GH1/GH5 NOE cross-peak was used as reference for the calculation of contribution of intra-residue diaxial NOE (2.5 Å from x-ray diffraction data (Sheldric, 1976)). For Gal (1->4) Xyl linkage, the G H1/X H4 cross-peak partially overlaps with G H1/G H3 cross-peak. The contribution of the later was calculated and subtracted from the observed G H1/G H3 +XH4 volume. Similarly, quantification of the transglycosidic NOE between G[prime] H1 and G H3 for Gal[prime] (1->3) Gal linkage was carried out by subtracting the contribution of G[prime] H1/ G[prime] H3 cross peak from the G[prime] H1-G[prime] H3 + G H3 cross peak. In an analogous manner, the contributions of G[prime] H1/G[prime] H3 and G[prime] H1/GH3 were subtracted from the combined U H1/GH3 + G[prime] H1/G[prime] H3 and G[prime] H1/GH3 NOE cross peak to derive NOE cross-peak volume for U H1/G[prime]H3. Since intra-residue NOEs do not contain information about the glycosidic orientation, they will not be discussed any further here.

Table I. 1H and 13C NMR chemical shifts for the constituent monosaccharide residues and the peptide backbone of OSHP (1), TSDP (2), and unglycosylated hexapeptide (3)a
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            
aAt pH 3.7, 278 K. The residue notation used is given in Figure 1.
bThe prime denotes the H3 proton in the IUPAC convention.


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 3) and ROESY spectrum (data not shown) of 1 exhibited the presence of strong d[alpha]N (i, i+1) contacts with no evidence of d[alpha]N (i, i+2) or long-range noncontiguous NOE's. Figure 4 compares the amide region of the ROESY and NOESY spectra. Interestingly, the 400 ms NOESY exhibited sequential dNN (i, i+1) NOEs (Figure 4B) which were absent in the ROESY spectrum (Figure 4A). This suggests that the dNN (i, i+1) NOEs arise from spin-diffusion (along the NH(i)->[alpha]H(i)->NH(i+1) pathway) in the NOESY spectrum (Figure 4B). The presence of strong d[alpha]N (i, i+1) NOE together with the general absence of dNN (i, i+1) (except for a weak S5/G6 contact) and general absence of d[beta]N (i, i+1) (except for S1/G2) in the ROESY spectrum are consistent with an extended type of backbone conformation (Dyson and Wright, 1991), perhaps with some flexibility due to glycines. We were unable to locate any nonsequential NOEs.


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 5) had an rms deviation of 0.48 Å for tetrasaccharide heavy atoms and 0.99 Å for all atoms with respect to their average. The NOEs calculated from simulated average tetrasaccharide structure (averages over all the 13 structures) showed good agreement with the experimental NOEs (Table III). The glycosidic dihedral angles for the energy minimized average structure A are [phis]1 = -85.5° and [psi]1 = 100.2°; [phis]2 = -68.9° and [psi]2 = 112.1°; [phis]3 = -62.7° and [psi]3 = -149.8°; and [phis]4 = -63.2° and [psi]4 = -147.0°. It is evident that U-G[prime], G[prime]-G, and G-X linkages adopt conformations compatible with -sc/-ac, -sc/-ac, and -sc/+ac, respectively. These values are quite similar to those reported for G-X linkage in G[prime]GXS (Choe et al., 1991), but differ particularly for G[prime]-G linkage, where comparable NOESY intensities were noted for the G[prime] H1/G H3 and G[prime] H1/G H4 cross-peaks, and values of -168.1° ([phis]3) and -171.6° ([psi]3) were calculated. A careful reexamination of the earlier NOESY data obtained on a WH-400 for G[prime]GXS showed that artifacts due to strong t1 ridges and symmetrization led to comparable intensities. The current study revises the earlier result (Choe et al., 1991) on G[prime]-G linkage. To check for the above mentioned experimental errors, we remeasured 400 ms NOESY spectra for G[prime]GXS at 600 MHz on a Bruker AM-600 system. The interglycosidic NOESY contacts observed for G[prime]GXS are in complete agreement with those in the present studies on 1 and 2 (i.e., strong G[prime] H1/G H3 and weak G[prime] H1/G H4 intensities). Because of the absence of nonsequential NOESY constraints along the peptide backbone, its conformation in 1 is not as well defined (not shown).

Table II. 1H-1H coupling constants for the constituent monosaccharide residues and peptide backbone of OSHP (1), TSDP (2), and unglycosylated hexapeptide (3)a
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

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. Average calculated torsion angles ([phis] and [psi]) for interglycosidic bonds in TSDPa
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)
aValues in parentheses indicate the maximum range.

Table IV. Comparison of experimental interglycosidic and interglycopeptide proton-proton distances (Å) in TSDP from 2D NOESY analysis with those calculated for the energy minimized average structure
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.

Materials and methods

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.

Acknowledgments

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.

Abbreviations

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°).

References

Andreotti ,A.H. and Kahne,D. (1993) Effect of glycosylation on peptide backbone conformation. J. Am. Chem. Soc., 115, 3352-3353.

Basus ,V. (1989) Proton nuclear magnetic resonance assignments. Methods Enzymol., 177, 132-149. MEDLINE Abstract

Baumann ,H., Jansson,P.-E. and Kenne,L. (1988) N.M.R. and conformational studies on some, 1,3-linked disaccharides. J. Chem. Soc. Perkin Trans., 1, 209-217.

Baumann ,H., Erbing,B., Jansson,P.-E. and Kenne,L. (1989) N.M.R. and conformational studies of some 3-O-, 4-O-, and, 3,4-di-O-glycopyranosyl substituted methyl [alpha]-O-galactopyranosides. J. Chem. Soc. Perkin Trans., 1, 2153-2165.

Bruzik ,K.S. and Nyholm,P.G. (1997) NMR study of the conformation of galactocerebroside in bilayers and solution: galactose reorientation during the metastable-stable gel transition. Biochemistry, 36, 566-575. MEDLINE Abstract

Bystrov ,V.F. (1976) Spin-spin coupling and the conformational states of peptide systems. Prog. NMR Spectrosc., 10, 41-81.

Choe ,B.-Y., Ekborg,G.C., Roden,L., Harvey,S.C. and Krishna,N.R., (1991) High-resolution NMR and molecular modeling studies on O-[beta]-d-Gal(1->3)-O-[beta]-d-Gal(1->4)-O-[beta]-d-Xyl-(1->0)-l-Ser, a carbohydrate-protein linkage region fragment form connective tissue proteoglycans. J. Am. Chem. Soc., 113, 3743-3749.

Curto ,E.V., Sakai,T.T., Jablonsky,M.J., Rio-Anneheim,S., Jacquinet,J.-C. and Krishna,N.R. (1996) Complete 1H NMR assignments of synthetic glycopeptides from carbohydrate-protein linkage regions of serglycins. Glycoconjugate J., 13, 599-607.

Dabrowski ,J. (1994) In Croasmun,W.R. and Carlson,R.K. (eds.), Two-Dimensional NMR Spectroscopy, Applications for Chemists and Biochemists, 2nd Edition. VCH, New York, pp. 741-783.

De Waard ,P., Vliegenthart,J.F.G., Harada,T. and Sugahara,K. (1992) Structural studies on sulfated oligosaccharides derived from the carbohydrate-protein linkage region of chondroitin 6-sulfate proteoglycans of shark cartilage II, seven compounds containing 2 or 3 sulfate residues. J. Biol. Chem., 267, 6036-6043. MEDLINE Abstract

Dyson ,H.J. and Wright,P.E. (1991) Defining solution conformations of small linear peptide. Annu. Rev. Biophys. Biophys. Chem., 50, 519-537.

Esko ,J.D. and Zhong,L. (1996) Influence of core protein sequence of glycosaminoglycan assembly. Curr. Opin. Struct. Biol., 6, 663-670. MEDLINE Abstract

Fritz ,T.A., Agrawal,P.K., Esko,J.D. and Krishna,N.R. (1997) Partial purification and substrate specificity of [alpha]-N-acetylglucosaminyltransferase I; synthesis, NMR spectroscopic characterization and in vivo assays of two aryl tetrasaccharide. Glycobiology, 7, 587-595. MEDLINE Abstract

Gerken ,T.A., Butenhof,K.J. and Shogren,R. (1989) Effects of glycosylation on the conformation and dynamics of O-linked glycoproteins: carbon-13 NMR studies of ovine submaxillary mucin. Biochemistry, 28, 5536-5543. MEDLINE Abstract

Gervais ,V., Zerial,A. and Oschkinat,H. (1997) NMR investigations of the role of the sugar moiety in glycosylated recombinant human granulocyte-colony stimulating factor. Eur. J. Biochem., 247, 386-395. MEDLINE Abstract

Goto ,F. and Ogawa, T. (1993) Recent aspects of glycoconjugates synthesis. A synthetic approach to the linkage region of proteoglycans. Pure Appl. Chem., 65, 793-801.

Howard ,K.P. and Prestegard,J.H. (1995) Membrane and solution conformations of monogalactosyldiacylglycerol using NMR/molecular modeling methods. J. Am. Chem. Soc., 117, 5031-5040.

Hricovini ,M., Tvaroska,I., Mirsch,J. and Duben,A.J. (1990) Solution behavior of methyl [beta]-xylobioside: conformational flexibility revealed by N.M.R. measurements and theoretical calculations. Carbohydr. Res., 198, 193-203. MEDLINE Abstract

Huang ,X., Barch,J.J.,Jr., Lung,F.T., Roller,P.P., Nora,P.L., Mushick,J. and Garrity,R.R. (1997) Glycosylation affects both the three-dimensional structure and antibody binding properties of the HIV-IIIIB GP120 peptide RP135. Biochemistry, 36, 10846-10856. MEDLINE Abstract

Kessler ,H., Matter,H., Gemmecker,G., Kottenhahn,M. and Bates,J.W. (1992) Structure and dynamics of a synthetic O-glycosylated cyclopeptide in solution determined by NMR spectroscopy and MD calculations. J. Am. Chem. Soc., 114, 4805-4818.

Kitagawa ,H., Tanaka,Y., Tsuchida,K., Goto,F., Ogawa,T., Lidholt,K., Lindhal,U. and Sugahara,K. (1995) N-Acetylgalactosamine(GalNAc) transfer to the common carbohydrate-protein linkage region of sulfated glycosaminoglycans. Identification of UDP-GalNAc:chondro-oligosaccharide [alpha]-N-acetylgalactosaminyltransferase in fetal bovine serum. J. Biol. Chem., 270, 22190-22195. MEDLINE Abstract

Kjéllen ,L. and Lindahl,U. (1991) Proteoglycans: structures and interactions. Annu. Rev. Biochem., 60, 443-475. MEDLINE Abstract

Krishna ,N.R., Choe,B.-Y., Prabhakaran,M., Ekborg,G.C., Roden,L. and Harvey,S.C. (1990) Nuclear magnetic resonance and molecular model studies on O-[beta]-d-galactopyranosyl-(1->4)-O-[beta]-d-xylopyranosyl-(1->0)-l-Ser, a carbohydrate-protein linkage region fragment from connective tissue proteoglycans. J. Biol. Chem., 265, 18256-18262. MEDLINE Abstract

Lee ,W., Moore,C.H., Watt,D.D. and Krishna,N.R. (1994) Solution structure of the variant-3 neurotoxin from Centruroides sculpturatus Ewing. Eur. J. Biochem., 218, 89-95.

Lemieux ,R.U., Koto,S. and Voisin,D. (1979) In Szarek,W.A. and Horton,D. (eds.), The Anomeric Effect, Origin and Consequnces. ACS Symposium Series 87. American Chemical Society, Washington, DC, pp. 17-29,

Liang ,R., Andreotti,A.H. and Kahne,D. (1995) Sensitivity of glycopeptide conformation to carbohydrate chain length. J. Am. Chem. Soc., 117, 10395-10396.

Liu ,J., Desai,U.R., Han,X.-J., Toida,T. and Linhardt,R.J. (1995) Strategy for the sequence analysis of heparin. Glycobiology, 5, 765-774. MEDLINE Abstract

Manzi ,A., Salimath,P.V., Spiro,R.C., Keifer,P.A. and Freeze,H.H. (1995) Identification of a novel glycosaminoglycan core-like molecule I. 500 MHz 1H NMR analysis using a nano-NMR probe indicate the presence of terminal [alpha]-GalNAc residue capping 4-methylumbelliferyl-[beta]-d-xylosides. J. Biol. Chem., 270, 9154 -9163. MEDLINE Abstract

Mer ,G., Hietter,H. and Lefevre,J.-F. (1996) Stabilization of proteins by glycosylation examined by NMR analysis of a fucosylated proteinase inhibitor. Nature Struct. Biol., 3, 45-53.

Neumann ,K.W., Tamura,J. and Ogawa, T. (1995) A stereocontrolled synthetic approach of glycopeptides corresponding to the carbohydrate-protein linkage region of cell-surface proteoglycans. Bioorg. Med. Chem., 3, 1637-1650. MEDLINE Abstract

Neumann ,K.W., Tamura,J.I. and Ogawa,T. (1996) Synthesis of a novel glycosaminoglycan pentasaccharide serine having an N-acetylgalactosamine residue [alpha]-linked to the core linkage tetrasaccharide. Glycoconjugate J., 13, 933-936.

Nielsson ,M., Westaman,J. and Svahn,C.-M. (1993) Synthesis of tri- and tetrasaccharides present in the linkage region of heparin and heparan sulphate. J. Carbohydr. Chem., 12, 23-37.

Paulsen ,H. (1990) Syntheses, conformations and x-ray structure analyses of the saccharide chains from the core regions of glycoproteins. Angew. Chem. Int. Ed. Engl., 29, 823-839.

Paulsen ,H., Busch,R., Sinnwell,V. and Pollex-Krüger,A. (1991) Konformationsanalytishe untersuchugen von D-xylosehaltigen O-glycopeptidsequuenzen. Carbohydr. Res., 214, 227-234. MEDLINE Abstract

Poole ,A.R. (1986) Proteoglycans in health and disease: structures and functions. Biochem. J., 236, 1-14. MEDLINE Abstract

Rio ,S., Beau,J.-M. and Jacquinet,J.-C. (1993) Total synthesis of the carbohydrate-protein linkage region common to several mammalian proteoglycans. Carbohydr. Res., 244, 295-313. MEDLINE Abstract

Roden ,L. (1980) Structure and metabolism of connective tissue proteoglycans. In Lennarz,W.J. (ed.), The Biochemistry of Glycoproteins and Proteoglycans. Plenum, New York, pp. 267-371.

Saha ,V.K., Griffith,L.S., Rademann,J., Geyer,A. and Scmidt,A.R. (1997) An N-acetylglucosamine containing glycopeptide-synthesis and structure assignment. Carbohydr. Res., 304, 21-28.

Sheldric ,B. (1976) The crstal structures of the [alpha]- and [beta]-anomers of d-galactose. Acta Crystallogr., B32, 1016-1020.

Silbert ,J.E. and Sugamaran,G. (1995) Intercellular membranes in the synthesis, transport, and metabolism of proteoglycans. Biochem. Biophys. Acta, 1241, 371-384.

Sugahara ,K., Ohi,Y., Harada,T., De Waard,P. and Vliegenthart,J.F.G. (1992) Structural studies on sulfated oligosaccharides derived from the carbohydrate-protein linkage region of chondroitin 6-sulfate proteoglycans of shark cartilage I, six compounds containing 0 or 1 sulfate and/or phosphate residue. J. Biol. Chem., 267, 6027-6035. MEDLINE Abstract

Sugahara ,K., Tohno-oka,R., Yamada,S., Khoo,K.-H., Morris,H.R. and Dell,A. (1994) Structural studies on the oligosaccharides isolated from bovine kidney heparan sulphate and characterization of bacterial heparitinases used as substrates. Glycobiology, 4, 535-544. MEDLINE Abstract

Sugahara ,K., Tsuda,H., Yoshida, K., Yamada,S., De Beer,T. and Vliegenthart,J.F.G. (1995) Structure determination of the octa- and decasaccharide sequences isolated from the carbohydrate-protein linkage region of porcine intestine heparin. J. Biol. Chem., 270, 22914-22923. MEDLINE Abstract

Van Halbeek ,H., Dorland,L., Veldink,G.A., Vliegenthart,J.F.G., Garegg,P.J., Noorberg,T. and Lindberg,B. (1982) A 500-MHz proton-magnetic-resonance study of several fragments of the carbohydrate-protein linkage region commonly occurring in proteoglycans. Eur. J. Biochem., 127, 1-6. MEDLINE Abstract

Vuister ,G.W., Boelens,R. and Kaptein,R. (1988) Nonselective three-dimensional NMR spectroscopy. The 3D NOE-HOHAHA experiment. J. Magn. Reson., 80, 176-185.

Wüthrich ,K. (1986) NMR of Proteins and Nucleic Acids. Wiley, New York.

Zsiska ,M. and Meyer,B. (1993) Influence of sulfate and carboxylate groups on the conformation of chondroitin sulfate related disaccharides. Carbohydr. Res., 243, 225-258. MEDLINE Abstract


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