Conformational studies on the MUC1 tandem repeat glycopeptides: implication for the enzymatic O-glycosylation of the mucin protein core

Leo Kinarsky2, Ganesh Suryanarayanan2, Om Prakash3, Hans Paulsen4, Henrik Clausen5, Franz-Georg Hanisch6, Michael A. Hollingsworth2 and Simon Sherman1,2

2 Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198-6805; 3 Department of Biochemistry, Kansas State University, Manhattan, KS 66506; 4 Institute of Organic Chemistry, University of Hamburg, 20146 Hamburg, Germany; 5 Faculty of Health Sciences, School of Dentistry, University of Copenhagen, Copenhagen DK-2200, Denmark; 6 Institute of Biochemistry, Medical Faculty, University of Cologne, 50931 Cologne, Germany

Received on May 19, 2003; revised on August 1, 2003; accepted on August 4, 2003


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
The tandem repeat of the MUC1 protein core is a major site of O-glycosylation that is catalyzed by several polypeptide GalNAc-transferases. To define structural features of the peptide substrates that contribute to acceptor substrate efficiency, solution structures of the 21-residue peptide AHGVTSAPDTRPAPGSTAPPA (AHG21) from the MUC1 protein core and four isoforms, glycosylated with {alpha}-N-acetylgalactosamine on corresponding Thr residues, AHG21 (T5), AHG21 (T10), AHG21 (T17), and AHG21 (T5,T17), were investigated by NMR spectroscopy and computational methods. NMR studies revealed that sugar attachment affected the conformational equilibrium of the peptide backbone near the glycosylated Thr residues. The clustering of the low-energy conformations for nonglycosylated and glycosylated counterparts within the VTSA, DTR, and GSTA fragments (including all sites of potential glycosylation catalyzed by GalNAc-T1, -T2, and -T4 transferases) showed that the glycosylated peptides display distinct structural propensities that may explain, in part, the differences in substrate specificities exhibited by these polypeptide GalNAc-transferases.

Key words: glycopeptide / NMR / O-glycosylation / substrate specificity


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Mucin-type O-glycosylation is initiated by the transfer of N-acetylgalactosamine (GalNAc) from UDP-GalNAc to the side chain of serine and/or threonine. This reaction is catalyzed by the family of UDP-N-acetylgalactosamine: polypeptide N-acetylgalactosaminyl transferases (GalNAc-transferases) (Lis and Sharon, 1993Go; Clausen and Bennett, 1996Go). Multiple isoenzymes of GalNAc-transferases exist in all species examined to date. There are published reports describing 12 human GalNAc-transferases (Ten Hagen et al., 2003Go) that demonstrate distinct (with some overlapping) kinetic properties and specificities for peptide substrates (Bennett et al., 1996Go; Hagen et al., 1997Go; Nishimori et al., 1994Go; Wandall et al., 1997Go; Hanisch et al., 1999Go; Tetaert et al., 2001aGo,bGo).

The primary step of O-glycosylation involves essential recognition events between the glycosyltransferase and protein substrate. A number of early studies investigated substrate specificity but achieved limited success. Initial studies were complicated by the variable expression of different GalNAc-transferases in different cell lines and the complexities of evaluating substrate specificity in vivo, where a number of distinct enzymes react with the substrate. It is now generally accepted that different GalNAc-transferases employ distinct parameters of substrate recognition; however, the mechanisms whereby the distinct enzymes undertake substrate recognition and catalytic activity are poorly understood (Clausen and Bennett, 1996Go; Gooley and Williams, 1994Go; Elhammer et al., 1993Go, 1999Go). No consensus peptide-sequence motif for acceptor sites has been identified for any of the GalNAc-transferases (Lis and Sharon, 1993Go; Clausen and Bennett, 1996Go; Ten Hagen et al., 2003Go). Although some enzymes show site-selective specificities, the same enzymes demonstrate overlapping specificities with other enzymes. Moreover, several enzymes (e.g., GalNAc-T4 and -T7) catalyze the glycosylation of some sites only after flanking serine or threonine residues have been glycosylated (Bennett et al., 1999aGo,bGo; Hassan et al., 2000Go; Hanisch et al., 2001Go). Previous studies have shown that the amino acid sequence and environment near the site of glycosylation may significantly affect enzyme activity (Nishimori et al., 1994Go; Wandall et al., 1997Go; Elhammer et al., 1993Go, 1999Go; Gerken et al., 1997Go, 2002Go). However, detailed structural information on the substrate parameters that determine the optimal recognition and binding by the different GalNAc-transferases is largely unknown.

The aim of this study was to define distinct conformational features of the human mucin (MUC1) peptide substrates that contribute to acceptor substrate efficiency. Synthetic peptides from the tandem repeat of the MUC1 core protein, a major site of mucin-type O-glycosylation, have been widely used for kinetic and structural studies of the transferase specificities (Nishimori et al., 1994Go; Liu et al., 1995Go; Fontenot et al., 1995Go; Wandall et al., 1997Go; Hanisch et al., 1999Go; Kirnarsky et al., 1998Go, 2000Go). Previously, based on nuclear magnetic resonance (NMR) studies of relatively short, nonglycosylated MUC1 peptides, we proposed a structural model of the acceptor substrate recognition by the transferases GalNAc-T1 and -T3 (Kirnarsky et al., 1998Go). This model defined a structural motif containing extended conformations of the peptide backbone at and around the glycosylation site as one important determinant for enzyme–substrate recognition (Kirnarsky et al., 1998Go; Elhammer et al., 1993Go, 1999Go; Gerken et al., 1997Go).

In the present study, we used NMR spectroscopy and computational methods for the structural analysis of a series of glycosylated and nonglycosylated 21-residue MUC1 peptides, AHGVTSAPDTRPAPGSTAPPA (AHG21). Some peptides were monoglycosylated with a GalNAc at positions Thr5, Thr10, or Thr17. Other peptides were diglycosylated at positions Thr5 and Thr17. Structural data on differentially glycosylated AHG21 peptides, in conjunction with kinetic data from three purified recombinant transferases, GalNAc-T1, -T2, and -T4 (Wandall et al., 1997Go; Hanisch et al., 1999Go, 2001Go; Hassan et al., 2000Go), allowed us to define distinct conformational propensities of the peptide backbone at the glycosylation site that we postulate serve as the primary target for the substrate recognition by these GalNAc-transferases.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
NMR analysis
Intraresidue resonances for each amino acid residue were assigned from the total correlation spectroscopy (TOCSY) and DQF-COSY, double quantum filtered correlation spectroscopy (DQF-COSY) spectra (Table I). Sequential assignments were made using the nuclear Overhauser enhancement spectroscopy (NOESY) and rotating frame nuclear Overhauser enhancement spectroscopy (ROESY) spectra to define d{alpha}N(i, i + 1) and dNN(i, i + 1) connectivities. Backbone proton–proton coupling constants 3JN{alpha}r were measured primarily from 1D spectra and verified with DQF-COSY spectra. The coupling constants ranged from 6 to 8.5 Hz, values that are consistent with an absence of ordered portions of a secondary structure. The NOE connectivities and the ranges for torsional angles of the nonglycosylated and glycosylated peptides, AHG21, AHG21 (T5), AHG21 (T10), AHG21 (T17), and AHG21 (T5, T17), are shown in Figure 1. A large number of strong consecutive d{alpha}N(i, i + 1) connectivities suggested a predominance of extended backbone conformations. Strong d{alpha}{delta} connectivities for Ala7–Pro8, Arg11–Pro12, Ala13–Pro14, Ala18–Pro19, and Pro19–Pro20 were diagnostic for a trans conformation of the peptide backbone. In addition, the trans form for the Pro19–Pro20 bond was supported by the absence of d{alpha}{alpha} contacts.


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Table I. Chemical shifts (ppm) for the nonglycosylated peptide AHG21 in H2O:D2O (pH = 4.5) at 10°C

 


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Fig. 1. Summary of the NMR data. (A) AHG21, (B) AHG21 (T5), (C) AHG21 (T10), (D) AHG21 (T17), (E) AHG21 (T5, T17). The seqplot command from the DYANA program was used to draw all plots (Güntert et al., 1997Go). The intensities of d{alpha}N, dNN, and dßN connectivities are represented by the thickness of the respective line as strong, medium, weak, or very weak. In case of proline, NH refers to {delta}H. An asterisk indicates ambiguous cross-peaks. For {phi} and {psi}. angles, triangles pointing upward or downward indicate restraints that are compatible with a {alpha}-helix or ß-strands; a star incloses both {alpha}- and ß-conformations; and a filled circle represents a restraint that excludes both {alpha}- and ß-conformations. For the {chi}1 angle, filled squares of three different sizes represent restraints that allow for one, two, or all three of the {chi}1 rotamers (-60°, 60°, 180°).

 
Very similar patterns of chemical shifts, connectivities, and coupling constants were observed for most amino acid residues of the nonglycosylated and glycosylated peptides. There were significant differences, however, between the glycosylated and nonglycosylated threonine residues and two vicinal residues at positions g-1 and g + 1, where g denotes the position of the glycosylation site (Table II). At all three sites used for glycosylation, the glycosylated threonine and preceding residue demonstrated downfield deviations from the random-coil values for alpha proton chemical shifts. These downfield shifts may indicate slight conformational changes to a more extended ß-strand-like structure. The {alpha}H chemical shifts for most of amino acid residues were close to the values of a random-coil structure in water. Neither peptide showed distinct evidence of a stabilized secondary structure. These NMR data are characteristic for linear peptides in aqueous solution in preferably extended conformations (Dyson and Wright, 1991Go).


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Table II. Chemical shifts (ppm) and coupling constants (CC) (Hz) for the threonines to be glycosylated and flanking residues

 
Sequential dNN connectivities were observed near all three threonine residues but under different conditions. All peptides with nonglycosylated T5 demonstrated weak dNN connectivities between Gly3–Val4, whereas additional dNN connectivities between Val4–Thr5* were observed in glycopeptides AHG21 (T5) and AHG21 (T5, T17) (Figure 1). Nonglycosylated Thr10 demonstrated medium dNN connectivities to both flanking residues, Asp9 and Arg11. On glycosylation of Thr10, the dNN connectivity between Asp9 and Thr10* was not detected. For the Gly15–Ser16–Thr17*–Ala18 fragment, both peptides, AHG21 (T5, T17) and AHG21 (T17), demonstrated sequential dNN connectivities between all these residues. The dNN connectivity between Gly15 and Ser16 was detected within the peptides with nonglycosylated T17 (Figure 1).

Carbohydrate–peptide and carbohydrate–carbohydrate interactions
On the addition of GalNAc, several NOE cross-peaks related to sugar–peptide interactions were observed near the glycosylated threonines (Table III). All three glycosylated threonines demonstrated medium cross-peaks between the peptide backbone amide proton and the amide proton of the N-acetyl group of GalNAc. As shown previously, this is consistent with an orientation of the monosaccharide perpendicular to the peptide backbone (Kirnarsky et al., 2000Go). For glycosylated Thr5, the methyl group of GalNAc showed weak contact with the alpha proton of the following Ser6. No sugar contacts were detected with adjacent residues for Thr10* and Thr17*. For all glycosylated threonines, weak or very weak NOE cross-peaks were observed between the H5 proton of the sugar ring and the methyl group of the side chain of the threonine. All three glycosylated threonines showed strong to medium contacts between the methyl group of GalNAc and the H2 and H3 sugar protons. We could not detect any cumulative structural effects caused by the simultaneous addition of two GalNAc residues at AHG21 (T5, T17), but we cannot rule out the possibility that such effects result from a higher density of glycosylation.


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Table III. Carbohydrate–peptide and carbohydrate–carbohydrate NOEs

 
Computational results
The limited set of NMR-derived constraints for these relatively small, linear peptides produced wide conformational variations of the structures generated by DYANA. After energy minimization, 125 structures (25 best conformers for each peptide) were selected for all peptides: AHG21, AHG21 (T5), AHG21 (T10), AHG21 (T17), and AHG21 (T5, T17). Those structures were grouped in structural clusters using root-mean-square deviation (RMSD) criterion for polypeptide backbone heavy atoms. Three peptide fragments, VTSA, DTR, and GSTA were selected for cluster analysis to determine the effects of glycosylation on the conformational propensities of the peptide backbone near the glycosylation site. These fragments included all sites of potential glycosylation and the flanking residues. The distinct structural clusters composed of conformers of either nonglycosylated or glycosylated peptides, or mixed clusters, represented distinct conformational propensities of the peptide backbone.

For the VTSA fragment, several distinct clusters were obtained using the RMSD criterion of 0.7 Å (Table IV). The most populated cluster (PBID) included about 30% of all structures and was composed of a mix of conformers of nonglycosylated and glycosylated at Thr5 peptides. Another mixed cluster (DPP/BD) contained about 15% of all structures. Two different clusters (PBDI and PPID), which represented 14% and 8% of all conformers, were populated only by structures without the sugar at the Thr5 position. Also, two distinct clusters (D/IBBP and DPBP), which represented 12% and 10% of all conformers, contained only conformers of peptides glycosylated at Thr5. A few smaller clusters (less than 5%) were not considered. Thus about half of all structures for the VTSA fragment fell in the clusters that included conformers of both nonglycosylated at Thr5 and glycosylated peptides, and another half of the structures were assigned to separate clusters with conformers of peptides either glycosylated or nonglycosylated at the Thr5 position.


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Table IV. Conformations of the averaged structures for the most populated structural clusters within the VTSA fragment of the AHG21 peptides

 


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Fig. 2. Conformational clusters on the Ramachandran plot. The B area contains extended ß-strand conformations; P, polyproline II–like structures; I, inverse {gamma}-turn; R and L, right and left {alpha}-helix, respectively; D, named by analogy with Zimmerman et al. (1977)Go. {Phi} and {psi} torsional angles are in degrees.

 
The backbone conformations of the averaged structures for all clusters are shown in the decreasing order of cluster population. Conformations of the nonglycosylated Thr5 for the averaged structures of the two most populated clusters were assigned to the B area on the Ramachandran plot (Figure 2; see details in Materials and methods). Conformations of Thr5 within the two other clusters were assigned to the P area of the Ramachandran plot, which is typical for the polyproline II conformation that has been suggested as the most probable conformation for the 25-residue MUC1 peptide (Spencer et al., 1999Go). After glycosylation, preferred conformations of the Thr5* were mostly distributed between the same B and P areas. For the vicinal Ser6 residue, the I and D areas of the Ramachandran plot were most populated, if Thr5 was not glycosylated (Table IV). Region I included an inverse {gamma}-turn structure that was previously described for shorter peptides within the VTSA and DTR fragments (Kirnarsky et al., 2000Go). The notation P/B or D/I designate conformations that are located between the corresponding areas of the Ramachandran plot. Glycosylation of Thr5 significantly affected conformational propensities of the vicinal Ser6 residue (Figure 3). On glycosylation, the two most populated clusters demonstrated extended conformations of the averaged structures that fell in the B area of the Ramachandran plot. Two smaller structural groups occupied P/B and I areas (Table IV).



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Fig. 3. Fragment VTSA. The most populated clusters with (A) nonglycosylated and (B) glycosylated Thr5.

 
For the DTR fragment, several clusters were defined with the RMSD = 0.7Å (Table V). For the AHG21(T10) peptide, glycosylated at position Thr10, two distinct clusters (RBP and IPP) were obtained with 13% and 7% of all conformers, respectively. (Note: 25 conformers glycosylated at Thr10 represent 20% of all conformers.) Conformations of the peptides with the nonglycosylated Thr10 residue made up five clusters with three larger clusters (IDP, IDB, and LDP) populated by 41%, 21%, and 12% of all conformers, correspondingly. The backbone conformations of the two most populated structural clusters with the nonglycosylated Thr10 demonstrated a very similar structural motif that was comprised of conformations populating the I and D regions for Asp9 and Thr10, correspondingly. The major difference between these two clusters was the conformational variation of the Arg11 (P or B). Many conformers demonstrated inverse {gamma}-turn-like conformations, which included the characteristic hydrogen bonding between COi-1 and HNi+1 groups for both the Thr10 and the preceding Asp9, as described previously (Kirnarsky et al., 2000Go). On glycosylation, the preferred conformations of Thr10* were more extended (B or P areas with {psi}>150°).


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Table V. Conformations of the averaged structures for the structural clusters within the DTR fragment of the AHG21 peptides

 
For the GSTA fragment, seven distinct clusters were obtained with the RMSD = 0.9 Å (Table VI). The larger RMSD resulted from the apparent conformational flexibility of the Gly15 residue. The largest mixed cluster (DBPP) contained 34% (26% and 8%) of all conformers, whereas the two smaller clusters (LDPB and LDB/D/IB) were made up of either only nonglycosylated at Thr 17 or glycosylated at this position conformers. Four small structural clusters were also observed: two consisted of either nonglycosylated at Thr17 (RIBP and RPPB) or glycosylated conformers (LPDP and RDDP). The preferred conformations for the nonglycosylated Thr17 fell into the P area of the Ramachandran plot. The B area was significantly less populated. The vicinal Ser16 residue demonstrated a preference of the extended conformations that occupied the B area of the Ramachandran plot ({psi}>110°). On glycosylation of the Thr17, the most populated conformations of the Ser16 were more folded, as evidenced by a shift to the D area ({psi}<110°).


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Table VI. Conformations of the averaged structures for the most populated structural clusters within the GSTA fragment of the AHG21 peptides

 
Glycosylation at Thr17 significantly affected mutual orientation of the side chains of the Thr residue and vicinal Ser16. The most populated clusters comprised of the nonglycosylated at the Thr17 position conformers (DBPP) and the glycosylated at Thr17 conformers (LDB/D/IB) are shown in Figure 4.



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Fig. 4. Fragment STAP. The most populated clusters with (A) nonglycosylated and (B) glycosylated Thr17.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Parameters that influence the substrate specificity and catalytic efficiency of the polypeptide GalNAc-transferases are poorly understood. These enzymes catalyze mucin-type O-glycosylation of serine and threonine residues, and have shown distinct (with some overlapping) specificities for peptide substrates, such as the MUC1 tandem repeat that was the subject of this investigation.

Previously, based on the NMR studies of the short peptides from the MUC1 protein core, we proposed a structural model that could explain some molecular details of the initial recognition of the peptide substrates by the GalNAc-T1 and -T3. It was hypothesized that polypeptide GalNAc-transferases interact with substrates at three positions: the residue to be glycosylated and two proximal domains at N- and C-termini. The extended backbone conformation of the substrate that provided the proper orientation of the potential donors and acceptors of the hydrogen bonding was predicted to be important for the substrate efficiency (Kirnarsky et al., 1998Go). Although this model was based on a very limited set of structural and kinetic in vitro data for the short, nine-residue peptides, it was generally consistent with the extended acceptor substrate conformation complementing the extended binding site of the GalNAc-transferase proposed by Elhammer and co-workers based on the in vivo data analysis (Elhammer et al., 1993Go, 1999Go). Also, we demonstrated previously that glycosylation of the threonine residue within the VTSA region affected peptide backbone conformations of the 15-residue MUC1 protein core fragment (Kirnarsky et al., 2000Go).

In the present study we determined the structural propensities of the polypeptide backbone near all sites of O-glycosylation that might be critical for the initial recognition by the distinct GalNAc-transferase and defined the influence of threonine glycosylation on the flanking serine residues within the VTSA and GSTA fragments. NMR-derived structural data on differently glycosylated 21- residue MUC1 peptides were compared with kinetic data for purified recombinant transferases GalNAc-T1, -T2, and -T4 (Wandall et al., 1997Go; Hassan et al., 2000Go; Hanisch et al., 1999Go, 2001Go). These kinetic data were obtained with the 21-residue AHG21 peptide, as well as with the larger MUC1-derived substrates, TAP24 and TAP25. GalNAc-T1 and -T2 have distinct specificities for these peptides with a partial overlap (Wandall et al., 1997Go). GalNAc-T4 requires prior glycosylation of the peptide substrate for activity and catalyzes glycosylation of two sites in the MUC1 peptides that are not used by other known GalNAc-transferases (Bennett et al., 1998Go). The requirement for prior glycosylation of peptide substrates for activity of GalNAc-T4 (and for substrate specificity exhibited by other GalNAc-transferases) has led to the postulation of two nonmutually exclusive hypotheses: (1) Glycosylation of peptide substrates induces conformations that can be recognized by the catalytic site of an appropriate GalNAc-transferase (e.g., GalNAc-T4); or (2) glycosylated residues on peptide substrates activate novel functions for GalNAc-transferases through the "lectin domain," which is separate from the catalytic site (through allosteric means or other ways such as localizing the enzyme to the substrate). We have tested the first hypothesis (glycosylation of peptide substrates alters their conformation so that conformations are created that can be recognized by the catalytic site of an appropriate GalNAc-transferase) in the studies reported in this manuscript. Specifically, we endeavored to detect alterations in the conformational propensity of MUC1 peptides that are glycosylated at positions that enable the peptides to serve as substrates for GalNAc-T4. Our NMR data for the AHG21 glycopeptides showed that O-glycosylation influenced chemical shifts and coupling constants only for glycosylated threonines and the nearest flanking residues. Therefore we focused our analysis on structural propensities within the peptide fragments VTSA, DTR, and GSTA.

The catalytic efficiencies of different GalNAc- transferases for glycosylation of different sites on MUC1 peptides can be ranked in the following order: GalNAc-T1:Thr5>Thr17>>Ser16; GalNAc-T2: Thr17>Thr5> Ser16; and GalNAc-T4:Thr10>Ser16 and Ser6, if the peptide was previously glycosylated at any other site (Wandall et al., 1997Go; Hassan et al., 2000Go; Hanisch et al., 2001Go). The catalytic efficiencies of these transferases for specific glycosylation sites on MUC1 peptides were correlated with the structural propensities of the peptide backbone within the AHG21 peptides obtained from the NMR data. The conformational preferences for the nonglycosylated Thr5, Thr17, and Thr10 residues were ranked in the following order:Thr5:B>P; Thr17:P>B; and Thr10:D (Tables IVGoVI). A comparison of the kinetic and structural data suggests that the kinetic efficiencies observed for the GalNAc-T1, -T2, and -T4 can be correlated with specific conformations for the residue to be glycosylated. Thus the extended backbone conformations assigned to the B area of the Ramachandran plot are preferable for the GalNAc-T1; polyproline II–like conformations assigned to the P area are primarily recognized by the GalNAc-T2; and relatively folded conformations assigned to the D area of the Ramachandran plot are required for the efficient recognition and binding by the GalNAc-T4. The amino acid sequence context and the proper conformations of the flanking residues may significantly contribute to the substrate efficiency.

For GalNAc-T1, the extended backbone conformations at and around the residue to be glycosylated are consistent with previously proposed extended structural motifs for substrate recognition (Elhammer et al., 1993Go, 1999Go; Gerken et al., 1997Go; Kirnarsky et al., 1998Go). The N-terminal extension and the presence of the proline residue at the g + 3 position are likely to contribute significantly to catalytic efficiency with GalNAc-T1. The ability of GalNAc-T1 to glycosylate the first threonine of the TAP25 peptides with valine substitutions (Figure 1, H and L in Hassan et al., 2000Go) does not contradict this observation because these peptides have been synthesized with a biotin group at the N-terminus (Hassan et al., 2000Go) that can serve structurally as an N-terminal extension. The inactivity of the GalNAc-T1 toward the Ser6, which preferably exhibits the extended (B) conformations within the VTSA fragment if the Thr5 is glycosylated, can be explained by the lack of a proline at the g + 3 position.

For GalNAc-T2, we proposed the polyproline II–like conformation of the peptide substrate backbone at the glycosylation site as the structural determinants for the recognition and binding. These conformations were among the most populated structures of the nonglycosylated threonine within the GSTA region of the AHG21 peptides. The ability of GalNAc-T2 to glycosylate the N-terminal threonine in the TAP24 and TAP25 peptides (Hassan et al., 2000Go) has shown that N-terminal elongation at the glycosylation site of the substrate is not critical for GalNAc-T2 activity, in contrast to GalNAc-T1. The proline residue at position g + 3 is likely to benefit the enzyme-substrate binding affinity, however, with MUC5AC motif peptides (Tetaert et al., 2001aGo,bGo), GalNAc-T2 glycosylated the threonine residue without a proline at the g + 3 position. The lack of the GalNAc-T2 activity toward the serine within the VTSA region (Wandall et al., 1997Go; Hanisch et al., 1999Go, 2001Go) may result from a combination of two factors: the relative paucity of polyproline II conformations of the serine for both glycosylated and nonglycosylated peptides and the absence of a proline residue at position g + 3. The preference for a proline residue at the g + 3 position, demonstrated by several GalNAc-transferases, may indicate the existence of a structurally (and evolutionary) conserved subsite within the active sites of these enzymes that is responsible for specific interactions with proline residues.

For substrates catalyzed by GalNAc-T4, the most preferred conformations of threonine/serine to be glycosylated were assigned to the D area on the Ramachandran plot. These conformations were abundant for the threonine residue within the DTR fragment and for the serine in the GSTA region, if the adjacent threonine was glycosylated. Both residues have been described as the effective acceptor sites for the GalNAc-T4 (Hassan et al., 2000Go; Hanisch et al., 2001Go). For serine residues within the VTSA fragment, the D area was significantly more populated if the threonine was nonglycosylated as compared to peptides glycosylated at Thr5. Hassan and co-workers have shown, however, that GalNAc-T4 may glycosylate the serine residue within the VTSA fragment of the longer peptides, TAP24 and TAP25, if all other sites (including Thr1) were glycosylated (see Hassan et al., 2000Go: Figure 1, A, G, and L). It is likely that the N-terminal extension, TAPP, especially when all threonines are glycosylated, promotes conformational changes that are beneficial for GalNAc-T4 activity.

Thus the length of the peptide substrate might affect substrate acceptability in two different ways. First, certain physicochemical parameters (volume, charge, hydrophobicity, etc.) of the amino acid residues around the glycosylation site might promote substrate recognition by providing distinct subsites that facilitate association with the catalytic site of the enzyme. Second, the length of the proximal portions of the acceptor substrate, especially to the N- terminal side of the glycosylation site, might significantly affect its structural flexibility and conformational propensities. Indeed, our NMR data on the VTSA fragment that was differently positioned within MUC1 peptides GVT9 (Kirnarsky et al., 1998Go), PPA15 (Kirnarsky et al., 2000Go), and AHG21, showed slightly different patterns of sequential connectivities, including dNN connectivities. The PPA15 peptide, which contained two conformationally constrained prolines at the N-terminus (PPAHGVTSA), demonstrated less flexibility in the VTSA fragment (Kirnarsky et al., 2000Go) than was observed for the AHG21 peptides.

Our model for enzyme–substrate specificity proposes that the relatively broad substrate specificity of polypeptide GalNAc-transferases is a result of the recognition of the distinct substrate backbone conformations rather than the specific side chains (Kirnarsky et al., 1998Go). It was predicted that the backbone of the central part of the acceptor substrate forms a set of hydrogen bonds with the enzyme, whose alignment is facilitated by the adjacent domains on the substrates. The amide proton of the acceptor residue may serve as a donor for hydrogen bonding with the enzyme to properly align the side chain hydroxyl moiety of the threonine/serine for glycosylation. The carbonyl oxygen of the reactive residue and several flanking residues may also be involved in hydrogen bonding with the GalNAc-transferase.

It should be noted that all three preferred conformations: the extended ß-strand-like structure, the polyproline II–like conformation, and the inverse {gamma}-turn-like conformation, demonstrate significant differences in the orientation of these proposed donor/acceptor groups for hydrogen bonding. Indeed, even though these conformations are relatively close on the Ramachandran plot, where all occupied the same upper left quadrant, the superimposition of the N–C{alpha}, C{alpha}–Cß and C{alpha}–C' bonds of the acceptor residue, taken in B, P, D, or I conformations, show sharply different relative orientations of the N–H and C'–O groups. The distinct orientations are a result of 60°–70° differences for the {phi}, {psi} angles between these conformational areas.

Glycosylation affected mutual orientation of the side chains of the flanking Thr and Ser residues that could be measured by a virtual torsional angle {Theta} (i, i + 1) between vectors Cß–C{alpha}(i) ... C{alpha}–Cß(i + 1). As can be seen from the Figure 5 side chains of the Thr5–Ser6 and Ser16–Thr17 oriented diametrically opposite to each other when Thr was glycosylated. The virtual torsional angle {Theta}(5,6) for the averaged structure with the nonglycosylated Thr5 was about 120°, whereas for the Thr5* it was ~165°. For the pair Ser16–Thr17, the virtual torsional angle {Theta}(16,17) changed from 85° for the nonglycosylated Thr17 to 165° for the Thr17*. The orientation of the side chains into diametrically opposite directions is typical for extended ß-like conformations proposed by different authors for the substrate- transferase binding (Elhammer et al., 1993Go; Gerken et al., 1997Go). Thus O-glycosylation of threonine promoted a ß-like structure for the flanking Thr–Ser residues within the MUC1 tandem repeat that might be beneficial for the subsequent glycosylation of Ser.



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Fig. 5. Superimposition of the energy-minimized averaged structures of the most populated clusters with the nonglycosylated (blue) and glycosylated (red) Thr residues. (A) VTSA fragment: clusters PBID (Figure 3A) and D/IBBP (Figure 3B); (B) STAP fragment: clusters DBPP (Figure 4A) and LDB/D/IB (Figure 4B).

 
It should be noted that our working hypothesis assumes a simple "lock-and-key" mechanism of interactions between the active site of enzyme and the low-energy conformers of substrate that presumably are in a best fit conformation. From other point, according to induced fit or conformational selection models, formation of protein–ligand complexes may require significant conformational flexibility of both molecules (Koshland, 1958; Berger et al., 1999Go). Therefore, subsequent experimental studies on the formation of enzyme–substrate complex should provide useful insight into the nature of GalNAc-transferase-substrate interactions.

In summary, the studies presented here suggest that distinct conformations of the substrate backbone contribute to specific interactions of acceptor substrates with three distinct GalNAc-transferases. The finding of different preferred conformations for the acceptor sites that are differently glycosylated by the GalNAc-T1, -T2, and -T4 supports the hypothesis that there are unique active site conformations for each member of this enzyme family.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Peptide synthesis
AHG21 glycopeptides:

where T*–Thr-GalNAc, were synthesized and purified by reverse phase chromatography on preparative and analytical high-performance liquid chromatography columns in acetonitrile/0.1% aqueous trifluoroacetic acid gradients, as previously described for glycopeptides A1 to A9 (Karsten et al., 1998Go). The purity of the glycopeptides was confirmed by matrix-assisted laser desorption ionization mass spectrometry.

NMR spectroscopy
The NMR spectroscopy was performed as described previously (Kirnarsky et al., 1998Go, 2000Go). Briefly, all high- resolution 1D and 2D 1H-NMR experimental data were acquired with a 11.75 T Varian UNITYplus spectrometer (Varian, Palo Alto, CA), operating at 499.96 MHz for 1H, with a 5-mm triple-resonance inverse detection probe. Using a peptide concentration of 3 mM, spectra were recorded at 10°C in 90% H2O/10% D2O and processed and analyzed using Varian software VNMR 6.1B on a Silicon Graphics Indigo2 XZ workstation. The invariant nature of NMR chemical shifts and line widths on 10-fold dilution indicated that the peptides were monomeric in solution at the 3 mM concentration used for 2D NMR analysis. Standard NMR pulse sequences were used for 2D DQF-COSY, TOCSY, NOESY, and ROESY experiments. Water peak suppression was obtained by low-power irradiation of the H2O peak during the relaxation delay (1.2 s). A total of 512 increments of 8K data points were collected for the DQF-COSY spectra, and a total of 256 increments of 4K data points were recorded for all other experiments. All data sets were obtained in hypercomplex, phase-sensitive mode.

Proton resonance assignments were confirmed by comparison of cross-peaks in a NOESY spectrum with those in a TOCSY spectrum (Wüthrich, 1986Go) acquired under similar experimental conditions. NOESY experiments were performed with 100, 200, and 400 ms mixing times. ROESY spectra were acquired with a mixing time of 300 ms. TOCSY spectra were recorded using MLEV-17 (Bax and Davis, 1985Go) for isotropic mixing for 90 ms at a B1 field strength of 8 KHz. The H2O peak (4.91 ppm at 10°C) was used as a reference peak for chemical shift assignments. Coupling constants (3JN{alpha}) were measured directly from 1D and DQF-COSY spectra. All experiments were zero-filled to 4K data points in the t1 dimension; when necessary, spectral resolution was enhanced by Lorenzian-Gaussian apodization. A mixing time of 200 ms was used for the calculation of inter-proton upper limit distance restraints. The NOE peaks were divided into four groups with upper limits of 2.5, 3.0, 3.5, and 4.0 Å.

Structure determination protocol
All structural data were generated and visualized using the SYBYL 6.6 software package (TRIPOS Associates, St. Louis, MO). A model of the O-glycosylated Thr residue was created within SYBYL using the Amber-based Kollman force field and Glycam parameters (Woods et al., 1995Go; Woods and Chappelle, 2000Go). 3D structures of the AHG21 peptides were generated from NMR data sets using a structure determination protocol described previously (Kirnarsky et al., 1998Go, 2000Go). Briefly, the FiSiNOE-3 (Shats and Sherman, 1996Go) and HABAS (Güntert et al., 1989Go) programs were used to define angle constraints and stereospecific assignments. These data, in conjunction with the distance constraints and coupling constants, were used as input data for the DYANA program (Güntert et al., 1997Go) within SYBYL to generate 3D structures consistent with the NMR data. The standard selection of minimization levels and parameters for the DYANA program implemented within the SYBYL 6.6 were used to generate 100 structures for each peptide that were consistent with the NMR data set (distance constraints violations were less than 0.1 Å; torsion angle constraints violations were less than 5°). All DYANA-generated structures were subjected to constrained energy minimization within SYBYL using the Powell method with a maximum of 2000 minimization cycles. Calculations were performed using the Kollman "all-atom" force field, Kollman charges with distance-dependent dielectric constant, and the GLYCAM parameters for the carbohydrate moiety.

After energy minimization, the 25 best structures of each peptide were pooled together and all structures were clustered using the RMSD criterion for backbone-heavy atoms, starting from the lowest-energy structure. To evaluate conformational parameters of the amino acid residues within the cluster, averaged structures of the clusters were calculated, and each residue was compared among the clusters. To simplify comparisons, conformational angles of the averaged structures were converted to a notation based on conformational areas of the Ramachandran plot. These conformational areas were defined by clustering the protein conformations from high-resolution X-ray data (Rubinstein et al., 2001Go) and denoted as B, P, I, D, R, and L (Figure 2). Note that the P area of the Figure 2 includes clusters p and c that were defined in Rubinstein et al. (2001)Go.


    Acknowledgements
 
We thank Dmitry Shats for technical assistance with manuscript preparation. These studies were supported by NIH Grants CA84106 (to S.S.) and CA69234 (to M.A.H). The Bioinformatics Core Facility of the UNMC Eppley Cancer Center supported by the Cancer Center Support Grant P30 CA36727 was used in these studies.


    Footnotes
 
1 To whom correspondence should be addressed; e-mail: ssherm{at}unmc.edu Back


    Abbreviations
 
DQF-COSY, double quantum filtered correlation spectroscopy; MUC1, human mucin; NMR, nuclear magnetic resonance; NOESY, nuclear Overhauser enhancement spectroscopy; RMSD, root-mean-square deviation; ROESY, rotating frame nuclear Overhauser enhancement spectroscopy; TOCSY, total correlation spectroscopy


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